The role of neuregulin receptors in cell differentiation and the response to inflammatory cytokines in the intestinal epithelium

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Request accessible transcript

Transcript (if available)

Content The role of neuregulin receptors in cell differentiation
and the response to inflammatory cytokines in the
intestinal epithelium.
by
Dana Almohazey
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
in Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CRANIOFACIAL BIOLOGY)
December 2016 Dana Almohazey Copyright 2016
Acknowledgment
I would like to thank my amazing mentor Dr. Mark Frey. I could not have done it without your
guidance, support, and constant encouragement. Thank you for being there for all the ups and
downs and all the “unnecessary” (I realize that now) freak-outs. You took a risk taking me in
even though I had no previous experience in a research lab. You believed in me even more than
I did in myself and for that I am forever grateful. I would also like to thank the rest of my PhD
committee: Dr. Andy Ouellette, Dr. David Warburton, Dr. Michael Paine, and Dr. and Dr. Wei Shi.
Thank you for all your feedback, guidance, and advice. It truly helped shape up my projects.
I would like to thank my absolutely awesome lab mates and friends, “The Frey Lab”: Edie Bucar,
Jessie Bernard, Jon Hsieh, Mike Schumacher, and our neighbor and honorary member of the
Frey lab, Soula Danopoulos. Our crazy antics never ceases to amaze me. You guys offered
both laughs and advice whenever I needed it. I could not have wished for better people to work
with. You guys are my support system.
I would like to thank my rock and my childhood friend, Arwa Nusair. I enjoyed our frequent chats
and, unfortunately, not so frequent visits. After a decade, we are finally gonna be in the same
time zone again. I would also like to thank my best friend Rasha Al Fajhan. I miss you sweetie.
Last but definitely not the least, I would like to thank my mom and dad. Without your support
and unconditional love, I would not have gotten to where I am now. I love you beyond words. I
would also like to thank my sisters, brothers, nieces, and nephew: Rasha, Mariam, Fahad, Aziz,
Leena, Lama, Deema, and Ibrahim. I miss you guys soooo much. ii
Table of contents:
Acknowledgment ………………………………………………………………………………….…
List of Tables …………………………………………………………………………………….……
List of Figures ………………………………………………………………………….……………..
Abstract ……………………………………………………………………………………………….
Introduction ………………………………………………………………………………….………..
1. Small intestinal architecture …………………………………………………….….…
1.1 Histology ………………………………………………………………….….
1.2 Intestinal stem cells and epithelial lineages ……………..………………
1.3 Quiescent vs rapidly cycling ISC ……………………………….…………
1.4 Signaling in the ISC niche ………………………………………….………
2. Paneth cells ……………………………………………………………………..……..
2.1 Discovery …………………………………………………………………….
2.2 Antimicrobial properties …………………………………………………….
2.3 Paneth cells as an ISC niche ………………………………………………
2.4 Paneth cell and diseases …………………………………………………..
3. ErbB family and their ligands …………………………………………………..……..
3.1 ErbB receptor tyrosine kinase family ……………………………………
3.2 ErbB ligands ………………………………………………………………..
3.3 ErbB3 dysregulation in diseases …………………………………………
4. Myofibroblasts ……………………………………………………………….…………
Chapter 1: The ErbB3 receptor tyrosine kinase negatively regulates Paneth cells by PI3K-
dependent suppression of Atoh1……………………………………………………………………
Abstract …………………………………………………………………………………..…..
Introduction ……………………………………………………………………………..……
iii
ii
v
vi
1
3
3
3
3
5
7
8
8
8
12
13
14
14
16
19
19
23
23
25
Materials and Methods …………………………………………………………..…………
Results …………………………………………………………………………………….…
Discussion …………………………………………………………………………..……….
Chapter 2: Inflammation Mediated Regulation of NRG4 in the Intestinal Subepithelial
Myofibroblasts…………………………………………………………………………………………
Abstract ………………………………………………………………………………..……..
Introduction ………………………………………………………………………..…………
Materials and Methods ………………………………………………………………..……
Results ……………………………………………………………………………………..…
Discussion …………………………………………………………………………..……….
Conclusion ……………………………………………………………………………………………
Abbreviations used ………………………………………………………………………………….
References ……………………………………………………………………………………………
iv
27
33
45
50
50
51
53
56
62
68
72
74
List of Tables:
Table 1: Paneth cell antimicrobial peptides………………………………………………………. v
10
List of Figures:
FIGURE 1. Schematic of the intestinal epithelial crypt-villus unit…………………………..
FIGURE 2: Paneth cells…………………………………………………………………………
FIGURE 3: ErbB receptors and ligands……………………………………………………….
FIGURE 4: ErbB receptors and their tyrosine phosphorylation docking sites…………….
FIGURE 5: ErbB signaling pathways………………………………………………………….
FIGURE 6: The role of myofibroblasts and fibroblasts in inflammation……………………
FIGURE 7. ErbB3-null intestinal crypts have more Paneth cells versus wild type……….
FIGURE 8. ErbB3-null intestines express Paneth cell markers by postnatal day 7………
FIGURE 9. ErbB3-null intestinal crypts have more MMP7/MUC2
+
intermediate
cells versus wild type………………………………………………………...
FIGURE 10. Atoh1 binds to ErbB3 promoter region and down-regulates its expression..
FIGURE 11. NRG1β exposure reduced expression of Paneth and stem cell, but not
goblet cell, markers……………………………………………………………….
FIGURE 12. ErbB4 signaling does not affect homeostatic Paneth cell or ISC marker
levels……………………………………………………………………………….
FIGURE 13. ErbB3 regulates Paneth cells through the PI3K/Akt pathway……………….
FIGURE 14. TNF downregulates NRG4 expression in myofibroblasts……………………
FIGURE 15. Neither JNK or MAPK signaling are required for TNF downregulation of
NRG4………………………………………………………………………………
FIGURE 16. TNF-induced downregulation of NRG4 is through the JAK/STAT pathway..
FIGURE 17. The inhibitory effect of TNF is likely not through Stat3………………………
FIGURE 18: ErbB3 and JAK/STAT pathway as potential therapeutic targets in IBD……
FIGURE 19: ErbB3-IEKO mice have have different microbial profile than their control
littermates…………………………………………………………………………
vi
4
9
15
17
18
21
34
36
37
39
40
42
43
57
58
59
61
70
71
Abstract
Crohn’s disease (CD) and neonatal necrotizing enterocolitis are characterized by epithelial
injury, defects in Paneth cells (PCs), and a disrupted subepithelial myofibroblast layer. Our lab is
interested in investigating novel treatments for these diseases aimed at protecting the
epithelium by studying the role of the neuregulin receptors, ErbB3 and ErbB4, and how they are
regulated during inflammation. PCs support the intestinal stem cells (ISC) with growth factors
and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and
defensins. The specific pathways regulating Paneth cell development and function are not fully
understood. Here we tested the role of the neuregulin receptor ErbB3 in control of Paneth cell
differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious
appearance of Paneth cells as early as postnatal day 7, and substantially increased the number
of mature Paneth cells in adult mouse ileum. ErbB3 loss had no effect on other secretory
lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated
levels of the Atoh1 transcription factor, which is required for secretory fate determination, while
Atoh1
+
cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal
progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting
these pathways in HT29 cells increased levels of ATOH1 and the Paneth cell marker LYZ.
Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner.
Interestingly, expression of the neuregulin ligands for ErbB3 and ErbB4 in the intestinal
subepithelial myofibroblasts (ISEMFs) is altered during inflammation. We previously showed
that neuregulin 4 (NRG4- ErbB4 ligand) is downregulated in CD patients, but not neuregulin 1
(NRG1- ErbB3/4 ligand). Here, we show that TNF (tumor necrosis factor), an inflammatory
cytokine, causes a reduction in NRG4 expression in ISEMFs. To understand the mechanism, we
investigated pathways known to interact with TNF. Inhibiting the JAK/STAT pathway, but not JNK
or MAPK, abrogated the reduction in NRG4. Our results show that TNF activation of the JAK/
1
STAT pathway causes a downregulation of NRG4 expression in ISEMFs. Inflammatory
cytokines result in a decrease in NRG4 with no change in NRG1, thus potentially favoring the
activation of ErbB3 over ErbB4 during inflammation. The dominance of ErbB3 activation could
lead to a reduction in PCs, thus compromising the epithelium. These projects are aimed at
understanding the mechanism of two defects in CD with the hope of finding novel therapeutic
options.
2
Introduction
Parts of the introduction were published in: Stem cells, tissue engineering, and regenerative
medicine, Warburton, David, 1949-. New Jersey : World Scientific, 2014. NLM ID: 101634466
[Book]. Chapter: The intestinal stem cell niche and its regulation by ErbB growth factor
receptors, Dana Almohazey and Mark R. Frey.
The intestine is a fascinating organ with a turnover of 3-5 days making it a great model to test
stem cell regeneration. Our lab is interested in finding novel ways to treat Inflammatory bowel
disease which is characterized by damages to the epithelium of the gastrointestinal tract. Here, I
will discuss my project’s organ of interest and what we know about the ErbB receptor tyrosine
kinases in the intestine.

1. Small intestinal architecture
1.1 Histology
The small intestine is the part of the gastrointestinal tract where most of the nutrient and water
absorption occurs. It has three regions: the duodenum, jejunum, and ileum. Histologically, the
small intestine is composed of the mucosa that contains the epithelium and the lamina propria,
followed by multiple layers of smooth muscle and connective tissue known as the submucosa,
muscularis externa, and serosa (Kerr, 2010). The intestinal epithelium is made up of a single
epithelial cell layer that is structured into peaks and troughs known as villi and crypts of
Lieberkühn, respectively (Fig 1).
1.2 Intestinal stem cells and epithelial lineages
The small intestine has two important roles: one is being part of the digestive process, while the
other is as a protective barrier from harmful substances and microorganisms passing through 3
FIGURE 1. Schematic of the intestinal epithelial crypt-villus unit. The stem cell compartments
are comprised of narrow, Lrg5+ cells anchored between Paneth cells at the base of the crypt
(“crypt base columnar” cells) and cells at the “+4” position (panel from (Almohazey and Frey,
2014). Inset: scanning electron microscopy image of the small intestine (panel from (Barker,
2014). 4
+4 cell
position
Paneth Cell
Goblet Cell
ISC
CBC
Cell
Shedding
Intestinal
Lumen
Enteroendocrine/
Enterocyte
Mesenchymal
Cell
Lamina
Propria
Muscularis
Mucosa
Tuft Cell
Crypt of
Lieberkühn
Villus
Nature Reviews | Molecular Cell Biology
a Small intestine
b Colon
Migration
Migration
Crypt
Proliferating TA cell
Proliferating TA
cell
Paneth cell
+4 stem cell
LGR5
+
stem cell
Enteroendocrine
cell
Enteroendocrine
cell
Tu cell
Enterocyte
Crypt
Villus
Goblet cell
Anoikis
Crypt
LGR5
+
stem cell
LGR5
+
stem cell
Tu cell
Goblet cell
Surface
epithelium
Absorptive
enterocyte
Absorptive
enterocytes
Paneth
cell
LGR5
+
CBC
stem cell
Tu cell
Goblet cell
Goblet cells
Enteroendocrine cell
Enteroendocrine cell
+4 stem cell
TA cells
TA cells
Tu cell
Absorptive
enterocyte
Villus
Crypt
Chimeric mice
Mice that are comprised of
two or more populations
of genetically distinct cells.
Adult stem cell origins in the intestine. Proliferating
cells displaying stem cell characteristics are present in
ex vivo cultures of E14 fetal intestinal epithelium
20–22
.
Analysis of genetic marker expression patterns in
newborn chimeric mice has shown that nascent crypts are
polyclonal, as they develop from fetal stem cells derived
from both parents
23,24
. During crypt morphogenesis in
the first 2 weeks of life, these early stem cell populations
Figure 1 | Epithelial self-renewal in the intestinal epithelium. a | In the small intestine (the structural organization of which
is shown in the scanning electron micrograph in the left panel), LGR5
+
(Leu-rich repeat-containing G protein-coupled
receptor 5-expressing) crypt base columnar (CBC) stem cells are intercalated with Paneth cells at the crypt base (middle
panel). These stem cells continuously generate rapidly proliferating transit-amplifying (TA) cells, which occupy the remainder
of the crypt. TA cells differentiate into the various functional cells on the villi (enterocytes, tuft cells, goblet cells and
enteroendocrine cells) to replace the epithelial cells being lost via anoikis at the villus tip. The +4 ‘reserve’ stem cells (which
occupy the fourth position from the crypt base) can restore the LGR5
+
CBC stem cell compartment following injury. This
differentiating hierarchy is shown in the tree on the right panel. Epithelial turnover occurs every 3–5 days. New Paneth cells
are supplied from the TA cells every 3–6 weeks. b | In the colon (the structural organization of which is shown in the scanning
electron micrograph in left panel), LGR5
+
stem cells at the crypt base generate rapidly proliferating TA cells in the lower half of
the crypt (middle panel). TA cells subsequently differentiate into the mature lineages of the surface epithelium (goblet cells,
enterocytes, enteroendocrine cells and tuft cells), as shown in the lineage tree on the right panel. Epithelial turnover occurs
every 5–7 days. Images in parts a and b are reproduced, with permission, from REF . 123 © Wiley (1986)
REVIEWS
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 15 | JANUARY 2014 | 21
the intestinal lumen. To keep up with these functions, the epithelium has to regenerate at an
extremely high rate with a turnover of about 3-5 days in mice (Cheng and Leblond, 1974). The
intestinal stem cells (ISCs) that are in charge of this turnover are anchored at or near the base
of the crypts. They divide to produce proliferative daughter cells, which then differentiate into
either an absorptive or a secretory cell lineage. The absorptive cell lineage is composed entirely
of enterocytes that make up the highest percentage of the epithelial lining, while the secretory
cell lineage is composed of tuft cells, goblet cells, enteroendocrine cells, and Paneth cells (PCs)
(Fig 1).
1.3 Quiescent vs rapidly cycling ISC
There are two well-described subsets of stem cells in the small intestinal epithelial barrier
(Barker et al., 2012; Potten, 1977): the rapidly cycling crypt based columnar cells (CBCs)
sandwiched between the Paneth cells at the base of the crypts, and the “reserve” +4 stem cells
which as the name implies are located (on average) four cells from the base of the crypt right
above the Paneth cells (Fig 1). Lineage tracing studies have revealed that these two
populations are both multi-potent (Barker et al., 2007; Montgomery et al., 2011; Potten, 1998),
and are likely able to interconvert through as yet poorly-defined mechanism(s). However, they
express distinct patterns of gene expression.
The +4 cells were the first-identified ISCs, originally defined as the label-retaining cells of the
gut epithelium. They express Bmi1 (primarily in the proximal small intestine), mTert, Hopx, and
leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) (Montgomery et al., 2011;
Sangiorgi and Capecchi, 2008; Takeda et al., 2011). Lrig1 has recently been a focus of
attention. It is highly expressed on ISCs (Powell et al., 2012) is a negative regulator of ErbB
5
receptor tyrosine kinases (see section 3), and is a tumor suppressor (Wang et al., 2013; Wong
et al., 2012).
In 2007, Barker and colleagues identified the CBCs as a rapidly-cycling stem cell population
(Barker et al., 2007). They express Lgr5 (leucine-rich-repeat-containing G-protein-coupled
receptor 5), a Wnt target gene and Wnt signaling amplifier which is a stem cell marker for
multiple tissues. The same group later identified Ascl2, another Wnt target gene, as another
CBC marker (van der Flier et al., 2009); its conditional deletion leads to loss of Lgr5+ CBCs. A
third major marker of this cell population is Olfm4, which is highly expressed in human small
intestinal and colonic CBCs, but only in the small intestinal CBCs of the mouse. Although its
function is not fully established, it is believed to behave in a Notch-dependent manner
(VanDussen et al., 2012). The observation that two of these markers are Wnt target genes
highlights how crucial the Wnt pathway is in the development and regulation of the stem cell
niche (see section 1.4).
Interestingly, recent studies have suggested significant overlap between the CBCs and +4 cells.
The Clevers group has published studies suggesting that there are Lgr5+ CBC cells that also
express Bmi1, Tert, Hopx, and Lrig1 (Munoz et al., 2012), which are generally thought of as +4
cell markers. Furthermore, the CBCs can lineage trace the entire crypt compartment, including
the +4 cells. However, +4 label retaining cells do appear to act as a reserve to replenish the
Lgr5+ cells when needed. In mice, this occurs only after injury/depletion of CBCs such as with
radiation or chemotherapy (Buczacki et al., 2013). The various reports of overlap in markers and
function between the CBC cells and +4 cells seem to point to a model of at least two inter-
convertible stem cell populations, likely maintained by the intestinal epithelium as a partially
redundant system to address different possible homeostatic, injury, and disease states in this
rapidly renewing tissue.
6
1.4 Signaling in the ISC niche
The regeneration and homeostasis of the ISC niche is governed by multiple signaling pathways.
The most widely-studied, and perhaps most influential during development, is Wnt/β-catenin. In
response to exogenous Wnt ligand binding to its receptor frizzled, β-catenin is stabilized in the
cytoplasm and then undergoes translocation to the nucleus, where it binds to the TCF/LEF co-
activator and causes the transcription of target genes (Korswagen, 2002). As discussed above,
at least two key ISC markers (Lgr5 and Ascl2) are Wnt target genes. Transgenic expression of
yes-associated protein 1, which inhibits Wnt signaling through binding the disheveled mediator,
leads to ISC loss and intestinal epithelial atrophy. Furthermore, it has been recently shown that
Lgr5 is a receptor for the Wnt-amplifying ligand R-spondin1. Lgr5 is physically associated with
frizzled and LRP5/6 (co-receptors of Wnt). Thus, Lgr5 is both a Wnt/β-catenin signaling target
and an upstream regulator of Wnt signaling (Carmon et al., 2011; de Lau et al., 2011), and tight
regulation of Wnt signaling is essential for maintenance of an appropriately sized stem cell pool.
Emerging evidence of crosstalk between the receptor tyrosine kinase epidermal growth factor
receptor (EGFR, a.k.a. ErbB1) and Wnt signaling pathways [reviewed in Ref. (Hu and Li, 2010)],
as well as evidence that the related family member ErbB4 cooperates with yes-associated
protein 1 in transcriptional regulation (Komuro et al., 2003) indicate a role for ErbB growth factor
receptors as modulators of Wnt signaling and thus, ISC development and homeostasis. Notch
signaling is another pathway that is implicated in the crypt homeostasis. Notch and EGFR
interact at the level of intracellular signaling in several tissues (e.g., lung epithelium) (Kang et
al., 2011). Notch has been shown to drive the differentiation and influence the cell fate towards
either a secretory or absorptive state (van Es et al., 2005), which the Samuelson laboratory
showed occurs at least in part through repression of the lineage-specifying transcription factor
7
Math1/Atoh1 (VanDussen et al., 2012). The target genes of this pathway are still under study;
however, Olfm4 (a CBC marker) is clearly a Notch target gene.
2. Paneth cells
2.1 Discovery
Paneth cells (PCs) were discovered by Gustav Schwalbe and Josef Paneth in the 1800s and
described as columnar epithelial cells with prominent cytoplasmic granules (Clevers and Bevins,
2013). In addition to these granules, Paneth cells are characterized by their comprehensive
endoplasmic reticulum and Golgi network (Fig 2). In humans, Paneth cells develop by 11 to 12
weeks of gestation (Moxey and Trier, 1978), while in mice by postnatal day 10 (Bry et al., 1994;
Ouellette et al., 1989). In both humans and mice, Paneth cells initially appear in low numbers
and become more common as development proceeds. Paneth cells are primarily in the small
intestine especially in mice, though they have been observed in modest numbers in the human
proximal colon or more broadly in some inflammatory conditions. Unlike the other cell types that
differentiate and migrate upwards along the crypt-villus axis, Paneth cells persist in the crypt
base along with the Lgr5+ ISCs. They also turn over much more slowly than other intestinal
epithelial cells, with a life span of about 57 days (Ireland et al., 2005).
2.2 Antimicrobial properties:
Paneth cells are considered to be part of the innate intestinal immune defense as they secrete
several antimicrobial peptides (AMPs) (Table 1). These AMPs are packaged into cytoplasmic
granules near the apical region and are released into the lumen in response to stimuli such as
bacteria or its products, and cholinergic agonists (Ayabe et al., 2002; Satoh et al., 1992). These
AMPs include: defensins, the regenerating islet-derived protein 3-alpha (REG3α), lysozyme C, 8
FIGURE 2: A.Transmission electron micrograph of small intestinal Paneth cells. Note the
characteristic granules (G) and endoplasmic reticulum (ER) (panel adapted from (Stappenbeck,
2010). B. An immunofluorescent image of a small intestinal crypt stained for: lysozyme (Paneth
cell marker, green), E-Cadherin (epithelial marker, red), and nuclei (blue). 9
MucosalImmunology | VOLUME 3 NUMBER 1 | JANUARY 2010 9
nature publishing group COMMENTARIES
highly secretory cells, Paneth cells addi-
tionally contain a robust rough endoplas-
mic reticulum and Golgi apparatus that
act as part of the “ factory ” to produce
these proteins.
The production of antimicrobial
proteins by Paneth cells seems to have
functional consequences in both health
and disease. These antimicrobial pro-
teins likely shape the composition and
abundance of the species of indigenous
microbes that reside within the intestinal
lumen. Cryptdins produced by Paneth
cells have also been shown to have a role
in the clearance of pathogens. In one
example, mice that were engineered to
overexpress one member of the cryptdin
family of proteins were much less sus-
ceptible to oral infection by Salmonella
typhimurium .
4

Paneth cells also secrete proteins that
infl uence other functions in the intestine.
Th e Wnt ligands that they produce likely
aff ect stem cells in the crypt base. Th ey
also produce factors that infl uence villus
vasculature development. Paneth cell also
produce infl ammatory cytokines such as
tumor necrosis factor- ! (reviewed by
Stappenbeck
2
) . I t i s n o t k n o w n w h e t h e r
these factors are secreted apically or baso-
laterally. Th e ability to now grow intesti-
nal crypts in culture will aid in the study
of the mechanism of secretion of this cell
type (for example, see Sato
5
).
Not only do Paneth cells potentially
alter the composition of the microbiome
in the gut lumen but these microbes can
influence Paneth cell gene expression
and function as well (reviewed by Stap-
penbeck
2
) . F o r e x a m p l e , o n e o f t h e m a j o r
bacterial species in the small intestine,
Bacteroides thetaiotaomicron , s t i m u l a t e s
the expression of an antimicrobial pro-
tein, angiogenin-4, in Paneth cells. Th is
bacterium also stimulates angiogenesis in
the mesenchymal core of small intestinal
villi through Paneth cells. The Paneth
cell – intestinal bacterial cross-talk is
particularly interesting in light of recent
fi ndings that show that Paneth cells can
limit bacterial penetration of intestinal
microbes across the epithelial barrier.
Th is function depends on Toll-like recep-
tor – Myd88 signaling in Paneth cells.
The fact that Paneth cells regulate
the ability of intestinal bacteria to pass
through the epithelial barrier suggests
that autophagy may be important in this
cell type. Autophagy is a process that
occurs in all cells and its major function
is the recycling of intracellular compo-
nents.
6
Th e machinery that is required
for this macro-autophagocytic process
is complex and requires >20 different
proteins to generate the classic dou-
ble membrane-bound structures in the
cytosol. Th ese autophagocytic vesicles are
then delivered to lysosomes for recycling
of individual components. In some cell
types, loss of autophagy function can lead
to alterations in proliferation, cell death,
and loss of the ability to clear intracel-
lular pathogens. Paneth cells have been
observed to form autophagocytic vesicles
when cells are stressed by processes such
as irradiation.
7

Another reason to consider the role of
autophagy in Paneth cells (as well as in
other intestinal cell types) is that patients
with one form of infl ammatory bowel
disease, Crohn ’ s disease, have polymor-
phisms in specifi c genes, including the
autophagy gene Atg16L1 . T h i s p r o -
tein forms a complex with two other
autophagy proteins, Atg5 and Atg12,
which together function to extend the
membrane of a nascent autophagocytic
vesicle. Loss of autophagy function in
fi broblasts through the manipulation of
Atg16L1 function suggests that bacterial
clearance is impaired because of loss of
autophagy.
8
However, loss-of-function
studies in mice for Atg16L1 did not fi nd
a role in the clearance of enteric patho-
genic bacteria, including Salmonella and
Listeria.
9

Instead, mice with loss of function for
Atg16L1 showed defects in the secretory
function of Paneth cells. Autophagy-
defi cient Paneth cells properly commit-
ted to diff erentiation and did not seem to
undergo premature apoptosis. However,
diminished autophagy function in Paneth
cells led to defects in the packaging of
antimicrobial proteins into granules and
eventually their proper export into the
lumen of the gut is defective.
9
I n s t e a d
of a robust endoplasmic reticulum and
secretory vesicles, the autophagy-defi -
cient Paneth cell cytoplasm contained
few granules and was typically fi lled with
small vesicles ( Figure 2 ) . Th e nature of
these cytoplasmic vesicles is still unclear.
To test for the possibility that they might
be endocytic vesicles that sample lumi-
nal bacteria, we stained for bacteria on
tissue sections. However, we have thus
far found no evidence that these vesi-
cles contain bacteria. Microarray stud-
ies of these Paneth cells showed that in
Figure 1 “ Good ” Paneth cells. T ransmission electron micrograph of small intestinal Paneth cells
from a wild-type mouse. Note the electron-dense secretory granules and layered endoplasmic
reticulum.
A. B.
Lysozyme
E-Cadherin
DAPI
G
ER
ANG4, angiogenin 4; CRS, cryptdin-related sequence; sPLA2, secretory group IIA
phospholipase A2 (also known as PLA2G2A) (Bevins and Salzman, 2011).
10
Table 1: Paneth cell antimicrobials
Name Biochemical
Classification
Antibacterial activities Transcriptionally
induced
α-defensins Antimicrobial
peptides
Against Gram-positive
and Gram-negative
bacteria
No
Lysozyme C β-1,4-
glycosidase
Against Gram-positive
and (to a lesser extent)
Gram-negative bacteria
No
sPLA2 Phospholipid-
sn-2 esterase
Against Gram-positive
bacteria only
No
REG3α in humans
and REG3γ in
mice
Antimicrobial C-
type lectin
Against Gram-positive
bacteria only
Yes
ANG4 (in mice) Ribonuclease Against Gram-positive
and Gram-negative
bacteria
Yes
CRS peptides (in
mice)
Antimicrobial
peptides
Against Gram-positive
and Gram-negative
bacteria
No
phospholipase A2 (PLA2), cryptdin-related sequence (CRS- only in mice), and Angiogenin 4
(ANG4- only in mice). Bellow is a brief description of the most abundant/best studied AMPs.
Defensins: α-defensins (cryptdins in mice) are the most abundant AMPs within the small
intestine (Wehkamp et al., 2006). They are constitutively expressed regardless of microbial
stimuli as they are present in germ free mice (Ayabe et al., 2002). In humans, there are only two
defensins expressed in Paneth cells: human defensin-5 (HD5) and -6 (HD6) (Jones and Bevins,
1992, 1993; Porter et al., 1997b). While the antimicrobial properties of HD5 have been known
for nearly two decades (Porter et al., 1997a), HD6 has been more elusive. Interestingly, HD6
binds to bacterial surface proteins, self-assembles to form nanonets, and thus traps the targeted
bacteria (Chu et al., 2012). Alpha defensins are synthesized as an inactive propeptide and
require proteolytic cleavage to into the active form. In mice, this proteolytic processing is
achieved by Matrix metalloprotease 7 (MMP7- also known as matrilysin) (Ayabe et al., 2002;
Wilson et al., 1999). However in humans, this step is accomplished by trypsin and MMP7 is not
detected in human Paneth cells (Ghosh et al., 2002).
REG3α: The regenerating islet-derived protein 3-alpha (REG3α) (REG3γ in mice) is a C-type
lectin (Christa et al., 1996). It shares a similarity with defensins as it is proteolytically cleaved to
its active form by trypsin (Mukherjee et al., 2009). However, unlike some constitutively
expressed AMPs such as defensins and lysozyme, REG3α is transcriptionally induced by the
Toll-like receptor (TLR) and its adaptor molecule MYD88 (Brandl et al., 2007; Vaishnava et al.,
2008).
Lysozyme C: Lysozyme C enzyme was detected in Paneth cells, macrophages, neutrophils,
and in secretions (Bevins and Salzman, 2011; Peeters and Vantrappen, 1975; Speece, 1964).
11
While humans have a single form of the enzyme encoded by the gene LYZ, mice have two
isoforms (Cross et al., 1988). The gene Lyz1 or LyzP encodes lysozyme C, type P, expressed in
Paneth cells, and Lyz2 or LyzM encodes lysozyme C, type M, in macrophages(Bevins and
Salzman, 2011; Cross et al., 1988). Lysozyme C is also expressed in germ free mice suggesting
that its expression is not dependent on a bacterial trigger (Satoh et al., 1988). Lysozyme is
commonly used as a Paneth cell marker in the intestine, due to the availability of antibodies and
the relatively proportional expression (i.e., lysozyme RNA and protein levels are a good
surrogate for number of Paneth cells).
2.3 Paneth cells as an ISC niche:
Based on the physical proximity of Paneth cells and the intestinal stem cells, investigators
hypothesized an interdependent relation. Surprisingly, multiple Paneth cell ablation mouse
models did not show any dramatic disruption of the crypt architecture (Garabedian et al., 1997;
Shroyer et al., 2007; Shroyer et al., 2005). However, it was later shown that these mouse
models still had a very limited number of Paneth cells and ISCs that were clustered together
(Sato et al., 2011), supporting the initial hypothesis. It was further confirmed when sorted Lgr5+
intestinal stem cells grew more efficiently when cultured with Paneth cells than when cultured
alone. These observations could be attributed to the wide range of growth factors that Paneth
cells secrete, such as Wnt3, Wnt11, EGF, TGFα, and the Notch ligand Dll4 (Sato et al., 2011).
However, it was recently shown that ablation of Foxl1+ fibroblasts led to a dramatic loss of
proliferation and a reduction of ISC makers with no effects on Paneth cells (Aoki et al., 2016).
Taken together, these reports suggest that while Paneth cells are not completely essential for
the stem cell niche, they certainly contribute in normal biology and in the response to stress.
12
2.4 Paneth cells and diseases:
Inflammatory bowel disease (IBD) is characterized by breaches in the epithelial barrier and a
chronic inflammation. Crohn’s disease (CD) is one of two distinct diseases that fall under the
IBD umbrella (the other being ulcerative colitis). CD most commonly affects the ileum, although
it can affect any region of the gastrointestinal tract. Symptoms include diarrhea or constipation,
rectal bleeding, and abdominal pain (Baumgart and Sandborn, 2012). Unfortunately, there is no
cure but current treatments are limited to surgical removal of affected regions or arresting the
heightened inflammatory response. Patients often present with a reduction in Paneth cell
numbers or their antimicrobial peptides, such as HD5 and HD6, implying that Paneth cell
abnormalities may have a role in initiating CD or its progression (Lewin, 1969; Wehkamp et al.,
2005). Furthermore, risk alleles that are commonly seen in CD patients, such as NOD2,
ATG16L1, and XBP1, directly affect Paneth cell function (Hampe et al., 2007; Hugot et al., 2001;
Kaser et al., 2008). Paneth cell-specific deletion in mice of Xbp1, a transcription factor involved
in the unfolded protein response, resulted in abnormal Paneth cell granules and the
development of spontaneous enteritis (Adolph et al., 2013). Furthermore, CD patients harboring
the ATG16L1 risk allele that affects autophagy present with abnormal Paneth cell granules,
which is indicative of impaired function (Cadwell et al., 2008; VanDussen et al., 2014). All these
findings taken together suggest that CD might be, on some level, a Paneth cell disease.
Paneth cells are observed in the healthy proximal colon of the human, albeit at lower levels than
in the small intestine. However in IBD, Paneth cell metaplasia is detected throughout the entire
colon (Cunliffe et al., 2001; Lewin, 1969; Simmonds et al., 2014; Tanaka et al., 2001).
Interestingly, HD5 and, to a lesser extent, lysozyme were detected in upper gastrointestinal
metaplasia at high frequency after chronic Helicobacter pylori infection (Shen et al., 2005). It is
likely that Paneth cell metaplasia is a coping mechanism against infections.
13
Neonatal necrotizing enterocolitis (NEC) is another inflammatory disorder that is believed to be
associated with dysfunctional Paneth cells. NEC occurs in preterm infants and is associated
with a reduction in Paneth cell numbers and its AMPs (Coutinho et al., 1998; Zhang et al.,
2012a). Although mRNA expression of HD5 and HD6 are upregulated in some NEC patients,
this increase did not correlate with protein levels hinting at either a disrupted translation or an
increased AMP secretion (Salzman et al., 1998). Interestingly, a murine NEC model which
employs Paneth cell ablation and bacterial infection does not result in NEC when performed
before the development of mature Paneth cells (Zhang et al., 2012a). Since PCs secrete
proinflammatory cytokines such as TNF (Schmauder-Chock et al., 1994), it is possible that
defective Paneth cells both defend against and, when defective, contribute to the pathogenesis
of NEC.
3. ErbB family and their ligands:
3.1 ErbB receptor tyrosine kinase family:
The ErbB tyrosine kinases make up a family of type I transmembrane growth factor receptors
[for review, see Refs. (Frey and Brent Polk, 2014; Linggi and Carpenter, 2006)]. Family
members include the prototypic ErbB1, epidermal growth factor receptor (EGFR), as well as
ErbB2/HER2, ErbB3, and ErbB4, and splice variants of the same (e.g., up to at least four and
possibly more distinct ErbB4 products with substantially different biochemistry have been
described). These receptors recognize a suite of ligands including epidermal growth factor
(EGF), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, and the heregulin/
neuregulin family (Fig 3). Ligand binding is associated with receptor dimerization, either as
homo- or heterodimers, increased tyrosine kinase activity, and auto-phosphorylation on C-
terminal tyrosine residues. Phosphotyrosines on the C-terminus then provide docking sites for 14
FIGURE 3: ErbB receptors and ligands. With varying efficiency and specificity, ErbB1 (EGFR),
ErbB3, and ErbB4 bind a suite of ligands including epidermal growth factor

(EGF), amphiregulin
(AR), transforming growth factor (TGF)-α, betacellulin (BTC), heparin-binding EGF-like growth
factor (HB-EGF), epiregulin (EPR), and neuregulins (NRG) 1–4. ErbB2 has no known ligand. L,
leucine-rich region; CR, cysteine-rich domain; TM, transmembrane region; JM, juxtamembrane
region; CT, carboxy-terminus (panel from (Almohazey and Frey, 2014). 15
Extracellular Ligand Binding
Domain
Transmembrane Region
Cytoplasmic Tyrosine Kinase
Domain
L1
L2
Kinase Tail
CT
ErbB1
(EGFR)
ErbB4 ErbB2 ErbB3
L1
L2
CT
Kinase Tail
L1
L2
Kinase Tail
CT
L1
L2
Kinase Tail
CT
EGF
AR
TGF-α
BTC
HB-EGF
EPR
NRG1
NRG2
NRG3
NRG4
CR2 CR2 CR2 CR2
TM
JM JM JM JM
TM TM TM
CR1 CR1 CR1 CR1
downstream effectors (Fig 4, 5). Unique biological differences between the family members
presumably contribute to fine regulation of signaling. For example, ErbB2 has no known ligand,
and is thought to function only as an “amplifier” in heterodimers with other ErbBs, while ErbB3
binds ligand but has greatly attenuated kinase activity (and indeed was believed for some years
to be kinase-dead), and thus is also likely to serve primarily as a modifier of other receptors’
signaling. Variability in the downstream C-terminal phosphotyrosine residues also adds
complexity; for example, ErbB3 has a more robust panel of phosphatidylinositol 3-kinase
binding sites relative to other ErbBs, while ErbB4 has a restricted panel of functional
downstream effector binding sites compared to EGFR, ErbB2, or ErbB3 (Fig 4, 5) (Kaushansky
et al., 2008). The following chapters will be focused on studying the neuregulin receptors, ErbB3
and ErbB4.
3.2 ErbB ligands:
Different ErbB ligands show distinct specificities and affinities for different ErbB receptors
(Wilson et al., 2009), and stimulate diverse dimerization patterns, downstream signaling, and
cellular responses. For example, EGF binds only EGFR, and in intestinal epithelial cells
promotes a broad range of responses including proliferation, migration, and survival (Yamaoka
et al., 2011); in contrast, neuregulin-4 (NRG4) binds only ErbB4 and preferentially promotes
intestinal cell survival but not proliferation or migration (Bernard et al., 2012). Some ligands bind
multiple receptors: for example, HB-EGF is shared by EGFR and ErbB4, while neuregulin-1β is
recognized by both ErbB3 and ErbB4. Thus, fine-tuning of the cellular response by an
appropriate balance of available growth factors is theoretically possible, though this has not
been investigated in any depth.
16
FIGURE 4: ErbB receptors and their tyrosine phosphorylation docking sites for regulatory
molecules involved in several signaling pathways (panel from (Wilson et al., 2009). 17
Differences in the sites of ligand-induced EGFR tyrosine phosphor-
ylation may underlie divergent ligand-induced EGFR coupling to
signalingeffectorsandbiologicalresponses.Forexample,EGFstimulates
abundant EGFR phosphorylation at Tyr1045 whereas AR does not
(Gilmore et al., 2008). Phosphorylation of Tyr1045 creates a canonical
binding site for the E3 ubiquitin ligase c-cbl, leading to EGFR
ubiquitination and degradation by the 26S proteasome (Levkowitz
etal.,1998;Levkowitzetal.,1999).Thus,unlikeAR,EGFstimulatesc-cbl
bindingtoEGFR,EGFRubiquitination,andEGFRdegradation(Sternetal.,
2008).Wehypothesizethatdifferentialligand-inducedEGFRphosphor-
ylation at Tyr1045 and differential EGFR coupling to cbl-dependent
ubiquitination and turnover leads to distinctions in the duration of
ligand-induced EGFR signaling, thereby accounting for the inability of
EGFtostimulatemotilityandinvasivenessincellsthatdorespondtoAR.
Differencesinthesitesofligand-inducedreceptorphosphorylation
may also account for divergence in the intrinsic activity of ErbB4
ligands. The ErbB4 ligands BTC, NRG1β, NRG2β, and NRG3 differen-
tially stimulate ErbB4 coupling to survival and proliferation in CEM/
ErbB4cellsanddifferentiallystimulateErbB4couplingtoShc,p85,Akt,
andErk1/2.Thisisaccompaniedbydistinctionsinthesitesofligand-
induced ErbB4 tyrosine phosphorylation (Sweeney et al., 2000).
NRG1β stimulates ErbB4 phosphorylation at nineteen tyrosine
residues (Kaushansky et al., 2008); these residues are candidates for
sites of ligand-specific tyrosine phosphorylation. However, ligand-
specific sites of ErbB4 tyrosine phosphorylation have yet to be iden-
tifiedandthebiologicalrelevanceofdifferencesinthesesitesofErbB4
phosphorylationhasyettobeestablished.
3.2.Differences intheconformationof theliganded
receptorextracellulardomainmayaccountfordistinctpatterns
ofErbBreceptortyrosinephosphorylationanddownstream signaling
ThebindingofanEGFfamilymembertoitscognateErbBreceptor
stabilizes the receptor extracellular domains in an extended con-
formation that exposes a dimerization arm in subdomain II, thereby
facilitating dimerization of the extracellular region (Burgess et al.,
Fig. 3.LigandstimulationofErbBreceptortyrosinephosphorylationcreatesdockingsitesfornumeroussignalingeffectors.PutativesitesofEGFR,ErbB2,ErbB3,andErbB4tyrosine
phosphorylationaredenoted,aswellassignalingeffectorspredictedorshowntobindtothesesitesofphosphorylation(Rotinetal.,1992;Cohenetal.,1996;Zrihan-Lichtetal.,1998;
Zrihan-Licht et al.,1998; Keilhack et al.,1998; Hellyer et al., 2001; Schulze et al., 2005; Kaushansky et al., 2008). The ErbB receptors are not drawn to scale.
4 K.J. Wilson et al. / Pharmacology & Therapeutics 122 (2009) 1–8
FIGURE 5: ErbB signaling pathways (panel from (Yarden and Sliwkowski, 2001).
18
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | FEBRUARY 2001 | 129
REVIEWS
A network of networks?
The ErbB network might integrate not only its own
inputs but also heterologous signals, including hor-
mones, neurotransmitters, lymphokines and stress
inducers
29
(FIG. 1).Many of these trans-regulatory inter-
actions are mediated by protein kinases that directly
phosphorylate ErbBs, thereby affecting their kinase
activity or endocytic transport
29
.The most extensively
studied mechanism involves activation of G-protein-
coupled receptors (GPCRs) by agonists such as
lysophosphatidic acid (LPA), carbachol (which specifi-
cally activates muscarinic acetylcholine receptors) or
thrombin (FIG. 2).
Experiments done with mutants and inhibitors of
ErbBs imply that the mitogenic activity of some GPCR
agonists requires transactivation of ErbB proteins.
These agents increase tyrosine phosphorylation of
ErbB1 and ErbB2, either by increasing their intrinsic
apoptosis (FIG. 1).Output depends on cellular context,as
well as the specific ligand and ErbB dimer. This has
been best shown in terms of mitogenic and transform-
ing responses: homodimeric receptor combinations are
less mitogenic and transforming than the correspond-
ing heterodimeric combinations, and ErbB2-containing
heterodimers are the most potent complexes
21–23
(FIG. 3).
Perhaps the best example of the ability of the ErbB
module to tune mitogenic signalling is provided by the
ErbB2–ErbB3 heterodimer: although neither ErbB2
nor ErbB3 alone can be activated by ligand, the het-
erodimer is the most transforming
24,25
and mitogenic
21
receptor complex. The ErbB2–ErbB3 heterodimer also
increases cell motility on stimulation with a ligand
26
;
but the other NRG receptor, ErbB4, which exists in sev-
eral isoforms, has been associated with processes vary-
ing from cellular chemotaxis
27
to proliferation and dif-
ferentiation
28
.
Figure 1 | The ErbB signalling network. a | Ligands and the ten dimeric receptor combinations comprise the input layer.
Numbers in each ligand block indicate the respective high-affinity ErbB receptors
8
. For simplicity, specificities of receptor binding
are shown only for epidermal growth factor (EGF) and neuregulin 4 (NRG4). ErbB2 binds no ligand with high affinity, and ErbB3
homodimers are catalytically inactive (crossed kinase domains). Trans-regulation by G-protein-coupled receptors (such as those
for lysophosphatidic acid (LPA), thrombin and endothelin (ET)), and cytokine receptors is shown by wide arrows. b | Signalling to
the adaptor/enzyme layer is shown only for two receptor dimers: the weakly mitogenic ErbB1 homodimer, and the relatively
potent ErbB2–ErbB3 heterodimer. Only some of the pathways and transcription factors are represented in this layer. c | How
they are translated to specific types of output is poorly understood at present. (Abl, a proto-oncogenic tyrosine kinase whose
targets are poorly understood; Akt, a serine/threonine kinase that phosphorylates the anti-apoptotic protein Bad and the
ribosomal S6 kinase (S6K); GAP , GTPase activating protein; HB-EGF, heparin-binding EGF; Jak, janus kinase; PKC, protein
kinase C; PLCγ, phospholipase Cγ; Shp2, Src homology domain-2-containing protein tyrosine phosphatase 2; Stat, signal
transducer and activator of transcription; RAF–MEK–MAPK and PAK–JNKK–JNK, two cascades of serine/threonine kinases that
regulate the activity of a number of transcription factors.)
MAPK
MEK
RAF
JNK
JNKK
PAK
Shp2
GAP
Shc
Nck
Vav Grb7
Rac
Crk
Jak
Grb2
Sos
PI(3)K
Cbl
Src
PLCγ
Ras–GDP
Ras–GTP
Bad
Akt
S6K Abl PKC
Stat Egr1 Elk Fos Myc Sp1
Jun
LPA,
thrombin,
ET, etc.
TGF-α
(1)
EGF
(1)
Epiregulin
(1,4)
β-cellulin
(1)
Amphi-
regulin
(1)
HB-EGF
(1,4)
NRG1
(3,4)
α β
NRG2
(4)
α β
NRG3
(4) Cytokines
NRG4
(4)
Apoptosis Migration Growth Adhesion Differentiation
Input
layer
Output
layer
Signal-processing
layer
Ligands
Receptor
dimers
Adaptors
and
enzymes
Cascades
Transcription
factors
13
1
2
11
22 4
2
1 4
3
2
44
3 4
3 3
a
b
c
MESODERM
The middle germ layer of the
developing embryo. It gives rise
to the musculoskeletal, vascular
and urinogenital systems, and
to connective tissue (including
that of the dermis).
ECTODERM
The outermost germ layer of
the developing embryo. It gives
rise to the epidermis and the
nerves.
AKT PATHWAY
Akt (or protein kinase B) is a
serine/threonine protein kinase
activated by the
phosphatidylinositol-3-OH
kinase pathway that activates
survival responses.
© 2001 Macmillan Magazines Ltd
3.3 ErbB3 dysregulation in disease:
ErbB3 is overexpressed in a wide variety of cancers occasionally as a heterodimerization
partner to ErbB2 or EGFR [for review, see Refs. (Ma et al., 2014; Sithanandam and Anderson,
2008)]. Its activation has been implicated as an alternative survival route in BRAF/MEK
inhibitors-resistant melanoma tumors (Abel et al., 2013; Capparelli et al., 2015; Fattore et al.,
2015). In breast cancer cell lines, ErbB3 resisted suppression by tyrosine kinase inhibitors (TKI).
While TKI treatment blocked the activation of EGFR and ErbB2, it resulted in an increased P-
ErbB3 and P-Akt, which might account for resistance to TKI therapy in cancer patients (Sergina
et al., 2007). In the intestines, ErbB3 is overexpressed in colorectal cancer (CRC) (Jaiswal et
al., 2013; Maurer et al., 1998; Ocana et al., 2013) with mutations affecting ligand binding and
heterodimerization in 11% of CRC patients (Jaiswal et al., 2013). Although ErbB3 has been well
studied in cancerous state, little is known about its role during homeostasis or inflammation. In
chapter 1, I will describe our studies that begin to define the role of ErbB3 in lineage allocation
in the intestine.
In mice, ErbB3 global deletion is embryonically lethal (at E13.5) as a result of cardiac and neural
defects (Erickson et al., 1997). Conditional deletion of ErbB3 in the intestines resulted in a
worsened outcome in chemically induced colitis and a improvement in a CRC mouse model
(Lee et al., 2009; Zhang et al., 2012b). These reports suggest a dual action of ErbB3: while it is
required in the recovery phase probably by inducing pro-survival pathways, it might represent a
bad prognostic marker in cancer.
4. Intestinal Subepithelial Myofibroblasts— a source of ErbB ligands:
The mesenchymal cell population in the lamina propria consists of: myofibroblasts, fibroblasts,
pericytes, bone marrow derived stromal stem cells, endothelial cells, and smooth muscle cells.
19
There are two types of myofibroblasts: the Intestinal subepithelial myofibroblast (ISEMF, aka
pericryptal myofibroblast) and the interstitial cells of Cajal (ICC). Postnatally, ISEMFs are
replenished by differentiation of fibroblasts, dedifferentiation from smooth muscle cells, epithelial
or endothelial-to-mesenchymal transition, and bone marrow derived stem cells. They possess
characteristics of both a fibroblast (F) and a smooth muscle (SM) cell. They are positive for α-
SMA (F—, SM+), and Vimentin (F+, SM—), but negative for Desmin (F—, SM+). ISEMFs are
located underneath the epithelial crypts of the small and large intestines (Fig 1) and in close
proximity to blood vessels and the lymphatic system. Their location gives them a unique role as
a communication link between epithelial and immune cells. They are involved in wound healing,
production of extracellular matrix proteins, tumor development, inflammation, and even in
providing a niche for the intestinal stem cells in the epithelial crypts. A functional impairment in
ISEMFs will lead to the dysregulation of any of these processes [for reviews see Refs (Mifflin et
al., 2011; Owens and Simmons, 2013; Powell et al., 2011; Speca et al., 2012).
ISEMFs play a major role in the inflammatory response (Fig 6). The ISEMF layer is disrupted in
IBD patients, probably due to these cells’ susceptibility to proinflammatory cytokines (Francoeur
et al., 2009). Moreover, TNF-overexpressing mice with a selective expression of TNF receptor 1
in ISEMF develop CD-like symptoms (Armaka et al., 2008). In addition, TNF induces tissue
inhibitor of metalloproteinases-1 (TIMP-1) in ISEMFs, resulting in the accumulation of collagen
and therefore contributing to CD-associated fibrosis (Theiss et al., 2005). These reports provide
a strong link between ISEMF function and IBD; however, we are more interested in investigating
if ISEMF can somehow affect ErbB signaling and if so how? Our lab have identified ISEMF as a
source of the ErbB ligands (Bernard et al., 2012). We showed that ErbB4 activation is important
for the protection of the intestinal epithelium (Bernard et al., 2012; McElroy et al., 2014);
therefore, it is important to have a good supply of its ligand, NRG4. Interestingly, while ErbB4 is 20
FIGURE 6: The role of myofibroblasts and fibroblasts (MF) in inflammation. EMT: epithelial to
mesenchymal transition. BM: bone morrow (panel from (Pinchuk et al., 2010). 21
overexpressed in IBD, NRG4 is downregulated (Bernard et al., 2012). In chapter 2, we try to
tease out how NRG4 expression is affected in ISEMFs and how that might be one of the factors
leading to IBD. 22
Chapter 1: The ErbB3 receptor tyrosine kinase negatively
regulates Paneth cells by PI3K-dependent suppression of
Atoh1.
Abstract
Paneth cells (PCs), a secretory population located at the base of the intestinal crypt, support the
intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing
antimicrobial peptides, including lysozyme and defensins. Paneth cell dysfunction is associated
with disorders such as Crohn’s disease and necrotizing enterocolitis, but the specific pathways
regulating Paneth cell development and function are not fully understood. Here we tested the
role of the neuregulin receptor ErbB3 in control of Paneth cell differentiation and the ISC niche.
Intestinal epithelial ErbB3 knockout caused precocious appearance of Paneth cells as early as
postnatal day 7, and substantially increased the number of mature Paneth cells in adult mouse
ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the
ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor,
which is required for secretory fate determination, while Atoh1
+
cells had reduced ErbB3,
suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced
activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells
increased levels of ATOH1 and the Paneth cell marker LYZ. Conversely, ErbB3 activation
suppressed LYZ and ATOH1 in a PI3K-dependent manner. These data suggest that ErbB3
restricts Paneth cell numbers through PI3K-mediated suppression of Atoh1 levels leading to
inhibition of Paneth cell differentiation, with important implications for regulation of the ISC
niche.
23
This paper is in revision at Cell Death and Differentiation. Dana Almohazey
1,2
, Yuan-Hung Lo
3
,
Claire V. Vossler
1
, Alan J. Simmons
4
, Jonathan J. Hsieh
1
, Edie B. Bucar
1
, Michael A.
Schumacher
1
, Kathryn E. Hamilton
5
, Ken S. Lau
4
, Noah F. Shroyer
3
, and Mark R. Frey
1,6
.
1
The Saban Research Institute at Children’s Hospital Los Angeles, Los Angeles, CA
2
University of Southern California Herman Ostrow School of Dentistry, Los Angeles, CA
3
Department of Medicine, Section of Gastroenterology and Hepatology, Baylor College of
Medicine, Houston, TX
4
Epithelial Biology Center and Department of Cell and Developmental Biology, Vanderbilt
University Medical Center, Nashville, TN
5
Division of Gastroenterology, Department of Medicine, University of Pennsylvania Perelman
School of Medicine, Philadelphia, PA
6
Department of Pediatrics and Department of Biochemistry and Molecular Biology, University of
Southern California Keck School of Medicine, Los Angeles, CA
To whom correspondence should be addressed: Mark R. Frey, The Saban Research Institute at
Children’s Hospital Los Angeles, 4650 Sunset Blvd., MS#137, Los Angeles, CA, USA, 90027;
Tel.: (323) 361-7204; email: mfrey@usc.edu 24
Introduction
Paneth cells (PCs) are heavily granulated epithelial cells located at the base of the intestinal
crypt, intercalated between the crypt base columnar (Lgr5
+
) stem cells (Barker et al., 2007;
Cheng, 1974). Paneth cells serve at least two important roles in maintaining a healthy intestinal
epithelium. They are a major source of antimicrobial peptides, including lysozyme (encoded by
Lyz1 in mice) and -defensins (Jones and Bevins, 1992; Ouellette et al., 1989; Peeters and
Vantrappen, 1975). Additionally, recent studies on the intestinal stem cell (ISC) niche
demonstrated that Paneth cells secrete key growth factors [including Wnt ligands and epidermal
growth factor (EGF)] that support Lgr5
+
stem cells (Sato et al., 2011). Thus, Paneth cells both
provide the ISCs with critical growth factors and afford them protection from enteric bacteria.
Alterations in Paneth cell number and function have been associated with inflammatory
disorders such as Crohn’s disease (Cadwell et al., 2008; Lewin, 1969; Wehkamp et al., 2005)
and necrotizing enterocolitis (McElroy et al., 2013). Furthermore, mouse models with Paneth
cell dysfunction (Adolph et al., 2013) demonstrate a critical role for these cells in maintaining
intestinal homeostasis and preventing colitis. Thus, Paneth cells may provide a key to new
avenues of treatment for intestinal inflammation. While several key transcription factors involved
in Paneth cell differentiation have been outlined and a role for morphogens such as Wnt and
Notch has been demonstrated in their maintenance (Andreu et al., 2008; Heuberger et al., 2014;
VanDussen et al., 2012), little is known about other growth factor-mediated signaling pathways
that regulate Paneth cell development or function.
The ErbB3 neuregulin (NRG) receptor—a member of the EGF receptor (EGFR) related
ErbB tyrosine kinase family—is expressed in most epithelial tissues, but its physiological
functions specific to each tissue are not well defined. Along with ErbB4, ErbB3 is one of the two
25
receptors for neuregulins in mammalian cells. With a more restricted ligand affinity (preferred
recognition for NRG1 and 2) than ErbB4 (which recognizes NRG1-4) (Jones et al., 1999), six
YXXM phosphatidylinositol 3-kinase (PI3K) docking sites (Prigent and Gullick, 1994), and low
but physiologically relevant kinase activity (Guy et al., 1994; Shi et al., 2010), ErbB3 is
biochemically unique among the ErbB family, and is therefore likely to play roles in cell signaling
different than those of other ErbBs.
While ErbB3 is robustly expressed in the intestinal epithelium (Lee et al., 2009), its
physiological function in this tissue is not well-understood. In the colon, it promotes recovery
from chemically-induced colitis (Zhang et al., 2012b) but also supports the survival of
transformed cells (Lee et al., 2009), through mechanisms that remain undefined. Even less is
known about ErbB3’s role in the small intestine. As the other neuregulin receptor, ErbB4, is a
protective factor for Paneth cells (McElroy et al., 2014), we tested whether ErbB3 also regulates
these cells. In this study we report that, in contrast to ErbB4, ErbB3 signaling restricts the
number of mature Paneth cells in the small intestine. This response requires PI3K signaling,
which in turn suppresses expression of Atoh1, a transcription factor required for secretory
differentiation. These data suggest that ErbB3 plays an unexpected repressive role in secretory
differentiation in the intestine. Signaling through the two neuregulin receptors may thus provide
balanced, opposing effects on Paneth cells, ultimately fine-tuning this secretory population and
the intestinal stem cell niche. 26
Materials and Methods
Animals—All animal use was approved and monitored by the Children’s Hospital Los Angeles or
Baylor College of Medicine Institutional Animal Care and Use Committees (animal welfare
assurance numbers A3276-01 and A3823-01, respectively). C57Bl/6 (WT), ErbB4
flox/flox
(ErbB4-
FF) and ErbB4
flox/flox
; Villin-Cre (ErbB4-IEKO) (46), ErbB3
flox/flox
(ErbB3-FF) and ErbB3
flox/flox
;
Villin-Cre (ErbB3-IEKO) (Lee et al., 2009), Atoh1
flox/flox
;VilCre
ERT2
, Atoh1
GFP/GFP
, and Atoh1
Flag/Flag

(Rose et al., 2009; Shroyer et al., 2007) mice were kept in standard housing with a 12-hour light/
dark cycle at 21º to 22º C. To achieve deletion of Atoh1 from intestinal epithelium, Atoh1
flox/
flox
;VilCre
ERT2
mice were given an intraperitoneal injection of 1 mg/mouse tamoxifen (Sigma)
dissolved in corn oil for 3 consecutive days, and euthanized on day 5. All experiments utilized
matched littermate controls, and both male and female animals were included.
Immunofluorescent and Histochemical Analysis—Hematoxylin and Eosin staining was
performed on sections of formaldehyde (4%) fixed, paraffin-embedded ileum (terminal 3 cm).
Immunofluorescent staining of tissue sections and enteroids used standard techniques as
previously reported (Bernard et al., 2012). Antigen unmasking was achieved by heating the
slides in 10 mM Sodium Citrate, pH 6.
Antibodies used for immunostaining—rabbit or goat α-Lysozyme (1:150, Dako #A0099, 1:100,
Santa Cruz #sc-27958 as appropriate for multicolor stains), rabbit α-Mucin 2 (1:100, Santa Cruz
#sc-15334), rat α-MMP7 (1:50, Vanderbilt University Antibody and Protein Resource), rabbit α-
ErbB3 (1:100, Cell Signaling #12708), mouse α-E-Cadherin (1:200-, BD Bioscience #610181),
Alexa Fluor 647-conjugated donkey α-rabbit (1:500, Life Technologies #A-31573), Alexa Fluor
488-conjugated donkey α-goat (1:2000, Life Technologies #A-11055), Alexa Fluor 546-
conjugated goat α-rabbit (1:2000, Life Technologies #A-11035), Cy3-conjugated donkey α-
27
mouse (1:200, Jackson ImmunoResearch Laboratories #715-166-151), FITC-conjugated goat
α-rat (1:200, Jackson ImmunoResearch Laboratories # 112-095-003), DAPI-containing
mounting medium (Vector Laboratories #H-1500), and Hoechst 33342 (ThermoScientific
#62249).
Epithelial isolation—Mucosal scraping was collected as previously described (Perret et al.,
1977; Simmons et al., 2015). Briefly, the most distal 6 cm of the ileum was collected, cut open
longitudinally, washed, and spread with the mucosal side facing upwards. The mucosa was then
scraped by a razor blade positioned at a 45˚ angle until the tissue is transparent. Scraping
efficiency was confirmed visually under a microscope and by qPCR for Lyz1 expression in
scrapings versus submucosa (leftover tissue) compared to whole tissue.
RNA isolation and Real-time Quantitative PCR (qPCR)—RNA was collected from ileal mucosal
scrapings, cell cultures, or TRIzol-suspended sorted cells using column-based isolation and a
TissueLyser LT (Qiagen). For RNA-seq, RNA quality controls were performed on an Agilent
Bioanalyzer nano chip and RNA integrity number was at least 8.8. For qPCR, cDNA was
synthesized from 1 µg of RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied
Biosystems). qPCR was performed using TaqMAN or SYBR Green (Applied Biosystems)
assays, and relative expression determined by the 2
-∆∆Ct
method. Reference genes were HPRT,
Cdh1, or Gapdh as indicated in each experiment.
Single cell analysis—Ileal mucosal scrapings were collected, washed, and processed for
DISSECT-CyTOF as previously described (Simmons et al., 2015). Protein parameters were
quantified on CyTOF1 mass cytometer (Fluidigm) using antibody conjugates previously
28
validated for CyTOF. Cell populations were defined as: epithelial cells (CD45
−
/ CK20
+
), crypt
progenitors (CK20
low
/Lyz
−
), and Paneth cells (Lyz
+
).
Antibodies for DISSECT-CyTOF analysis—CD45, clone 30-F11; cytokeratin 20, clone D9Z1Z;
lysozyme and MMP7, same as for immunofluorescence above; P-Akt (S473), clone D9E; P-
ERK1/2 (T202/Y204), clone D13.14.4E; P-RSK (T359/S363), D1E9; P-CREB (S133), clone
87G3; P-Rb (S807/811), clone J112-906; P-S6 (S240/244), clone D68F8; P-ATF-2 (T71), clone
11G2; P-c-Jun (S73), clone D47G9; P-p38 (T180/Y182), clone D3F9; and P-STAT3 (Y705),
clone D3A7.
Characterization of Paneth cell granules—Sections from ErbB3 FF and ErbB3-IEKO mice were
stained for lysozyme, and granule morphologies were blindly scored in well-oriented crypts
using previously published criteria (Cadwell et al., 2008; VanDussen et al., 2014). Briefly, normal
apically localized lysozyme-positive granules were scored as Norm, Dis (disordered) was
basally located granules, Dep (depleted/diminished) was Paneth cells with <10 granules, and
Diff (diffuse) was Paneth cells with cytoplasmic (not in granules) lysozyme staining. Four mice
per phenotype were used with a range from 121 to 243 Paneth cells, which were quantified in at
least 40 crypts per mouse.
Protein Analysis and Western Blot—Protein was extracted from ileal mucosal scrapings using
previously described protocols (Bernard et al., 2012). Samples were quantified by Dc protein
assay (Pierce) and 30 μg each separated on SDS-polyacrylamide gels (4-12%, Invitrogen),
transferred onto nitrocellulose membranes, and immunoblotted followed by densitometric
quantification using the LI-COR Odyssey infrared detection system.
29
Antibodies for western blot analysis—From Cell Signaling Technologies, rabbit α-total and -
phospho-ErbB3 (#12708, 4791), rabbit α-total and -phospho-EGFR (#4267, 3777), rabbit α-
phospho-ErbB4 (#4757), total and phospho-ErbB2, mouse α-total Akt (#2920) and rabbit α-
phospho-Akt (#4060), mouse α-total Erk1/2 (#9107) and rabbit α-phospho-Erk1/2 (#4370),
rabbit α-total and phospho-p38 MAPK (#9212, 9211), rabbit α-JNK2 (#4672) and mouse α-
phospho-JNK (#9255); from Santa Cruz Biotechnology, rabbit α-total ErbB4 (#sc-283); from LI-
COR, donkey α-rabbit IRDye 680LT (#926-68023) and donkey α-mouse IRDye 800CW
(#926-32212). Equal loading was monitored by blotting for mouse β-actin (Sigma Aldrich- Clone
AC-15 #A1978- 1:10,000).
ATOH1
+
Cell sorting—Crypts were dissociated as previously described (50) with TrypLE™
express (Invitrogen) supplemented with 10 μM Y-27632 (Sigma-Aldrich) and 1 mM N-
acetylcysteine (Sigma-Aldrich) for 5 min at 37 °C, strained to remove clumps, pelleted and
resuspended in 5% BSA, 1 mM EDTA and 10 μM Y27632 in PBS. Cell viability was assessed
using 7-AAD. ATOH1
+
and 7-AAD-negative single cells were sorted into 500 μl TRIzol reagent
(Invitrogen) for RNA sequencing.
Chromatin Immunoprecipitation (ChIP)—Purified Atoh1
GFP/GFP
or Atoh1
Flag/Flag
ileal crypts were
crosslinked in RPMI 1640 with 1% formaldehyde, 50 mM HEPES pH 8.0, 1 mM EDTA, 1 mM
EGTA, and 100 mM NaCl for 30 min, quenched by glycine, and washed twice in ice-cold PBS.
Chromatin was sonicated to 300-1000 bp fragments in 10 mM Tris-HCl pH 8.0 with 1 mM EDTA
pH, 1 mM EGTA and protease inhibitor cocktail (Calbiochem, #539134), and incubated 10 min
in sonication buffer with 0.5% sarkosyl solution and cleared by centrifugation. Then 500 μl
sheared chromatin was mixed with 150 μl binding buffer (440 mM NaCl, 0.44% sodium
30
deoxycholate, 4.4% Trion-X-100) and incubated with Protein G Dynabeads (Invitrogen,
#100-03D) pre-bound to 2 μg anti-GFP (Novus, NB600-303) at 4°C overnight, washed five
times in 50 mM HEPES pH 8.0 with 1% Nonidet P-40, 1% sodium deoxycholate, 1 mM EDTA,
and 500 mM LiCl, and eluted at 65°C for 15 min in 50 mM Tris-HCl pH 8.0 with 10 mM EDTA
and 1% SDS. DNA was purified by phenol-chloroform extraction.
In Vitro Cultures, Growth Factors, and Inhibitors—Ileal enteroid cultures were prepared as
previously published (McElroy et al., 2014; Sato et al., 2011). Briefly, epithelial crypts were
isolated by EDTA Ca
2+

chelation and shaking, then embedded in Matrigel (Corning, 356237)
supplemented with EGF (PeproTech, 20ng/ml), Noggin (R&D, 100 ng/ml), and R-Spondin 1
(R&D, 500 ng/ml). HT29 human colorectal cancer cells (ATCC, line authenticated by short
tandem repeat typing at the USC Bioreagent & Cell Culture Core Facility and were found to be
negative for mycoplasma contamination) were maintained in DMEM with 10% FBS, 4 mM L-
glutamine, and 100 units/ml penicillin and streptomycin. Cells were shifted to 0.5% FBS
containing media for 24 h before experiments. Growth factor and inhibitors were from: NRG1β,
R&D; NRG4, Reprokine; U0126, Millipore; LY294002, Cell Signaling; and Rapamycin, MP
Biomedicals.
Gene expression assays and primers—TaqMAN assay numbers used were: Lyz1
(Mm00657323_m1); Lgr5 (Mm00438890_m1); Olfm4 (Mm01320260_m1); Muc2
(Mm01276696_m1); Chga (Mm00514341_m1); Egfr (Mm00433023_m1); ErbB2
(Mm00658541_m1); ErbB4 (Mm01256793_m1); Atoh1 (Mm00476035_s1); Cdh1
(Mm01247357_m1); HPRT (Hs02800695); LYZ (Hs00426232). SYBR primer sequences used
were:
Defa5 (5’-GCTCAACAATTCTCCAGGTGACCC-3’; 5’-AGCAGACCCTTCTTGGCCTC-3’);
31
Muc2 (5’-ATGCCCACCTCCTCAAAGAC-3’; 5’-GTAGTTTCCGTTGGAACAGTGAA-3’);
Lgr5 (5’-ACCTGTGGCTAGATGACAATGC-3’; 5’-TCCAAAGGCGTAGTCTGCTAT-3’);
Bmi1 (5’-TGGTTGTTCGATGCATTTCT-3’; 5’-CTTTCATTGTCTTTTCCGCC-3’);
Lrig1 (5’-CTCAAAAGCAGCAGAGTCGAT-3’; 5’-GGGAACTCGCTGGACTGC-3’);
Gapdh (5’-CACCCAGAAGACTGTGGATGGC-3’; 5’-TGCCTGCTTCACCACCTTCT-3’);
Lyz1 (5’-AGCCGATACTGGTGTAATGATGGCA-3’; 5’-CCATGCCACCCATGCTCGAAT-3’).
Statistics—All data represent three or more independent experiments, as indicated in the figure
legends. Cohort sizes of ErbB3
IEKO
and WT comparisons were defined based on previous
variability observed in lineage markers in order to give 80% power to detect 2-fold changes.
Samples from one mouse had poor RNA quality and measured parameters were definitive
outliers by ROUT test (Q=0.1%); this animal was excluded from analysis. Statistical analysis
was performed using Prism 5.0 software (GraphPad). Significance of results was tested by
ANOVA with Dunnett’s post-test or Student’s t test as appropriate. 32
Results
ErbB3-null intestinal crypts have more Paneth cells versus wild type. To test the role of
ErbB3 in regulation of Paneth cells, we generated mice lacking ErbB3 expression in the
intestinal epithelium (ErbB3-IEKO) by crossing the ErbB3
flox/flox
line (Lee et al., 2009) to Villin-
Cre transgenic animals. In wild type littermates, ErbB3 was expressed throughout the entire
epithelium including the crypts, while the ErbB3-IEKO mice did not express any (Fig 7A).
Histological and immunofluorescence analyses of the ErbB3-IEKO mice showed a significant
increase in Paneth cell numbers, marked by lysozyme, compared to control littermates (Fig 7B).
Similarly, qPCR on mucosal lysates revealed a doubling in the expression of Lyz1 (Fig 7C), and
Cytometry by Time of Flight (CyTOF) analysis on dissociated epithelium showed an increased
proportion of epithelial cells expressing the Paneth cell markers lysozyme and matrix
metalloproteinase-7 (MMP7) (Fig 7D). The additional Paneth cells were located, as normal, at
the crypt base with normal granule morphology (Fig 7E,F), which suggests normal function
(Cadwell et al., 2008; VanDussen et al., 2014). Consistent with the demonstrated role of Paneth
cells in supporting the ISC niche through Wnt production, there was a significant increase in the
Wnt-regulated ISC marker, Lgr5, but not the Notch-regulated Olfm4 (Fig 7G,H). Moreover,
ErbB3 deletion did not alter the expression of other secretory cell markers such as Muc2 (goblet
cells), Chga (enteroendocrine cells), or Dclk1 (tuft cells) (Fig 7I-K), suggesting a specific effect
on Paneth cells rather than an overall expansion of the secretory lineages. To check whether
effects of ErbB3 deletion might involve compensatory changes in other ErbB family members,
we assessed RNA and protein levels for Egfr, ErbB2, and ErbB4. Western blot and qPCR
analyses did not reveal any differences in other ErbB members in ErbB3-IEKO mice compared
to their control littermates (Fig 7L,M). Morphometric analysis on H&E sections supported the
idea that alterations in these mice are restricted to the crypt, showing a significant increase in 33
FIGURE 7. ErbB3-null intestinal crypts have more Paneth cells versus wild type. A,
Immunofluorescence analysis for ErbB3 on paraffin-embedded tissue sections from ErbB3-FF
and ErbB3-IEKO ileal crypts showing normal expression pattern and confirming knockout. B,
Immunofluorescence analysis for lysozyme (green), E-cadherin (red), and DAPI (blue) on
paraffin-embedded tissue sections from ErbB3-FF and ErbB3-IEKO ileums. Scale bars, 75 µm
(10x images) and 10 µm (40x images). C, qPCR analysis (Cdh1 as reference) of ileal epithelial
scrapings for Lyz1 (n=9 mice/genotype). D, CyTOF analysis for Paneth cells (lysozyme
+
or
MMP7
+
) as a percentage of epithelial cells (n=5 mice/genotype). E and F, Paneth cell location
(below or above +4 cell position) and Paneth cell granule characterization (Norm-normal, Dis-
disordered, Dep-depleted, and Diff-diffuse) in ≥40 crypts/mouse (n=4 mice/genotype). G-L,
qPCR analysis for indicated gene products (n=9 mice/genotype). M, Western blot analysis for
ErbB protein expression (representative of 3 mice/genotype examined). N, Crypt and villus
length and width (n≥4 mice/genotype). Error bars, +/- SEM. 34
crypt length and width in the ErbB3-IEKO animals versus littermate controls, but no change on
the villi (Fig 7N).
Paneth cells develop precociously in ErbB3-IEKO mice. The selective increase of Paneth
cells in the ErbB3-null intestinal epithelium could represent accelerated development of this
lineage. To test this possibility, we examined intestines at postnatal day (P) 7, at which point the
mouse ileum is normally devoid of mature lysozyme
+
Paneth cells (Bry et al., 1994; Ouellette et
al., 1989). We observed highly granulated cells near the intestinal crypt base in P7 ErbB3-IEKO
mice, but not littermate controls (Fig 8A). The presence of lysozyme
+
cells in the conditional
knockouts was confirmed by immunofluorescence (Fig 8B) and qPCR (Fig 8C). Although Lyz1
levels in ErbB3-IEKO P7 pups were not as high as in adults, it was clearly expressed and at a
similar level as in P13 WT mice (not shown). Thus, deletion of ErbB3 results in a premature
appearance of differentiated Paneth cells, with elevated numbers maintained into adulthood.
ErbB3-null intestinal crypts have more MMP7/MUC2
+
intermediate cells versus wild type.
Paneth cells and goblet cells share a common precursor in the secretory lineage which can be
identified by simultaneous expression of markers of both cell types (Troughton and Trier, 1969;
VanDussen et al., 2012). Immunofluorescence for MMP7 (Paneth cells) and MUC2 (goblet cells)
showed a significant increase in double-positive cells in ErbB3-IEKO mice (Fig 9A,B). We
speculate that most of these additional intermediate secretory precursor cells will preferentially
differentiate to Paneth cells, since we did not detect any overall alteration in Muc2 expression
(Fig 7I). These results suggest that the deletion of ErbB3 selectively drives the cell fate
commitment towards Paneth cell differentiation.
35
FIGURE 8. ErbB3-null intestines express Paneth cell markers by postnatal day 7. A, H&E
on paraffin-embedded tissue sections from ErbB3-FF and ErbB3-IEKO ileums collected at
postnatal day 7. B, Immunofluorescence analysis for lysozyme (green), E-cadherin (red), and
DAPI (blue). Scale bars, 50 µm (20x images) and 25 µm (40x images). Arrowheads, example
granulated/lysozyme
+
cells. C, qPCR analysis (Cdh1 as reference) of ileal whole tissue
homogenates for Lyz1 (n=6 mice/genotype). Error bars, +/- SEM. 36
FIGURE 9. ErbB3-null intestinal crypts have more MMP7/MUC2
+
intermediate cells versus
wild type. A, Immunofluorescence analysis for MMP7 (green), MUC2 (red), and DAPI (blue) on
paraffin-embedded sections from ErbB3-FF and ErbB3-IEKO ileums. Scale bars, 75 µm (10x
images) and 25 µm (40x images). Arrowheads, example double-positive cells. B, Quantification
of MMP7/MUC2
+
cells/crypt (n=9 mice/genotype). Error bars, +/- SEM.
37
Atoh1 binds to the ErbB3 promoter region and down-regulates its expression. Since our
data suggest that ErbB3 regulates secretory cell differentiation, we asked whether it has
functional interactions with Atoh1, a transcription factor which is expressed in MMP7/MUC2
+

intermediate cells and required for secretory cell fate determination (Shroyer et al., 2007; Yang
et al., 2001). ChIP analysis showed that Atoh1 binds to the ErbB3 promoter region (Fig 10A,B).
Furthermore, Atoh1
+
cells had reduced ErbB3 expression (Fig 10C). Conversely, ErbB3-IEKO
intestines showed a significant increase in Atoh1 expression (Fig 10D), suggesting a negative
feedback loop between Atoh1 and ErbB3.
NRG1β exposure reduced expression of Paneth and stem cell, but not goblet cell,
markers in enteroid cultures. To investigate the effects of activating ErbB3 in a readily-
tractable model, we used in vitro enteroid cultures (Sato et al., 2011). We treated ileal enteroids
from C57Bl/6 mice with NRG1β (10 ng/ml) for up to 5 days and collected RNA for analysis.
Consistent with the hypothesis that ErbB3 restricts Paneth cell numbers, NRG1β treatment
resulted in sustained reduction in expression of Lyz1 (Fig 11A), as well as Defa5, which
encodes another antimicrobial peptide expressed by Paneth cells. In contrast, Muc2 levels were
largely unchanged, similar to our in vivo results. We also observed a reduction in Lgr5 (Fig 11B),
and to a lesser extent in Lrig1 and Bmi1. This could represent the close dependence of the
Lgr5
+
ISCs on Paneth cells (Sato et al., 2011). A 24 h NRG1β exposure was sufficient to
provoke reduction in Paneth cell and ISC markers (Fig 11C). To test whether this represents a
reduction in Paneth cell numbers or a reduction of antimicrobial peptides per cell, we stained
enteroids with lysozyme and found that lysozyme-positive cell counts were reduced (Fig 11D).
ErbB4 is not required for NRG1β-stimulated Paneth cell loss. Our previous studies using
ErbB4 ligand in intestinal injury models and ErbB4-null enteroids (McElroy et al., 2014) suggest 38
FIGURE 10. Atoh1 binds to ErbB3 promoter region and down-regulates its expression. A,
CHIP-seq performed on sorted Atoh1-GFP
+
cells from Atoh1
GFP/GFP
mice. The peak density plots
show enrichment of Atoh1 peak (lower box). Erbb3 gene is labeled with exons as thick
rectangles (representative of 3 independent sorts). B, ErbB3 PCR performed on CHIP product
from Atoh1
Flag/Flag
mice (representative of 3 independent experiments). ErbB3 NC is a negative
control region 5kb upstream of Atoh1 binding region. C, RNA-seq analysis of Atoh1-IEKO and
sorted Atoh1-GFP
+
cells for ErbB3. D, qPCR analysis (Cdh1 as reference) of ileal epithelial
scrapings for Atoh1 in ErbB3-FF and ErbB3-IEKO (n=8 mice/genotype). Error bars, +/- SEM.
39
FIGURE 11. NRG1β exposure reduced expression of Paneth and stem cell, but not goblet
cell, markers. A and B, qPCR analysis (Gapdh as reference) of ileal enteroid cultures treated
with NRG1β (10 ng/ml) daily for 5 days for indicated gene products (n=3 independent
experiments). C and D, enteroids were treated with a single dose of NRG1β for 24 h. C, qPCR
analysis of indicated genes (n=3 independent experiments). Dashed line represents control. D,
Quantification of lysozyme
+
cells in control and NRG1β treated enteroids using in situ
immunofluorescence analysis for lysozyme (pink) and Hoechst (blue) (n=3 independent
experiments, ~200 crypts/condition). *, p<0.05; **, p<0.01; †, p<0.001 vs. control. Error bars, +/-
SEM.
40
that in contrast to ErbB3, ErbB4 is protective for Paneth cells. To compare the results of ErbB4
versus ErbB3 deletion in homeostasis, we examined ErbB4-IEKO mouse ileums. Surprisingly,
unchallenged ErbB4-IEKO mice displayed no alterations in either Paneth cell or ISC markers
(Fig 12A). To investigate whether ErbB4 is involved in NRG1β-induced Paneth cell loss in
enteroids, we treated C57Bl/6 enteroids with the ErbB4-specific ligand, NRG4, which did not
result in any change in Lyz1, Defa5, or Lgr5 (Fig 12B). Then, we treated ErbB4-IEKO enteroids
with NRG1β. In the ErbB4-null enteroids, NRG1β was still able to cause a reduction in Lyz1 and
Lgr5 (Fig 12C) comparable to what was seen in wild type cultures (Fig 11). Taken together with
our previous studies, these results suggest that ErbB4 primarily plays a protective role for
Paneth cells after an injury or other challenge, but does not substantially impact their
homeostatic maintenance. In contrast, ErbB3 restricts the numbers of Paneth cells during
normal development and homeostasis.
ErbB3 maintains baseline intestinal epithelial PI3K and MAPK signaling in vivo. ErbB3
signaling stimulates activation of PI3K-Akt and ERK1/2 mitogen-activated protein kinase
(MAPK) signaling in cultured intestinal cell lines (Bernard et al., 2012). Other investigators have
demonstrated that MAPK activation negatively regulates Paneth cell numbers (Heuberger et al.,
2014), which could be related to ErbB3 activity. In contrast, however, a role for PI3K signaling in
Paneth cells has not been investigated. To begin to identify which pathways might be
responsible for ErbB3 regulation of Paneth cells, we first performed western blot analysis on
lysates of mucosal scrapings. Most targets examined were unaltered in this analysis, with only
phospho-Akt levels significantly reduced in ErbB3-IEKO mice (Fig 13A,B). Using the more
sensitive CyTOF analysis which can also define signaling in cellular subsets, we determined
that both phospho-Akt and phospho-ERK1/2 were significantly reduced in cytokeratin (CK)
20
low
/Lysozyme
−
crypt progenitor cells from the ErbB3-IEKO mice as well as, to a lesser extent, 41
FIGURE 12. ErbB4 signaling does not affect homeostatic Paneth cell or ISC marker
levels. A, qPCR analysis (Cdh1 as reference) of ileal epithelial scrapings from ErbB4-FF and
ErbB4-IEKO animals for indicated gene products (n=13-14 mice/genotype). B, qPCR analysis
(Gapdh as reference) of wild type enteroids treated with ErbB4-specific ligand, NRG4 (100 ng/
ml) for 24 h (n=4 independent experiments). C, qPCR analysis (Gapdh as reference) of ErbB4-
IEKO enteroids treated with NRG1β for 24 h. B and C, Dashed line represents control. Error
bars, +/- SEM. *, p<0.05.
42
FIGURE 13. ErbB3 regulates Paneth cells through the PI3K/Akt pathway. A, Western blot
analysis of ileal epithelial scrapings from ErbB3-FF and ErbB3-IEKO mice (representative of 3
mice/genotype examined). B, Relative intensity of P-Akt/Akt in A. C, CyTOF analysis (median
fluorescence intensity) of indicated proteins in all epithelial cells (white bars), Paneth cells
(lysozyme
+
, grey bars), and crypt progenitors (CK20
low
/lysozyme
−
, black bars) (n=5 mice/
genotype). D, CyTOF analysis of P-Akt (S473), P-ERK1/2 (T202/Y204), and P-RSK (T359/
S363) in crypt progenitors (CK20
low
/lysozyme
−
). E-H, qPCR analysis (HPRT as reference) of
HT-29 cells for LYZ and ATOH1 after 24 h treatment with NRG1β (10 ng/ml) +/- PI3K inhibitor
LY294002 (LY, 2.5, 5, 10, and 20 µM), MEK1/2 inhibitor U0126 (U, 1.25, 2.5, 5, and 10 µM), and
mTORC1 inhibitor Rapamycin (RAPA, 100 nM). Inhibitors were added an hour before NRG1β
treatment (n≥3 independent experiments per panel). Dashed line represents control. Error bars,
+/- SEM. *, p<0.05; ** p<0.01; †, p<0.001; #, p<0.0001 vs. control.  43
in the epithelium overall (Fig 13C,D). In contrast, Paneth cells themselves showed only a non-
significant trend towards reduction in activity of these pathways (Fig 13C). These results are
consistent with the hypothesis that changes in Paneth cell numbers are a consequence of
altered MAPK and/or PI3K signaling in the stem cell/progenitor compartment.
ErbB3 regulates Paneth cells through the PI3K-Akt pathway. To define the involvement of
PI3K and MAPK in ErbB3 regulation of Paneth cell development, we used HT29 human
colorectal cancer cells. Unlike in the mouse, Paneth cells can be observed in the human colon
(although not as the same rates as in the small intestine), and colonic Paneth cell metaplasia
has been reported in inflammation and cancerous conditions (Lewin, 1969; Simmonds et al.,
2014; Tanaka et al., 2001). HT29 cells express characteristics of both secretory and absorptive
cell lineages and have been used to study mechanisms controlling Paneth/goblet cell balance
(Heuberger et al., 2014; Huet et al., 1987). NRG1β elicited a reduction in baseline LYZ and
ATOH1 expression in these cells (Fig 13E-G). Conversely, the PI3K inhibitor LY294002 and
MEK1/2 inhibitor U0126 caused dose-dependent increases in LYZ and ATOH1 (Fig 13F,G).
These data suggest that baseline activities of PI3K and MAPK are involved in restricting LYZ
and ATOH1 levels. The mTORC1 inhibitor Rapamycin had no effect (Fig 13H), indicating that
PI3K involvement is selectively through Akt or possibly mTORC2. Interestingly, PI3K inhibition
could induce LYZ and ATOH1 in the presence of NRG1β, while MAPK inhibition could not.
Thus, while PI3K and MAPK are both involved in the regulation of Paneth cells, only PI3K
signaling is required for ErbB3-mediated effects on this lineage. 44
Discussion
Herein we report that the ErbB3 neuregulin receptor limits the number of Paneth cells in the
intestinal crypt. ErbB3 deletion from the intestinal epithelium resulted in a significant increase in
morphologically normal Paneth cells in adult mice, as well as early appearance of these cells in
the developing postnatal intestine (Fig 7, 8). Consistent with the role Paneth cells can play in
supplying Wnt and growth factors to the ISC niche, the increase in Paneth cells was
accompanied by elevated Lgr5, a marker for Wnt-dependent rapidly-cycling ISCs, but no overall
change in markers for other intestinal cell lineages (Fig 7). We did observe an increase in
MMP7-MUC2 double-positive intermediate cells in ErbB3-IEKO mice (Fig 9), and demonstrated
that ErbB3 and the secretory lineage differentiation factor Atoh1 negatively regulate one another
(Fig 10). Furthermore, exposure to the ErbB3 ligand NRG1β down-regulated Paneth cell and
ISC markers in enteroid cultures and cultured cells (Fig 11-13). These effects appear to be
through altered PI3K-Akt activation (Fig 13). Taken together, our results position ErbB3 as an
important regulator of Paneth cell differentiation.
Our lab previously showed that the ErbB4 receptor tyrosine kinase protects Paneth cells
in injury models and enteroid cultures (McElroy et al., 2014). In this context, we note that
NRG1β (which binds both ErbB3 and ErbB4) stimulated Lyz1 loss even in ErbB4-null enteroids,
while WT enteroids treated with NRG4 (which activates ErbB4 but not ErbB3) did not show
differences in Paneth cell or ISC markers (Fig 12). Thus it appears that ErbB3 and ErbB4 have
very different, even opposing functions with regard to Paneth cell maintenance. This is
particularly interesting in that the balance of NRG1β versus NRG4 shifts during inflammation
(Bernard et al., 2012; Wang et al., 2014), which could lead to an altered Paneth cell population.
This could, in turn, impact the stem cell niche through the growth factors produced by the
45
Paneth cells, as well as predisposing to an altered microbiota by changing the quantity or quality
of antimicrobial peptides released by the Paneth cells.
While our results rule out a requirement for ErbB4 in ErbB3-mediated restriction of
Paneth cells, it will be important in future studies to determine whether heterodimerization with
either EGFR or ErbB2 is necessary. ErbB3 was initially thought to be kinase inactive and
therefore dependent on heterodimers with other ErbB members (Berger et al., 2004; Guy et al.,
1994; Pinkas-Kramarski et al., 1996), but more recent studies have shown that it has kinase
activity that, while low, is significant and sufficient for signaling. ErbB3 is competent to bind ATP
and autophosphorylate, although much more weakly than EGFR, and is able to form
homodimers (Shi et al., 2010; Steinkamp et al., 2014; Telesco et al., 2011). Another unique
feature of ErbB3 is that it has six YXXM docking sites (Hellyer et al., 1998; Prigent and Gullick,
1994). When the YXXM sequence is phosphorylated, it binds with the SH2 domain of the p85
PI3K regulatory subunit. The presence of six of these motifs makes ErbB3 a stronger activator
of the PI3K-Akt pathway than other ErbBs. Given the impact of PI3K signaling on LYZ and
ATOH1 expression (Fig 13), the difference between ErbB3 homodimers versus heterodimers
with EGFR or ErbB2 may thus be functional in fine-tuning this regulatory mechanism.
To date, relatively little is known about the physiology of ErbB3 in the intestine. Mice with
intestinal epithelial deletion of ErbB3 had impaired recovery when subjected to dextran sodium
sulphate (DSS)-induced colitis (20). The colitis response might seem at odds with our results,
since Paneth cell loss or dysfunction is often a feature of intestinal inflammation (Adolph et al.,
2013; Cadwell et al., 2008; Lewin, 1969; McElroy et al., 2013; Wehkamp et al., 2005). However,
the small intestine and the colon are of course different organs, and in mice the colon has few if
any Paneth cells under homeostatic conditions. Furthermore, as described by Threadgill and
46
colleagues, ErbB3 provides a strong survival signal for colonocytes (Lee et al., 2009). It may be
that in a direct injury-based model such as DSS, long-term effects on enterocyte survival due to
loss of ErbB3 in the whole epithelium contribute to worsened recovery, and downstream effects
from Paneth cells in the small bowel do not come into play. Alternatively, ErbB3-null Paneth
cells, despite having a normal appearance in homeostasis, may be more susceptible to injury
and inflammation. Ongoing work in the laboratory is focused on testing these possibilities.
ErbB3 likely has multiple different, context-dependent functions in the gastrointestinal tract.
Paneth cells secrete products such as EGF, Notch, and Wnt ligands, which support
Lgr5
+
ISCs (Sato et al., 2011). Other investigators have shown that GSK3β can either target
Atoh1 or β-catenin for degradation in human colorectal cancer (Tsuchiya et al., 2007). In ErbB3-
IEKO mice an increase in Paneth cell numbers, and therefore Wnt, might drive an accumulation
of Atoh1 that feeds into a loop resulting in a further increase in the secretory cell lineage.
Conditional Atoh1 knockout results in a loss of all intestinal secretory cell lineages (Shroyer et
al., 2007). Inversely, Notch inhibition had the opposite outcome as well as a reduction in the ISC
markers Olfm4 and Lgr5 (VanDussen et al., 2012). However, in neither of these cases was the
effect specific to Paneth cells as it is with ErbB3. Furthermore, our results show that Olfm4 did
not change in ErbB3-IEKO mice, suggesting that the increase in Atoh1 in ErbB3-IEKO is
independent to changes in Notch signaling.
NRG1β treatment of enteroid cultures specifically affected Paneth cell markers (Lyz1
and Defa5) and the CBC ISC marker Lgr5, while effects on the +4 ISC markers Bmi1 and Lrig1
were modest. Since Lrig1 negatively regulates the ErbB family (Laederich et al., 2004), its
gradual reappearance in these cultures after treatment might be a compensatory response to
ErbB3 activation. However, the regulatory mechanisms surrounding Lrig1 are complex; deletion
47
of Lrig1 in mice resulted in an increase in EGFR, ErbB2, and ErbB3 expression, but also an
increase in Paneth cells and ISCs (Wong et al., 2012). Although ErbB3 levels were higher in
these mice, there was no difference in ErbB3 activation. In contrast, there was an increase in P-
EGFR and P-ErbB2, which could be drivers of Paneth cell and ISC accumulation, consistent
with a need for EGF in enteroid cultures to prime and support their growth. ErbB3 may be
unique amongst the ErbB family in its ability to down-regulate Paneth cells, while other ErbBs
either protect (ErbB4) or induce them (EGFR and/or ErbB2). As we did not detect an expansion
of other cell lineages, our data support a model of direct effects on Paneth cell differentiation
rather than a secondary effect of proliferating ISCs. Furthermore, unlike other cell lineages,
Paneth cells can persist in expressing Lgr5 (Grun et al., 2015), raising the possibility that some
of the increase in Lgr5 expression in the ErbB3-IEKO mice is attributable to these cells. The
mechanisms through which ErbB3 activation targets Lgr5
+
ISC either directly or indirectly, while
out of the scope of this study, merit further investigation.
Other growth factors affect Paneth cells and ISCs, and likely interact with ErbB3
signaling. Shp2 and MAPK cooperate to shift the intestinal secretory cell balance away from
Paneth cells in favor of goblet cells (Heuberger et al., 2014), and Fgf10 similarly affects this
balance, again presumably through MAPK activation (Al Alam et al., 2015). In contrast, an
explicit role for PI3K signaling in regulating these cells has not been tested. NRG1β activates
both ERK MAPK and PI3K-Akt pathways in intestinal cells (Bernard et al., 2012), and we find
that crypt progenitor cells from ErbB3-IEKO mice have reduced activation of both pathways (Fig
13). Inactivation of PI3K or MAPK in HT29 cells resulted in a dose dependent increase in LYZ
and ATOH1 expression. Interestingly, the response to MAPK inhibitor could be overcome by
NRG1β, while PI3K inhibition was dominant, suggesting mechanistically distinct roles for these
two pathways in Paneth cell development. Others have shown that inhibition of MAPK signaling
48
leads to differentiation and cell cycle arrest (Aliaga et al., 1999; Lemieux et al., 2011), which
might explain an increase in Paneth cells but not the specificity for this lineage. Moreover, we
did not find any evidence for altered proliferation in the ErbB3-IEKO intestines (P-Rb results in
Fig 13C and data not shown).
In summary, our results demonstrate that ErbB3 restricts Paneth cell numbers via a
mechanism involving PI3K-Akt and Atoh1. Understanding the growth factor signaling
mechanisms that affect Paneth cell function is an intriguing and under-explored area with
potential therapeutic benefit. For example, transient blockade of ErbB3 activity might
conceivably be used to reverse the Paneth cell dropout seen in inflammatory bowel disease
patients (Lewin, 1969; Wehkamp et al., 2005). The balance of NRG1/NRG4 expression is
altered in IBD (Bernard et al., 2012), which could alter the equilibrium between ErbB3 and
ErbB4 vis-à-vis Paneth cell control. Furthermore, the ErbB3 ligand NRG1 is recurrently mutated
in patients with colitis-associated colorectal cancers compared to those with sporadic cancer
(Robles et al., 2016). This implies functional dysregulation of ErbB3 in the pre-cancerous
inflammatory state. Taken together with our study, these reports suggest that continuous
alteration of ErbB3 activity might be a risk factor in inflammatory bowel disease and possibly
inflammation-associated colon cancer. 49
Chapter 2: Inflammation Mediated Regulation of NRG4 in the
Intestinal Subepithelial Myofibroblasts.
Abstract
Tumor necrosis factor (TNF) is one of the major cytokines involved in driving the inflammatory
response in diseases such as inflammatory bowel disease (IBD). Neuregulin 4 (NRG4), which is
an ErbB4 ligand, is downregulated in IBD patients. It is secreted from the intestinal subepithelial
myofibroblasts (ISEMFs) located underneath the intestinal crypts. Our lab showed that NRG4
was able to protect the intestinal epithelium against TNF-induced apoptosis. In this study, we
tested if TNF is implicated in downregulating NRG4 leaving the epithelium susceptible to
damage and breaches in the epithelial barrier. TNF treatment of ISMEFs resulted in a reduction
in NRG4 expression. In an effort to understand the mechanism, we used pharmacological
inhibitors of JNK, MAPK, and JAK/STAT pathways. While inhibiting JNK and MAPK did not alter
the TNF effect, inhibiting JAK/STAT pathway abrogated the reduction in NRG4. Our results
show that TNF activation of the JAK/STAT pathway causes a downregulation of NRG4
expression in ISEMFs. This could point the way towards new treatment targets for IBD patients
that do not respond to anti-TNF therapy. 50
Introduction
Inflammatory bowel disease (IBD) is characterized by breaches in the epithelial barrier
accompanied by an exaggerated chronic immune response in genetically susceptible patients.
IBD has two subtypes: Crohn’s disease (CD) and ulcerative colitis (UC). While the exact causes
and the sequence of events leading to its progression is unknown, one of the causes is believed
to be a dysregulated interaction between the intestinal environment and the immune system.
The intestinal subepithelial myofibroblasts (ISEMFs) are located underneath the intestinal
epithelium and are in close proximity to the lymphatic and blood vessels. This strategic location
gives ISEMFs an important role in mediating the communication between the epithelium and
immune cells, making them a potential therapeutic target for IBD.
Tumor necrosis factor (TNF) is elevated in the serum and stool of IBD patients (Braegger et al.,
1992; Komatsu et al., 2001) suggesting that it might contribute in driving the inflammatory
response. This is further supported by the effective use of anti-TNF therapy in IBD patients.
Furthermore, Tnf overexpressing mice (Tnf
ΔARE/+
) present with CD-like symptoms (Kontoyiannis
et al., 1999). Interestingly, the specific expression of TNF receptor 1 in ISEMFs resulted in
symptoms similar to that observed in the Tnf
ΔARE/+
mice (Armaka et al., 2008). These findings
suggest that ISEMFs are involved in the pathogenesis of IBD.
The neuregulin receptor ErbB4 is one of the four ErbB receptor tyrosine kinases; this family also
includes: Epidermal Growth Factor receptor (EGFR) a.k.a. ErbB1, ErbB2, and ErbB3. We
previously showed that while ErbB4 is overexpressed in IBD patients, its specific ligand
neuregulin 4 (NRG4) is downregulated (Bernard et al., 2012; Frey et al., 2009). Since ErbB4
activation has a protective role in the intestinal epithelium (McElroy et al., 2014), a
downregulation of its specific ligand, NRG4, might be part of the disease etiology and
51
progression. In healthy mice, NRG4 was detected in the whole tissue but not the epithelium
(Bernard et al., 2012), suggesting that it is secreted from the subepithelial mucosa.
Here we are investigating the mechanisms controlling the inflammation-induced reduction of
NRG4. In this report, we show that TNF treatment resulted in a gradual downregulation of
NRG4 in ISEMFs. We also show that this TNF-mediated signaling is through the JAK/STAT
pathway. Although anti-TNF therapy is beneficial in some IBD patients, there is a substantial
subset of patients who either do not respond to it or develop a resistance to it. Therefore,
targeting pathways that are involved in the NRG4 downregulation may bolster epithelial repair
and provide an option for new therapeutic avenues. 52
Materials and Methods:
Human Samples— The procedures of this study were approved by the Institutional Review
Board at Children’s Hospital Los Angeles and written informed consent was obtained from each
subject.
Animals— All animal use was approved and monitored by the Children’s Hospital Los Angeles
Institutional Animal Care and Use Committees (animal welfare assurance number A3276-01).
C57Bl/6 mice were kept in standard housing with a 12-hour light/dark cycle at 21˚ to 22˚C.
Myofibroblast Isolation and Culture— Intestinal subepithelial myofibroblasts (ISEMFs) were
generously provided by Dr. Tracy Grikscheit. Human or murine small intestinal specimens were
used for the isolation of ISEMF (hISEMF and mISEMF, respectively). Isolation was performed
as previously described (Lahar et al., 2011). Briefly, the tissue was placed into a 15ml tube
containing cold Hank’s Buffered Salt Solution (HBSS). The tube was vigorously shaken and
then filtered. The tissue was then cut into small pieces and digested with dispase and
collagenase type IV in HBSS for 30min followed by the addition of 10% FBS in DMEM. The cells
were centrifuged, resuspended in 10% FBS, NEAA, and Antibiotic/Antimycotic in DMEM, and
then plated.
Cell Cultures and Treatment Conditions— ISEMF cells were cultured at 37˚C with 5% CO2 and
maintained in 10% FBS, 1X NEAA, and 1% PenStrep in DMEM. Cells were treated with human
or murine TNF (hTNF, mTNF) as appropriate (PeproTech); U0126 (Millipore); and SP600125
(Sigma-Aldrich).
53
RNA Isolation and Real-time Quantitative PCR (qPCR)— RNA was extracted from cell cultures
using E.Z.N.A. (OMEGA bio-tek). cDNA was synthesized from 1µg of RNA using a High-
Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed using
TaqMAN (Applied Biosystems) assays, and relative expression determined by the 2
-ΔΔCt

method. Reference gene was HPRT.
Gene Expression Assays and Primers— TAqMAN assay numbers used were: NRG4
(Hs00945535_m1), HPRT (Hs02800695_m1), Nrg4 (Mm00446254_m1), Stat3
(Mm01219775_m1), and Hprt (Mm01545399_m1).
Protein Assay and Western blot— Protein was extracted from cell cultures using previously
described protocols (Bernard et al., 2012). Samples were quantified by Dc protein assay
(Pierce) and 30µg of each sample was separated on SDS-polyacrylamide gel (4-12%,
Invitrogen), transferred onto nitrocellulose membrane, and immunoblotted and scanned using
the LI-COR Odyssey infrared detection system.

Antibodies for Western Blot Analysis— From Cell Signaling Technologies, rabbit α-JNK2
(#4672) and mouse α-phospho-JNK (#9255), mouse α-total Erk1/2 (#9107) and rabbit α-
phopho-Erk1/2 (#4370), rabbit α-phospho-STAT3 (#9145); from LI-COR, donkey α-rabbit IRDye
680LT (#926-68023) and donkey α-mouse IRDye 800CW (#926-32212). Equal loading was
monitored by blotting for mouse β-Actin (Sigma Aldrich- Clone AC-15 #A1978- 1:10,000).
RNA silencing— Non-targeting control and Stat3 siRNA (# L-040794-01) On-Target plus siRNA
SMART pools were purchased from Dharmacon and transfected into mouse ISEMF (50nM
54
siRNA) with Lipofectamine RNAiMax (Invitrogen- #13778-075) following the manufacture’s
protocol.
Statistics— All data represents three or more independent experiments, as indicated in the
figure legends. Statistical analysis was performed using Prism 7.0 software (GraphPad).
Significance of results was tested by ANOVA with Dunnett’s or Sidak’s post-test as appropriate. 55
Results
TNF downregulates NRG4 expression in myofibroblasts. We previously showed that while
ErbB4 is overexpressed in IBD, its specific ligand, NRG4, is downregulated (Bernard et al.,
2012; Frey et al., 2009). Since NRG4 is mainly secreted by subepithelial mucosa (Bernard et
al., 2012), we used ISEMFs for our experimental design. To test the role of the inflammatory
response in affecting NRG4, we treated hISEMFs with TNF at different concentrations and
different time points (Fig 14). Our results showed a gradual TNF-induced downregulation of
NRG4 expression with a maximum reduction after 24h although longer time points have not
been tested.
TNF-induced downregulation of NRG4 is through the JAK/STAT pathway. To identify which
downstream signaling pathways are activated by TNF resulting in NRG4 reduction, we treated
mISEMFs with inhibitors with the aim of blocking the downregulation. TNF has been shown to
interact with JNK and MAPK pathways [for review, see Ref. (Wajant et al., 2003)]. However,
neither the JNK inhibitor JNKi II, or the MEK1/2 inhibitor U0126 could overcome the inhibitory
effect of TNF (Fig 15). These results suggest that neither JNK or MAPK are involved in the TNF-
induced downregulation of NRG4.
The JAK/STAT pathway is involved in the signaling of several cytokines (e.g. interleukins and
interferon gamma) and is involved in inflammatory immune responses (Nguyen et al., 2013;
Pickert et al., 2009). Genome-wide association studies revealed that IBD patients (both CD and
UC) have mutations in JAK2 and its downstream molecule STAT3 (Anderson et al., 2009;
Barrett et al., 2008). Therefore, we explored JAK/STAT pathway as a potential TNF target by
using the JAK1/2 inhibitor Ruxolitinib. Our results show that Ruxolitinib was able to block the
TNF effect (Fig 16), implicating the the JAK/STAT pathway. 56
FIGURE 14. TNF downregulates NRG4 expression in myofibroblasts. A, qPCR analysis
(HPRT as reference) of hISEMFs for NRG4 expression treated with different concentrations of
TNF (1, 10, 100ng/ml) for 24h. B, qPCR analysis (HPRT as reference) of hISEMFs for NRG4
expression at different time points after treatment with hTNF (10ng/ml). Dashed line represents
control (n≥3 independent experiments). Error bars, +/- SEM. *, p<0.05; ** p<0.01; #, p<0.0001
vs. control. 57
A. B.
FIGURE 15. Neither JNK or MAPK signaling are required for TNF downregulation of
NRG4. A, qPCR analysis (Hprt as reference) of mISEMFs for Nrg4 expression after 24h
treatment with mTNF (100ng/ml) +/— JNK inhibitor SP600125 (10µM), and MEK1/2 inhibitor
U0126 (10µM) (n=4 independent experiments). Error bars, +/- SEM. *, p<0.05; ** p<0.01 vs.
control. B and C, Confirmation of inhibition using Western blot analysis on lysates from
mISEMFs treated with JNK inhibitor SP600125 (10µM), and MEK1/2 inhibitor U0126 (10µM) for
1h followed by treatment with mTNF (100ng/ml) for 10min (n=1). 58
FIGURE 16. TNF-induced downregulation of NRG4 is through the JAK/STAT pathway.
A, qPCR analysis (Hprt as reference) of mISEMFs for Nrg4 expression after 24h treatment with
mTNF (100ng/ml) +/— JAK1/2 inhibitor Ruxolitinib (Ruxo, 5 or 10µM) (n=5 independent
experiments). Error bars, +/- SEM. *, p<0.05; †, p<0.001 vs. control. ns, non significant. B,
Confirmation of inhibition using Western blot analysis on lysates from mISEMFs treated with
Ruxolitinib (Ruxo, 5 or 10µM) for 1h followed by treatment with mTNF (100ng/ml) for 10min
(n=1). C, qPCR analysis (HPRT as reference) of hISEMFs for NRG4 expression after 24h
treatment with mTNF (10ng/ml) +/— STAT3 inhibitor Stattic (10 or 20µM) (n=2 independent
experiments). 59
C.
B.
The inhibitory effect of TNF is likely not through Stat3. TNF has been shown to induce the
production of IL-6 (Legrand-Poels et al., 2000), which in turn activates its downstream target
STAT3 (Guschin et al., 1995). Since our results with Ruxolitinib implicated JAK/STAT signaling
in TNF-induced NRG4 suppression, we investigated STAT3 as a TNF target for the
downregulation of NRG4. The STAT3 inhibitor Stattic was effective in blocking TNF-induced
NRG4 loss (Fig 16C). However, it was also cytotoxic as has been previously reported (Schust et
al., 2006), which may have confounded the results. Therefore, we used siRNA transfection
technique to knockdown Stat3 in mISEMFs. Although the transfection resulted in about 50%
knockdown of Stat3 (Fig 17A), we did not observe an abrogation of the TNF-induced reduction
of Nrg4 (Fig 17B). These results either suggest that a low level of Stat3 is sufficient to elicit an
effect on the expression of Nrg4, or that other Stat members are involved in this mechanism. 60
FIGURE 17. The inhibitory effect of TNF is likely not through Stat3. A and B, qPCR analysis
(Hprt as reference) of transfected mISEMFs with non-targeting (NT) or Stat3 siRNA for Stat3
and Nrg4 expression +/— mTNF (100ng/ml) for 24h (n=3 independent experiments). Error bars,
+/- SEM. *, p<0.05; **, p<0.01 vs. control.  61
Discussion
TNF is one of the major contributing cytokines that trigger inflammation in IBD and necrotizing
enterocolitis (NEC). In this study, we report that TNF significantly reduced NRG4 expression in
ISEMFs (Fig 14). To further understand the mechanism, we showed that neither JNK or MAPK
inhibitors were able to block the effect of TNF on NRG4 (Fig 15). However, Ruxolitinib, which is
a JAK1/2 inhibitor, was able to abrogate TNF-induced downregulation of NRG4 (Fig 16). Which
Stat member is downstream of JAK activation remains to be investigated, but our results
suggest that Stat3 might not be involved as 50% knockdown of Stat3 did not alter NRG4
reduction (Fig 17). Taken together, these results present JAK/STAT signaling in ISEMF as a
potential therapeutic target in intestinal inflammatory disorders.
IBD patients have increased levels of serum and stool TNF (Braegger et al., 1992; Komatsu et
al., 2001). As we have previously shown, IBD patients have significantly lower levels of NRG4
expression (Bernard et al., 2012). Similarly, NRG4 was reduced in IL-10
-/-
mice, which is a
chronic colitis mouse model with a high serum TNF level (Bernard et al., 2012; Cohen et al.,
2004). These reports suggested that TNF might play a role in downregulating NRG4, which is
secreted from the submucosa (Bernard et al., 2012). Indeed, our results show that TNF
treatment of ISEMF resulted in a reduction in NRG4 expression (Fig 14). Since NRG4 has a
protective role against apoptosis and intestinal inflammation (Bernard et al., 2012; McElroy et
al., 2014), a reduced expression of NRG4 induced by TNF could be one of the factors
implicated in breaches of the epithelial barrier and thus exacerbating the inflammatory
response.
It is possible that TNF is indirectly affecting NRG4 expression. TNF transactivates ErbB4 by
releasing the surface-bound HB-EGF by activating TNF alpha converting enzyme (TACE or
62
ADAM17) through MAPK (Hilliard et al., 2011). Therefore, the reduced expression of NRG4
could be in response to ErbB4 transactivation by TNF. To test this hypothesis, treating ISEMFs
with an exogenous HB-EGF, NRG4, or TACE inhibitor and observing changes in NRG4
expression would definitely provide an answer; these experiments are currently in progress. In
addition, long term treatment with total parenteral nutrition (TPN), which has well documented
side effects (one of which is intestinal epithelial atrophy) results in high levels of TNF (Gogos et
al., 1992; Opara et al., 1995; Pironi et al., 1994). Interestingly, while TPN-treated mice had a low
expression of NRG4, it was restored to normal levels in TPN-treated ADAM17 (TACE) knockout
mice (Feng et al., 2015). These findings provide alternative theories of how TNF regulates
NRG4 expression.
TNF has antagonistic activity against the transforming growth factor-β (TGFβ) signaling
pathway. This pathway is activated by the ligand binding with the two receptor dimers of
TGFβRI and TGFβRII, forming a ligand/heterotetrameric receptor complex. Signal transduction
of the TGFβ pathway is carried out by the SMAD proteins, which consists of three subgroups:
the receptor-regulated SMADs (R-SMAD), the common mediator SMAD (Co-SMAD), and the
inhibitory SMADs (I-SMAD). R-SMADs include: SMAD1, SMAD2, SMAD3, SMAD5, and SMAD9
(aka SMAD8). While the Co-SMAD only consist of SMAD4, I-SMADs include SMAD6 and
SMAD7. The ligand receptor complex leads to the recruitment and phosphorylation of two R-
SMADs, followed by binding with the Co-SMAD (SMAD4). The SMAD trimer complex is then
translocated to the nucleus and initiates the transcription of its target genes [for review, see Ref.
(Schmierer and Hill, 2007)]. TNF antagonizes TGFβ/Smad signaling by blocking Smad3
transcription, and inducing Smad7 that inhibits the formation of Smad2/4 complex (Bitzer et al.,
2000; Hayashi et al., 1997; Verrecchia et al., 2000). Supporting these findings, IBD patients
(high levels of TNF) were found to have an increase in SMAD7 and a decrease in p-SMAD3
63
(Monteleone et al., 2001). Interestingly, even though TNF activates MAPK pathway (Kaiser et
al., 1999; Winston et al., 1995), inhibition of MAPK also blocks the formation of the Smad2/3/4
complex in hepatic myofibroblasts (Jiang et al., 2015). Our results showed that MAPK inhibition
resulted in a reduction in NRG4 similar to that in the TNF treatment (Fig 15) hinting at a
common mechanism that links TNF and inhibition of MAPK such as the TGFβ/Smad pathway.
All these reports with our findings suggest that while MAPK is not involved in NRG4
downregulation, TGFβ/Smad signaling might be.
TNF activates MAPK pathway (Kaiser et al., 1999; Winston et al., 1995). However, our results
showed that MAPK inhibition resulted in a reduction in NRG4 similar to that in the TNF
treatment (Fig 15). MAPK inhibition blocks the formation of the Smad2/3/4 complex in hepatic
myofibroblasts (Jiang et al., 2015). On the other hand, TNF is known to be an antagonist to
TGFβ/Smad signaling. TNF induces Smad7, one of the two inhibitory Smad proteins, that
blocks the formation of Smad2/4 complex (Bitzer et al., 2000; Hayashi et al., 1997). It also
induces c-Jun and JunB and thus blocking Smad3 transcription (Verrecchia et al., 2000). All
these reports with our findings suggest that while MAPK is not involved in NRG4
downregulation, TGFβ/Smad signaling might be.
The Janus Kinase/ Signal Transducers and Activators of Transcription (JAK/STAT) pathway is
involved in the signaling cascade of several cytokines [for review, see Refs. (Aaronson and
Horvath, 2002; Rawlings et al., 2004; Zundler and Neurath, 2016)]. There are four JAK
molecules: JAK1, JAK2, JAK3, and TYK2. The downstream target of JAK activation are the
STAT transcription factors. There are seven STAT molecules: STAT1, STAT2, STAT3, STAT4,
STAT5a, STAT5b, and STAT6. When a receptor is activated by a ligand, it dimerizes bringing the
two JAK molecules closer leading to their autophosphorylation, transphosphorylation of the
64
receptor's tyrosine residue, and recruitment of STAT molecules. STATs are then phosphorylated
to form homo- or heterodimers. The dimerized STAT complex then translocates to the nucleus,
binds to the DNA, and initiates the transcription of its target genes.
IBD patients were found to have single nucleotide polymorphisms (SNPs) in JAK2, TYK2,
STAT1, STAT3, and STAT4 genes implicating a dysregulated signaling in the JAK/STAT pathway
in IBD patients (Anderson et al., 2009; Barrett et al., 2008; Jostins et al., 2012; Wu et al., 2007).
Therefore, JAK/STAT pathway was investigated as a potential target for IBD therapy. JAK
inhibitors that are undergoing clinical trials as IBD treatments are: Tofacitinib (Phase III-
ClinicalTrials.gov- Identifier: NCT01470612), and Peficitinib (Phase II- ClinicalTrials.gov-
Identifier: NCT01959282) for ulcerative colitis, and Filgotinib (Phase II- ClinicalTrials.gov-
Identifier: NCT02048618) for Crohn’s disease. Our results show that inhibiting JAK1 and/or
JAK2 was able to block the TNF-induced reduction of NRG4. These results provide some
insight into the mechanism of action of JAK inhibitors in IBD and how they affect ISEMFs.
A link between functional abnormalities in STAT3 and IBD is well established. GWAS uncovered
an association between IBD (both CD and UC) and mutations in STAT3 gene (Anderson et al.,
2009; Barrett et al., 2008; Franke et al., 2008). STAT3 is constitutively activated in T cells and
macrophages of IBD patients. Its phosphorylation was found to be confined in regions with
active inflammation and correlated with disease severity (Lovato et al., 2003; Mudter et al.,
2005; Musso et al., 2005). In addition, TNF was found to induce Stat3 DNA binding activity in
intestinal myofibroblasts (Theiss et al., 2005). Therefore, Stat3 seemed like a good candidate.
Although only a 50% knockdown of Stat3 was achieved, it did not block the TNF effect on NRG4
expression (Fig17 B). It is possible that the remaining 50% is sufficient to induce an effect, but it
is also possible that another member of the Stat family is involved. We found that the STAT3
65
inhibitor Stattic blocked TNF-induced NRG4 downregulation, but it also induced cell death so
these results must be interpreted cautiously. As previously mentioned, other STAT members
were also mutated in IBD patients. Some of them were found to be downstream targets of TNF.
For example, TNF treated adipocytes resulted in a strong phosphorylation of STAT3 and STAT5
and to a lesser extent STAT1 and STAT6 (Guo et al., 1998). TNF was also found to
phosphorylate STAT3 and STAT5b in B cells (Miscia et al., 2002). Further studies need to
conducted to tease out which Stat member is involved in the TNF-dependent NRG4 response.
Interestingly, TNF treatment mildly reduced Stat3 RNA expression (Fig17 A). It could be due to a
negative feedback by the JAK/STAT negative regulators: suppressors of cytokine signaling
(SOCS), protein inhibitors of activated STATs (PIAS), and protein tyrosine phosphatases
(PTPs). But, it could also be explained by TNF’s antagonism to the TGFβ/Smad signaling. As
previously mentioned, TNF inhibits the formation of Smad2/3/4 complex. Supporting this idea,
hepatocytes from Smad3
-/-
mice had a reduced level of P-Stat3 (Kremer et al., 2014). Taken
together, these reports suggest that Stat3 might not be part of NRG4 downregulation cascade.
At this point, it is unclear which TNF receptor mediates the NRG4 reduction. However, it can be
tested by using the commercially available agonist antibodies to activate the receptors
selectively or by preparing ISEMFs from TnfR1
-/-
and TnfR2
-/-
mice (which are available in our
collaborator’s lab). Loss of or the persistence of NRG4 expression after TNF treatment would
confirm the involvement of either or both of the receptors.
To summarize, our results show that TNF causes a reduction in NRG4 expression. This effect
appears to be through the JAK/STAT pathway. However, a lot needs to be explored. For
example, Which TNFR does TNF bind to to trigger that response? Which STAT is downstream
66
of JAK activation? Are there other pathways involved? Answering these questions will take us a
step closer to utilizing JAK/STAT pathway in ISEMF as a novel therapeutic target to counteract
the TNF-triggered complications. 67
Conclusion:
Inflammatory bowel disease (IBD) affects more than 1.4 million patients in the US (Kappelman
et al., 2013), 2.5 to 3 million in Europe (Burisch et al., 2013), and has an increasing prevalence
worldwide especially in the pediatric population. It is often characterized by breaches in the
epithelial barrier and a dysregulated interaction between the microbiota and the immune system
leading to chronic disease. Current treatments largely targets the excessive inflammation (such
as anti-TNF therapy), which a subset of patients are resistant to while others develop resistance
to over time. We are investigating novel approaches to treating IBD that target the damaged
epithelial barrier, to which there are no current treatments.
My work shows that the ErbB3 receptor tyrosine kinase limits Paneth cell numbers, which are
either lost or functionally impaired in IBD patients. The mechanism of this response is by ErbB3
negatively regulating Atoh1 (a secretory lineage differentiation factor) through activating the
PI3K-Akt pathway.
I also demonstrated that conditional knockout of ErbB3 leads to an early development of Paneth
cells, which could be beneficial for neonatal necrotizing enterocolitis (NEC) that is characterized
by a low number of Paneth cells and low levels of its antimicrobial peptides (Schaart et al.,
2009; Zhang et al., 2012a). I also showed that TNF, which is one of the major contributors to
inflammation in IBD and NEC, greatly affects the neuregulin ligands that activate ErbB3 and
ErbB4. TNF exposure results in the downregulation of NRG4 (an ErbB4 specific ligand) in
intestinal subepithelial myofibroblasts (ISEMFs), which recapitulates what we found in IBD
patients (Bernard et al., 2012). Furthermore, since ErbB4 activation is important in protecting
68
the epithelium against inflammation (Bernard et al., 2012; McElroy et al., 2014), a reduction in
its ligand will compromise that role and further exacerbates the epithelial damage.
For NEC or IBD patients who developed resistance to current therapies, there is an urgent need
for novel treatment options. Bone marrow transplantation or stem cell cell therapy seems like an
effective alternative for non-responsive or severe IBD. It can replace impaired ISEMFs, immune
cells, and epithelial cells by EMT, however studies have shown that the benefits were only
temporary (Garcia-Bosch et al., 2010). We provide new targets such as ErbB3 blocking agents
to boost the number of Paneth cells, or JAK/STAT inhibitors to block the downregulation of
NRG4 and therefore offer epithelial protection (Fig 18). However, there is still more work to be
done. Initial studies suggest that there is an alteration in the microbiota of the ErbB3-null mice
(Fig 19), which will be further explored in our lab. We also need to know if these mice recover
from infections sooner than their control littermates. In other words, how do these abundant
Paneth cells react to a challenge? We need to further understand how does ErbB3 affect Atoh1
and why does it only lead to a specific increase in Paneth cells but non of the other secretory
cells. Which Stat is downstream of the TNF-induced downregulation of NRG4? All these
questions remain to be answered. 69
FIGURE 18: ErbB3 and JAK/STAT pathway as potential therapeutic targets in IBD. 70
FIGURE 19: ErbB3-IEKO mice have have different microbial profile than their control
littermates. 71
ErbB3 FF ErbB3 KO
IBD ErbB3 IEKO:
Firmicutes
Bacteroidetes
Proteobacteria
Actinobacteria
Abbreviations used
72
Cytokeratin
(Cytometry by Time of Flight)
Dextran Sodium Sulfate
EGF Receptor
Intestinal Epithelial Specific Knockout
Intestinal Stem Cell
Matrix Metalloproteinase-7
Neuregulin
Paneth Cell
Phosphatidylinositol 3-Kinase
Quantitative Real-Time PCR
c-Jun N-terminal kinase
Mitogen Activated Protein Kinase
Janus Kinase
Signal Transducers and Activators of Transcription
Human, Murine Intestinal Subepithelial Myofibroblast
Human, Murine Tumor Necrosis Factor
Ruxolitinib
Tumor Necrosis Factor Receptor
Transforming Growth Factor-β
Neuregulin
CK
CyTOF
DSS
EGFR
IEKO
ISC
MMP7
NRG
Paneth cell
PI3K
qPCR
JNK
MAPK
JAK
STAT
hISEMF, mISEMF
hTNF, mTNF
Ruxo
TnfR
TGFβ
NRG
Abbreviations used, cont.
73
IBD
CD
UC
SNP
TACE
HB-EGF
TPN
Inflammatory Bowel Disease
Crohn’s Disease
Ulcerative Colitis
Single Nucleotide Polymorphism
TNF Alpha Converting Enzyme
Heparin-Binding EGF-like growth factor
Total Parenteral Nutrition
References:
Aaronson, D.S., and Horvath, C.M. (2002). A road map for those who don't know JAK-STAT.
Science 296, 1653-1655.
Abel, E.V., Basile, K.J., Kugel, C.H., 3rd, Witkiewicz, A.K., Le, K., Amaravadi, R.K., Karakousis,
G.C., Xu, X., Xu, W., Schuchter, L.M., et al. (2013). Melanoma adapts to RAF/MEK inhibitors
through FOXD3-mediated upregulation of ERBB3. J Clin Invest 123, 2155-2168.
Adolph, T.E., Tomczak, M.F., Niederreiter, L., Ko, H.J., Bock, J., Martinez-Naves, E., Glickman,
J.N., Tschurtschenthaler, M., Hartwig, J., Hosomi, S., et al. (2013). Paneth cells as a site of
origin for intestinal inflammation. Nature 503, 272-276.
Al Alam, D., Danopoulos, S., Schall, K., Sala, F.G., Almohazey, D., Fernandez, G.E., Georgia,
S., Frey, M.R., Ford, H.R., Grikscheit, T., et al. (2015). Fibroblast growth factor 10 alters the
balance between goblet and Paneth cells in the adult mouse small intestine. Am J Physiol
Gastrointest Liver Physiol 308, G678-690.
Aliaga, J.C., Deschenes, C., Beaulieu, J.F., Calvo, E.L., and Rivard, N. (1999). Requirement of
the MAP kinase cascade for cell cycle progression and differentiation of human intestinal cells.
Am J Physiol 277, G631-641.
Almohazey, D., and Frey, M.R. (2014). The Intestinal Stem Cell Niche and Its Regulation by
ErbB Growth Factor Receptors. In Stem cells, tissue engineering, and regenerative medicine, D.
Warburton, ed. (United States, New Jersey: World Scientific).
Anderson, C.A., Massey, D.C., Barrett, J.C., Prescott, N.J., Tremelling, M., Fisher, S.A.,
Gwilliam, R., Jacob, J., Nimmo, E.R., Drummond, H., et al. (2009). Investigation of Crohn's
disease risk loci in ulcerative colitis further defines their molecular relationship.
Gastroenterology 136, 523-529 e523.
Andreu, P., Peignon, G., Slomianny, C., Taketo, M.M., Colnot, S., Robine, S., Lamarque, D.,
Laurent-Puig, P., Perret, C., and Romagnolo, B. (2008). A genetic study of the role of the Wnt/
beta-catenin signalling in Paneth cell differentiation. Dev Biol 324, 288-296.
Aoki, R., Shoshkes-Carmel, M., Gao, N., Shin, S., May, C.L., Golson, M.L., Zahm, A.M., Ray,
M., Wiser, C.L., Wright, C.V., et al. (2016). Foxl1-expressing mesenchymal cells constitute the
intestinal stem cell niche. Cell Mol Gastroenterol Hepatol 2, 175-188.
Armaka, M., Apostolaki, M., Jacques, P., Kontoyiannis, D.L., Elewaut, D., and Kollias, G. (2008).
Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory
joint and intestinal diseases. J Exp Med 205, 331-337.
74
Ayabe, T., Satchell, D.P., Pesendorfer, P., Tanabe, H., Wilson, C.L., Hagen, S.J., and Ouellette,
A.J. (2002). Activation of Paneth cell alpha-defensins in mouse small intestine. J Biol Chem 277,
5219-5228.
Barker, N. (2014). Adult intestinal stem cells: critical drivers of epithelial homeostasis and
regeneration. Nat Rev Mol Cell Biol 15, 19-33.
Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth,
A., Korving, J., Begthel, H., Peters, P.J., et al. (2007). Identification of stem cells in small
intestine and colon by marker gene Lgr5. Nature 449, 1003-1007.
Barker, N., van Oudenaarden, A., and Clevers, H. (2012). Identifying the stem cell of the
intestinal crypt: strategies and pitfalls. Cell Stem Cell 11, 452-460.
Barrett, J.C., Hansoul, S., Nicolae, D.L., Cho, J.H., Duerr, R.H., Rioux, J.D., Brant, S.R.,
Silverberg, M.S., Taylor, K.D., Barmada, M.M., et al. (2008). Genome-wide association defines
more than 30 distinct susceptibility loci for Crohn's disease. Nat Genet 40, 955-962.
Baumgart, D.C., and Sandborn, W.J. (2012). Crohn's disease. Lancet 380, 1590-1605.
Berger, M.B., Mendrola, J.M., and Lemmon, M.A. (2004). ErbB3/HER3 does not homodimerize
upon neuregulin binding at the cell surface. FEBS Lett 569, 332-336.
Bernard, J.K., McCann, S.P., Bhardwaj, V., Washington, M.K., and Frey, M.R. (2012).
Neuregulin-4 is a survival factor for colon epithelial cells both in culture and in vivo. Journal of
Biological Chemistry 287, 39850-39858.
Bevins, C.L., and Salzman, N.H. (2011). Paneth cells, antimicrobial peptides and maintenance
of intestinal homeostasis. Nat Rev Microbiol 9, 356-368.
Bitzer, M., von Gersdorff, G., Liang, D., Dominguez-Rosales, A., Beg, A.A., Rojkind, M., and
Bottinger, E.P. (2000). A mechanism of suppression of TGF-beta/SMAD signaling by NF-kappa
B/RelA. Genes Dev 14, 187-197.
Braegger, C.P., Nicholls, S., Murch, S.H., Stephens, S., and MacDonald, T.T. (1992). Tumour
necrosis factor alpha in stool as a marker of intestinal inflammation. Lancet 339, 89-91.
Brandl, K., Plitas, G., Schnabl, B., DeMatteo, R.P., and Pamer, E.G. (2007). MyD88-mediated
signals induce the bactericidal lectin RegIII gamma and protect mice against intestinal Listeria
monocytogenes infection. J Exp Med 204, 1891-1900.
75
Bry, L., Falk, P., Huttner, K., Ouellette, A., Midtvedt, T., and Gordon, J.I. (1994). Paneth cell-
differentaition in the developing intestine of normal and transgenic mice. Proceedings of the
National Academy of Sciences of the United States of America 91, 10335-10339.
Buczacki, S.J., Zecchini, H.I., Nicholson, A.M., Russell, R., Vermeulen, L., Kemp, R., and
Winton, D.J. (2013). Intestinal label-retaining cells are secretory precursors expressing Lgr5.
Nature 495, 65-69.
Burisch, J., Jess, T., Martinato, M., Lakatos, P.L., and EpiCom, E. (2013). The burden of
inflammatory bowel disease in Europe. J Crohns Colitis 7, 322-337.
Cadwell, K., Liu, J.Y., Brown, S.L., Miyoshi, H., Loh, J., Lennerz, J.K., Kishi, C., Kc, W., Carrero,
J.A., Hunt, S., et al. (2008). A key role for autophagy and the autophagy gene Atg16l1 in mouse
and human intestinal Paneth cells. Nature 456, 259-U262.
Capparelli, C., Rosenbaum, S., Berger, A.C., and Aplin, A.E. (2015). Fibroblast-derived
neuregulin 1 promotes compensatory ErbB3 receptor signaling in mutant BRAF melanoma. J
Biol Chem 290, 24267-24277.
Carmon, K.S., Gong, X., Lin, Q., Thomas, A., and Liu, Q. (2011). R-spondins function as ligands
of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc Natl Acad
Sci U S A 108, 11452-11457.
Cheng, H. (1974). Origin, differentiation and renewal of the four main epithelial cell types in the
mouse small intestine. IV. Paneth cells. Am J Anat 141, 521-535.
Cheng, H., and Leblond, C.P. (1974). Origin, differentiation and renewal of the four main
epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four
epithelial cell types. Am J Anat 141, 537-561.
Christa, L., Carnot, F., Simon, M.T., Levavasseur, F., Stinnakre, M.G., Lasserre, C., Thepot, D.,
Clement, B., Devinoy, E., and Brechot, C. (1996). HIP/PAP is an adhesive protein expressed in
hepatocarcinoma, normal Paneth, and pancreatic cells. Am J Physiol 271, G993-1002.
Chu, H., Pazgier, M., Jung, G., Nuccio, S.P., Castillo, P.A., de Jong, M.F., Winter, M.G., Winter,
S.E., Wehkamp, J., Shen, B., et al. (2012). Human alpha-defensin 6 promotes mucosal innate
immunity through self-assembled peptide nanonets. Science 337, 477-481.
Clevers, H.C., and Bevins, C.L. (2013). Paneth cells: maestros of the small intestinal crypts.
Annu Rev Physiol 75, 289-311.
76
Cohen, S.L., Moore, A.M., and Ward, W.E. (2004). Interleukin-10 knockout mouse: a model for
studying bone metabolism during intestinal inflammation. Inflamm Bowel Dis 10, 557-563.
Coutinho, H.B., da Mota, H.C., Coutinho, V.B., Robalinho, T.I., Furtado, A.F., Walker, E., King,
G., Mahida, Y.R., Sewell, H.F., and Wakelin, D. (1998). Absence of lysozyme (muramidase) in
the intestinal Paneth cells of newborn infants with necrotising enterocolitis. J Clin Pathol 51,
512-514.
Cross, M., Mangelsdorf, I., Wedel, A., and Renkawitz, R. (1988). Mouse lysozyme M gene:
isolation, characterization, and expression studies. Proc Natl Acad Sci U S A 85, 6232-6236.
Cunliffe, R.N., Rose, F.R., Keyte, J., Abberley, L., Chan, W.C., and Mahida, Y.R. (2001). Human
defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous
epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease. Gut
48, 176-185.
de Lau, W., Barker, N., Low, T.Y., Koo, B.K., Li, V.S., Teunissen, H., Kujala, P., Haegebarth, A.,
Peters, P.J., van de Wetering, M., et al. (2011). Lgr5 homologues associate with Wnt receptors
and mediate R-spondin signalling. Nature 476, 293-297.
Erickson, S.L., O'Shea, K.S., Ghaboosi, N., Loverro, L., Frantz, G., Bauer, M., Lu, L.H., and
Moore, M.W. (1997). ErbB3 is required for normal cerebellar and cardiac development: a
comparison with ErbB2-and heregulin-deficient mice. Development 124, 4999-5011.
Fattore, L., Malpicci, D., Marra, E., Belleudi, F., Noto, A., De Vitis, C., Pisanu, M.E., Coluccia, P.,
Camerlingo, R., Roscilli, G., et al. (2015). Combination of antibodies directed against different
ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in
melanoma. Oncotarget 6, 24823-24841.
Feng, Y., Tsai, Y.H., Xiao, W., Ralls, M.W., Stoeck, A., Wilson, C.L., Raines, E.W., Teitelbaum,
D.H., and Dempsey, P.J. (2015). Loss of ADAM17-Mediated Tumor Necrosis Factor Alpha
Signaling in Intestinal Cells Attenuates Mucosal Atrophy in a Mouse Model of Parenteral
Nutrition. Mol Cell Biol 35, 3604-3621.
Francoeur, C., Bouatrouss, Y., Seltana, A., Pinchuk, I.V., Vachon, P.H., Powell, D.W., Sawan, B.,
Seidman, E.G., and Beaulieu, J.F. (2009). Degeneration of the pericryptal myofibroblast sheath
by proinflammatory cytokines in inflammatory bowel diseases. Gastroenterology 136, 268-277
e263.
Franke, A., Balschun, T., Karlsen, T.H., Hedderich, J., May, S., Lu, T., Schuldt, D., Nikolaus, S.,
Rosenstiel, P., Krawczak, M., et al. (2008). Replication of signals from recent studies of Crohn's
disease identifies previously unknown disease loci for ulcerative colitis. Nat Genet 40, 713-715.
77
Frey, M.R., and Brent Polk, D. (2014). ErbB receptors and their growth factor ligands in pediatric
intestinal inflammation. Pediatr Res 75, 127-132.
Frey, M.R., Edelblum, K.L., Mullane, M.T., Liang, D., and Polk, D.B. (2009). The ErbB4 growth
factor receptor is required for colon epithelial cell survival in the presence of TNF.
Gastroenterology 136, 217-226.
Garabedian, E.M., Roberts, L.J., McNevin, M.S., and Gordon, J.I. (1997). Examining the role of
Paneth cells in the small intestine by lineage ablation in transgenic mice. J Biol Chem 272,
23729-23740.
Garcia-Bosch, O., Ricart, E., and Panes, J. (2010). Review article: stem cell therapies for
inflammatory bowel disease - efficacy and safety. Aliment Pharmacol Ther 32, 939-952.
Ghosh, D., Porter, E., Shen, B., Lee, S.K., Wilk, D., Drazba, J., Yadav, S.P., Crabb, J.W., Ganz,
T., and Bevins, C.L. (2002). Paneth cell trypsin is the processing enzyme for human defensin-5.
Nat Immunol 3, 583-590.
Gogos, C.A., Zoumbos, N.C., Makri, M., and Kalfarentzos, F. (1992). Tumor necrosis factor
production by human mononuclear cells during total parenteral nutrition containing long-chain
triglycerides. Nutrition 8, 26-29.
Grun, D., Lyubimova, A., Kester, L., Wiebrands, K., Basak, O., Sasaki, N., Clevers, H., and van
Oudenaarden, A. (2015). Single-cell messenger RNA sequencing reveals rare intestinal cell
types. Nature 525, 251-255.
Guo, D., Dunbar, J.D., Yang, C.H., Pfeffer, L.M., and Donner, D.B. (1998). Induction of Jak/STAT
signaling by activation of the type 1 TNF receptor. J Immunol 160, 2742-2750.
Guschin, D., Rogers, N., Briscoe, J., Witthuhn, B., Watling, D., Horn, F., Pellegrini, S.,
Yasukawa, K., Heinrich, P., Stark, G.R., et al. (1995). A major role for the protein tyrosine kinase
JAK1 in the JAK/STAT signal transduction pathway in response to interleukin-6. EMBO J 14,
1421-1429.
Guy, P.M., Platko, J.V., Cantley, L.C., Cerione, R.A., and Carraway, K.L., 3rd (1994). Insect cell-
expressed p180erbB3 possesses an impaired tyrosine kinase activity. Proc Natl Acad Sci U S A
91, 8132-8136.
Hampe, J., Franke, A., Rosenstiel, P., Till, A., Teuber, M., Huse, K., Albrecht, M., Mayr, G., De La
Vega, F.M., Briggs, J., et al. (2007). A genome-wide association scan of nonsynonymous SNPs
identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet 39, 207-211.
78
Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y.Y., Grinnell, B.W., Richardson, M.A., Topper,
J.N., Gimbrone, M.A., Jr., Wrana, J.L., et al. (1997). The MAD-related protein Smad7 associates
with the TGFbeta receptor and functions as an antagonist of TGFbeta signaling. Cell 89,
1165-1173.
Hellyer, N.J., Cheng, K., and Koland, J.G. (1998). ErbB3 (HER3) interaction with the p85
regulatory subunit of phosphoinositide 3-kinase. Biochem J 333 ( Pt 3), 757-763.
Heuberger, J., Kosel, F., Qi, J.J., Grossmann, K.S., Rajewsky, K., and Birchmeier, W. (2014).
Shp2/MAPK signaling controls goblet/paneth cell fate decisions in the intestine. Proceedings of
the National Academy of Sciences of the United States of America 111, 3472-3477.
Hilliard, V.C., Frey, M.R., Dempsey, P.J., Peek, R.M., Jr., and Polk, D.B. (2011). TNF-alpha
converting enzyme-mediated ErbB4 transactivation by TNF promotes colonic epithelial cell
survival. Am J Physiol Gastrointest Liver Physiol 301, G338-346.
Hu, T., and Li, C. (2010). Convergence between Wnt-beta-catenin and EGFR signaling in
cancer. Mol Cancer 9, 236.
Huet, C., Sahuquillomerino, C., Coudrier, E., and Louvard, D. (1987). Absorptive and mucus-
secreting subclones isolated from a multipotent intestinal-cell line (HT29) provide new models
for cell polarity and terminal differentiation. Journal of Cell Biology 105, 345-357.
Hugot, J.P., Chamaillard, M., Zouali, H., Lesage, S., Cezard, J.P., Belaiche, J., Almer, S., Tysk,
C., O'Morain, C.A., Gassull, M., et al. (2001). Association of NOD2 leucine-rich repeat variants
with susceptibility to Crohn's disease. Nature 411, 599-603.
Ireland, H., Houghton, C., Howard, L., and Winton, D.J. (2005). Cellular inheritance of a Cre-
activated reporter gene to determine Paneth cell longevity in the murine small intestine. Dev
Dyn 233, 1332-1336.
Jaiswal, B.S., Kljavin, N.M., Stawiski, E.W., Chan, E., Parikh, C., Durinck, S., Chaudhuri, S.,
Pujara, K., Guillory, J., Edgar, K.A., et al. (2013). Oncogenic ERBB3 mutations in human
cancers. Cancer Cell 23, 603-617.
Jiang, Y., Wu, C., Boye, A., Wu, J., Wang, J., Yang, X., and Yang, Y. (2015). MAPK inhibitors
modulate Smad2/3/4 complex cyto-nuclear translocation in myofibroblasts via Imp7/8 mediation.
Mol Cell Biochem 406, 255-262.
Jones, D.E., and Bevins, C.L. (1992). Paneth cells of the human small intestine express an
antimicrobial peptide gene. J Biol Chem 267, 23216-23225.
79
Jones, D.E., and Bevins, C.L. (1993). Defensin-6 mRNA in human Paneth cells: implications for
antimicrobial peptides in host defense of the human bowel. FEBS Lett 315, 187-192.
Jones, J.T., Akita, R.W., and Sliwkowski, M.X. (1999). Binding specificities and affinities of egf
domains for ErbB receptors. FEBS Lett 447, 227-231.
Jostins, L., Ripke, S., Weersma, R.K., Duerr, R.H., McGovern, D.P., Hui, K.Y., Lee, J.C.,
Schumm, L.P., Sharma, Y., Anderson, C.A., et al. (2012). Host-microbe interactions have
shaped the genetic architecture of inflammatory bowel disease. Nature 491, 119-124.
Kaiser, G.C., Yan, F., and Polk, D.B. (1999). Conversion of TNF alpha from antiproliferative to
proliferative ligand in mouse intestinal epithelial cells by regulating mitogen-activated protein
kinase. Exp Cell Res 249, 349-358.
Kang, J.H., Lee, E.H., Park, S.W., and Chung, I.Y. (2011). MUC5AC expression through
bidirectional communication of Notch and epidermal growth factor receptor pathways. J
Immunol 187, 222-229.
Kappelman, M.D., Moore, K.R., Allen, J.K., and Cook, S.F. (2013). Recent trends in the
prevalence of Crohn's disease and ulcerative colitis in a commercially insured US population.
Dig Dis Sci 58, 519-525.
Kaser, A., Lee, A.H., Franke, A., Glickman, J.N., Zeissig, S., Tilg, H., Nieuwenhuis, E.E.,
Higgins, D.E., Schreiber, S., Glimcher, L.H., et al. (2008). XBP1 links ER stress to intestinal
inflammation and confers genetic risk for human inflammatory bowel disease. Cell 134,
743-756.
Kaushansky, A., Gordus, A., Budnik, B.A., Lane, W.S., Rush, J., and MacBeath, G. (2008).
System-wide investigation of ErbB4 reveals 19 sites of Tyr phosphorylation that are unusually
selective in their recruitment properties. Chem Biol 15, 808-817.
Kerr, J.B. (2010). Gastrointestinal Tract. In Functional Histology (Australia: Elsevier).
Komatsu, M., Kobayashi, D., Saito, K., Furuya, D., Yagihashi, A., Araake, H., Tsuji, N.,
Sakamaki, S., Niitsu, Y., and Watanabe, N. (2001). Tumor necrosis factor-alpha in serum of
patients with inflammatory bowel disease as measured by a highly sensitive immuno-PCR. Clin
Chem 47, 1297-1301.
Komuro, A., Nagai, M., Navin, N.E., and Sudol, M. (2003). WW domain-containing protein YAP
associates with ErbB-4 and acts as a co-transcriptional activator for the carboxyl-terminal
fragment of ErbB-4 that translocates to the nucleus. J Biol Chem 278, 33334-33341.
80
Kontoyiannis, D., Pasparakis, M., Pizarro, T.T., Cominelli, F., and Kollias, G. (1999). Impaired
on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint
and gut-associated immunopathologies. Immunity 10, 387-398.
Korswagen, H.C. (2002). Canonical and non-canonical Wnt signaling pathways in
Caenorhabditis elegans: variations on a common signaling theme. Bioessays 24, 801-810.
Kremer, M., Son, G., Zhang, K., Moore, S.M., Norris, A., Manzini, G., Wheeler, M.D., and Hines,
I.N. (2014). Smad3 signaling in the regenerating liver: implications for the regulation of IL-6
expression. Transpl Int 27, 748-758.
Laederich, M.B., Funes-Duran, M., Yen, L., Ingalla, E., Wu, X., Carraway, K.L., 3rd, and
Sweeney, C. (2004). The leucine-rich repeat protein LRIG1 is a negative regulator of ErbB
family receptor tyrosine kinases. J Biol Chem 279, 47050-47056.
Lahar, N., Lei, N.Y., Wang, J., Jabaji, Z., Tung, S.C., Joshi, V., Lewis, M., Stelzner, M., Martin,
M.G., and Dunn, J.C. (2011). Intestinal subepithelial myofibroblasts support in vitro and in vivo
growth of human small intestinal epithelium. PLoS One 6, e26898.
Lee, D., Yu, M., Lee, E., Kim, H., Yang, Y., Kim, K., Pannicia, C., Kurie, J.M., and Threadgill,
D.W. (2009). Tumor-specific apoptosis caused by deletion of the ERBB3 pseudo-kinase in
mouse intestinal epithelium. J Clin Invest 119, 2702-2713.
Legrand-Poels, S., Schoonbroodt, S., and Piette, J. (2000). Regulation of interleukin-6 gene
expression by pro-inflammatory cytokines in a colon cancer cell line. Biochem J 349 Pt 3,
765-773.
Lemieux, E., Boucher, M.J., Mongrain, S., Boudreau, F., Asselin, C., and Rivard, N. (2011).
Constitutive activation of the MEK/ERK pathway inhibits intestinal epithelial cell differentiation.
Am J Physiol Gastrointest Liver Physiol 301, G719-730.
Lewin, K. (1969). The Paneth cell in disease. Gut 10, 804-811.
Linggi, B., and Carpenter, G. (2006). ErbB receptors: new insights on mechanisms and biology.
Trends Cell Biol 16, 649-656.
Lovato, P., Brender, C., Agnholt, J., Kelsen, J., Kaltoft, K., Svejgaard, A., Eriksen, K.W.,
Woetmann, A., and Odum, N. (2003). Constitutive STAT3 activation in intestinal T cells from
patients with Crohn's disease. J Biol Chem 278, 16777-16781.
Ma, J., Lyu, H., Huang, J., and Liu, B. (2014). Targeting of erbB3 receptor to overcome
resistance in cancer treatment. Mol Cancer 13, 105.
81
Maurer, C.A., Friess, H., Kretschmann, B., Zimmermann, A., Stauffer, A., Baer, H.U., Korc, M.,
and Buchler, M.W. (1998). Increased expression of erbB3 in colorectal cancer is associated with
concomitant increase in the level of erbB2. Hum Pathol 29, 771-777.
McElroy, S.J., Castle, S.L., Bernard, J.K., Almohazey, D., Hunter, C.J., Bell, B.A., Al Alam, D.,
Wang, L., Ford, H.R., and Frey, M.R. (2014). The ErbB4 ligand Neuregulin-4 protects against
experimental necrotizing enterocolitis. American Journal of Pathology 184, 2768-2778.
McElroy, S.J., Underwood, M.A., and Sherman, M.P. (2013). Paneth cells and necrotizing
enterocolitis: a novel hypothesis for disease pathogenesis. Neonatology 103, 10-20.
Mifflin, R.C., Pinchuk, I.V., Saada, J.I., and Powell, D.W. (2011). Intestinal myofibroblasts:
targets for stem cell therapy. Am J Physiol Gastrointest Liver Physiol 300, G684-696.
Miscia, S., Marchisio, M., Grilli, A., Di Valerio, V., Centurione, L., Sabatino, G., Garaci, F., Zauli,
G., Bonvini, E., and Di Baldassarre, A. (2002). Tumor necrosis factor alpha (TNF-alpha)
activates Jak1/Stat3-Stat5B signaling through TNFR-1 in human B cells. Cell Growth Differ 13,
13-18.
Monteleone, G., Kumberova, A., Croft, N.M., McKenzie, C., Steer, H.W., and MacDonald, T.T.
(2001). Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease. J
Clin Invest 108, 601-609.
Montgomery, R.K., Carlone, D.L., Richmond, C.A., Farilla, L., Kranendonk, M.E., Henderson,
D.E., Baffour-Awuah, N.Y., Ambruzs, D.M., Fogli, L.K., Algra, S., et al. (2011). Mouse
telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells.
Proc Natl Acad Sci U S A 108, 179-184.
Moxey, P.C., and Trier, J.S. (1978). Specialized cell types in the human fetal small intestine.
Anat Rec 191, 269-285.
Mudter, J., Weigmann, B., Bartsch, B., Kiesslich, R., Strand, D., Galle, P.R., Lehr, H.A., Schmidt,
J., and Neurath, M.F. (2005). Activation pattern of signal transducers and activators of
transcription (STAT) factors in inflammatory bowel diseases. Am J Gastroenterol 100, 64-72.
Mukherjee, S., Partch, C.L., Lehotzky, R.E., Whitham, C.V., Chu, H., Bevins, C.L., Gardner,
K.H., and Hooper, L.V. (2009). Regulation of C-type lectin antimicrobial activity by a flexible N-
terminal prosegment. J Biol Chem 284, 4881-4888.
Munoz, J., Stange, D.E., Schepers, A.G., van de Wetering, M., Koo, B.K., Itzkovitz, S.,
Volckmann, R., Kung, K.S., Koster, J., Radulescu, S., et al. (2012). The Lgr5 intestinal stem cell
signature: robust expression of proposed quiescent '+4' cell markers. EMBO J 31, 3079-3091.
82
Musso, A., Dentelli, P., Carlino, A., Chiusa, L., Repici, A., Sturm, A., Fiocchi, C., Rizzetto, M.,
Pegoraro, L., Sategna-Guidetti, C., et al. (2005). Signal transducers and activators of
transcription 3 signaling pathway: an essential mediator of inflammatory bowel disease and
other forms of intestinal inflammation. Inflamm Bowel Dis 11, 91-98.
Nguyen, A.V., Wu, Y.Y., Liu, Q., Wang, D., Nguyen, S., Loh, R., Pang, J., Friedman, K., Orlofsky,
A., Augenlicht, L., et al. (2013). STAT3 in epithelial cells regulates inflammation and tumor
progression to malignant state in colon. Neoplasia 15, 998-1008.
Ocana, A., Vera-Badillo, F., Seruga, B., Templeton, A., Pandiella, A., and Amir, E. (2013). HER3
overexpression and survival in solid tumors: a meta-analysis. J Natl Cancer Inst 105, 266-273.
Opara, E.I., Meguid, M.M., Yang, Z.J., Chai, J.K., and Veerabagu, M. (1995). Tumor necrosis
factor-alpha and total parenteral nutrition-induced anorexia. Surgery 118, 756-760; discussion
760-752.
Ouellette, A.J., Greco, R.M., James, M., Frederick, D., Naftilan, J., and Fallon, J.T. (1989).
Developmental regulation of cryptdin, a corticostatin defensin precursor messenger-RNA in
mouse small inestinal crypt epithelium. Journal of Cell Biology 108, 1687-1695.
Owens, B.M., and Simmons, A. (2013). Intestinal stromal cells in mucosal immunity and
homeostasis. Mucosal Immunol 6, 224-234.
Peeters, T., and Vantrappen, G. (1975). The Paneth cell: a source of intestinal lysozyme. Gut
16, 553-558.
Perret, V., Lev, R., and Pigman, W. (1977). Simple method for the preparation of single cell
suspensions from normal and tumorous rat colonic mucosa. Gut 18, 382-385.
Pickert, G., Neufert, C., Leppkes, M., Zheng, Y., Wittkopf, N., Warntjen, M., Lehr, H.A., Hirth, S.,
Weigmann, B., Wirtz, S., et al. (2009). STAT3 links IL-22 signaling in intestinal epithelial cells to
mucosal wound healing. J Exp Med 206, 1465-1472.
Pinchuk, I.V., Mifflin, R.C., Saada, J.I., and Powell, D.W. (2010). Intestinal mesenchymal cells.
Curr Gastroenterol Rep 12, 310-318.
Pinkas-Kramarski, R., Soussan, L., Waterman, H., Levkowitz, G., Alroy, I., Klapper, L., Lavi, S.,
Seger, R., Ratzkin, B.J., Sela, M., et al. (1996). Diversification of Neu differentiation factor and
epidermal growth factor signaling by combinatorial receptor interactions. EMBO J 15,
2452-2467.
83
Pironi, L., Paganelli, G.M., Miglioli, M., Biasco, G., Santucci, R., Ruggeri, E., Di Febo, G., and
Barbara, L. (1994). Morphologic and cytoproliferative patterns of duodenal mucosa in two
patients after long-term total parenteral nutrition: changes with oral refeeding and relation to
intestinal resection. JPEN J Parenter Enteral Nutr 18, 351-354.
Porter, E.M., Liu, L., Oren, A., Anton, P.A., and Ganz, T. (1997a). Localization of human
intestinal defensin 5 in Paneth cell granules. Infect Immun 65, 2389-2395.
Porter, E.M., van Dam, E., Valore, E.V., and Ganz, T. (1997b). Broad-spectrum antimicrobial
activity of human intestinal defensin 5. Infect Immun 65, 2396-2401.
Potten, C.S. (1977). Extreme sensitivity of some intestinal crypt cells to X and gamma
irradiation. Nature 269, 518-521.
Potten, C.S. (1998). Stem cells in gastrointestinal epithelium: numbers, characteristics and
death. Philos Trans R Soc Lond B Biol Sci 353, 821-830.
Powell, A.E., Wang, Y., Li, Y., Poulin, E.J., Means, A.L., Washington, M.K., Higginbotham, J.N.,
Juchheim, A., Prasad, N., Levy, S.E., et al. (2012). The pan-ErbB negative regulator Lrig1 is an
intestinal stem cell marker that functions as a tumor suppressor. Cell 149, 146-158.
Powell, D.W., Pinchuk, I.V., Saada, J.I., Chen, X., and Mifflin, R.C. (2011). Mesenchymal cells of
the intestinal lamina propria. Annu Rev Physiol 73, 213-237.
Prigent, S.A., and Gullick, W.J. (1994). Identification of c-erbB-3 binding sites for
phosphatidylinositol 3'-kinase and SHC using an EGF receptor/c-erbB-3 chimera. EMBO J 13,
2831-2841.
Rawlings, J.S., Rosler, K.M., and Harrison, D.A. (2004). The JAK/STAT signaling pathway. J Cell
Sci 117, 1281-1283.
Robles, A.I., Traverso, G., Zhang, M., Roberts, N.J., Khan, M.A., Joseph, C., Lauwers, G.Y.,
Selaru, F.M., Popoli, M., Pittman, M.E., et al. (2016). Whole-Exome Sequencing Analyses of
Inflammatory Bowel Disease-Associated Colorectal Cancers. Gastroenterology 150, 931-943.
Rose, M.F., Ren, J., Ahmad, K.A., Chao, H.T., Klisch, T.J., Flora, A., Greer, J.J., and Zoghbi,
H.Y. (2009). Math1 is essential for the development of hindbrain neurons critical for perinatal
breathing. Neuron 64, 341-354.
Salzman, N.H., Polin, R.A., Harris, M.C., Ruchelli, E., Hebra, A., Zirin-Butler, S., Jawad, A.,
Martin Porter, E., and Bevins, C.L. (1998). Enteric defensin expression in necrotizing
enterocolitis. Pediatr Res 44, 20-26.
84
Sangiorgi, E., and Capecchi, M.R. (2008). Bmi1 is expressed in vivo in intestinal stem cells. Nat
Genet 40, 915-920.
Sato, T., van Es, J.H., Snippert, H.J., Stange, D.E., Vries, R.G., van den Born, M., Barker, N.,
Shroyer, N.F., van de Wetering, M., and Clevers, H. (2011). Paneth cells constitute the niche for
Lgr5 stem cells in intestinal crypts. Nature 469, 415-418.
Satoh, Y., Ishikawa, K., Oomori, Y., Takeda, S., and Ono, K. (1992). Bethanechol and a G-
protein activator, NaF/AlCl3, induce secretory response in Paneth cells of mouse intestine. Cell
Tissue Res 269, 213-220.
Satoh, Y., Ishikawa, K., Tanaka, H., Oomori, Y., and Ono, K. (1988). Immunohistochemical
observations of lysozyme in the Paneth cells of specific-pathogen-free and germ-free mice. Acta
Histochem 83, 185-188.
Schaart, M.W., de Bruijn, A.C., Bouwman, D.M., de Krijger, R.R., van Goudoever, J.B., Tibboel,
D., and Renes, I.B. (2009). Epithelial functions of the residual bowel after surgery for necrotising
enterocolitis in human infants. J Pediatr Gastroenterol Nutr 49, 31-41.
Schmauder-Chock, E.A., Chock, S.P., and Patchen, M.L. (1994). Ultrastructural localization of
tumour necrosis factor-alpha. Histochem J 26, 142-151.
Schmierer, B., and Hill, C.S. (2007). TGFbeta-SMAD signal transduction: molecular specificity
and functional flexibility. Nat Rev Mol Cell Biol 8, 970-982.
Schust, J., Sperl, B., Hollis, A., Mayer, T.U., and Berg, T. (2006). Stattic: a small-molecule
inhibitor of STAT3 activation and dimerization. Chem Biol 13, 1235-1242.
Sergina, N.V., Rausch, M., Wang, D., Blair, J., Hann, B., Shokat, K.M., and Moasser, M.M.
(2007). Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3.
Nature 445, 437-441.
Shen, B., Porter, E.M., Reynoso, E., Shen, C., Ghosh, D., Connor, J.T., Drazba, J., Rho, H.K.,
Gramlich, T.L., Li, R., et al. (2005). Human defensin 5 expression in intestinal metaplasia of the
upper gastrointestinal tract. J Clin Pathol 58, 687-694.
Shi, F., Telesco, S.E., Liu, Y., Radhakrishnan, R., and Lemmon, M.A. (2010). ErbB3/HER3
intracellular domain is competent to bind ATP and catalyze autophosphorylation. Proc Natl Acad
Sci U S A 107, 7692-7697.
85
Shroyer, N.F., Helmrath, M.A., Wang, V.Y.C., Antalffy, B., Henning, S.J., and Zoghbi, H.Y. (2007).
Intestine-specific ablation of mouse atonal homolog 1 (Math1) reveals a role in cellular
homeostasis. Gastroenterology 132, 2478-2488.
Shroyer, N.F., Wallis, D., Venken, K.J., Bellen, H.J., and Zoghbi, H.Y. (2005). Gfi1 functions
downstream of Math1 to control intestinal secretory cell subtype allocation and differentiation.
Genes Dev 19, 2412-2417.
Simmonds, N., Furman, M., Karanika, E., Phillips, A., and Bates, A.W. (2014). Paneth cell
metaplasia in newly diagnosed inflammatory bowel disease in children. BMC Gastroenterol 14,
93.
Simmons, A.J., Banerjee, A., McKinley, E.T., Scurrah, C.R., Herring, C.A., Gewin, L.S.,
Masuzaki, R., Karp, S.J., Franklin, J.L., Gerdes, M.J., et al. (2015). Cytometry-based single-cell
analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-alpha-induced
apoptosis in vivo. Mol Syst Biol 11, 835.
Sithanandam, G., and Anderson, L.M. (2008). The ERBB3 receptor in cancer and cancer gene
therapy. Cancer Gene Ther 15, 413-448.
Speca, S., Giusti, I., Rieder, F., and Latella, G. (2012). Cellular and molecular mechanisms of
intestinal fibrosis. World J Gastroenterol 18, 3635-3661.
Speece, A.J. (1964). Histochemical Distribution of Lysozyme Activity in Organs of Normal Mice
and Radiation Chimeras. J Histochem Cytochem 12, 384-391.
Stappenbeck, T.S. (2010). The role of autophagy in Paneth cell differentiation and secretion.
Mucosal Immunol 3, 8-10.
Steinkamp, M.P., Low-Nam, S.T., Yang, S., Lidke, K.A., Lidke, D.S., and Wilson, B.S. (2014).
erbB3 is an active tyrosine kinase capable of homo- and heterointeractions. Mol Cell Biol 34,
965-977.
Takeda, N., Jain, R., LeBoeuf, M.R., Wang, Q., Lu, M.M., and Epstein, J.A. (2011).
Interconversion between intestinal stem cell populations in distinct niches. Science 334,
1420-1424.
Tanaka, M., Saito, H., Kusumi, T., Fukuda, S., Shimoyama, T., Sasaki, Y., Suto, K., Munakata,
A., and Kudo, H. (2001). Spatial distribution and histogenesis of colorectal Paneth cell
metaplasia in idiopathic inflammatory bowel disease. J Gastroenterol Hepatol 16, 1353-1359.
86
Telesco, S.E., Shih, A.J., Jia, F., and Radhakrishnan, R. (2011). A multiscale modeling approach
to investigate molecular mechanisms of pseudokinase activation and drug resistance in the
HER3/ErbB3 receptor tyrosine kinase signaling network. Mol Biosyst 7, 2066-2080.
Theiss, A.L., Simmons, J.G., Jobin, C., and Lund, P.K. (2005). Tumor necrosis factor (TNF)
alpha increases collagen accumulation and proliferation in intestinal myofibroblasts via TNF
receptor 2. J Biol Chem 280, 36099-36109.
Troughton, W.D., and Trier, J.S. (1969). Paneth and goblet cell renewal in mouse duodenal
crypts. Journal of Cell Biology 41, 251-268.
Tsuchiya, K., Nakamura, T., Okamoto, R., Kanai, T., and Watanabe, M. (2007). Reciprocal
targeting of Hath1 and beta-catenin by Wnt glycogen synthase kinase 3beta in human colon
cancer. Gastroenterology 132, 208-220.
Vaishnava, S., Behrendt, C.L., Ismail, A.S., Eckmann, L., and Hooper, L.V. (2008). Paneth cells
directly sense gut commensals and maintain homeostasis at the intestinal host-microbial
interface. Proc Natl Acad Sci U S A 105, 20858-20863.
van der Flier, L.G., van Gijn, M.E., Hatzis, P., Kujala, P., Haegebarth, A., Stange, D.E., Begthel,
H., van den Born, M., Guryev, V., Oving, I., et al. (2009). Transcription factor achaete scute-like
2 controls intestinal stem cell fate. Cell 136, 903-912.
van Es, J.H., van Gijn, M.E., Riccio, O., van den Born, M., Vooijs, M., Begthel, H., Cozijnsen,
M., Robine, S., Winton, D.J., Radtke, F., et al. (2005). Notch/gamma-secretase inhibition turns
proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435, 959-963.
VanDussen, K.L., Carulli, A.J., Keeley, T.M., Patel, S.R., Puthoff, B.J., Magness, S.T., Tran, I.T.,
Maillard, I., Siebel, C., Kolterud, A., et al. (2012). Notch signaling modulates proliferation and
differentiation of intestinal crypt base columnar stem cells. Development 139, 488-497.
VanDussen, K.L., Liu, T.C., Li, D.L., Towfic, F., Modiano, N., Winter, R., Haritunians, T., Taylor,
K.D., Dhall, D., Targan, S.R., et al. (2014). Genetic variants synthesize to produce Paneth cell
phenotypes that define subtypes of Crohn's disease. Gastroenterology 146, 200-209.
Verrecchia, F., Pessah, M., Atfi, A., and Mauviel, A. (2000). Tumor necrosis factor-alpha inhibits
transforming growth factor-beta /Smad signaling in human dermal fibroblasts via AP-1
activation. J Biol Chem 275, 30226-30231.
Wajant, H., Pfizenmaier, K., and Scheurich, P. (2003). Tumor necrosis factor signaling. Cell
Death Differ 10, 45-65.
87
Wang, G.X., Zhao, X.Y., Meng, Z.X., Kern, M., Dietrich, A., Chen, Z., Cozacov, Z., Zhou, D.,
Okunade, A.L., Su, X., et al. (2014). The brown fat-enriched secreted factor Nrg4 preserves
metabolic homeostasis through attenuation of hepatic lipogenesis. Nat Med 20, 1436-1443.
Wang, Y., Poulin, E.J., and Coffey, R.J. (2013). LRIG1 is a triple threat: ERBB negative
regulator, intestinal stem cell marker and tumour suppressor. Br J Cancer 108, 1765-1770.
Wehkamp, J., Chu, H., Shen, B., Feathers, R.W., Kays, R.J., Lee, S.K., and Bevins, C.L. (2006).
Paneth cell antimicrobial peptides: topographical distribution and quantification in human
gastrointestinal tissues. FEBS Lett 580, 5344-5350.
Wehkamp, J., Salzman, N.H., Porter, E., Nuding, S., Weichenthal, M., Petras, R.E., Shen, B.,
Schaeffeler, E., Schwab, M., Linzmeier, R., et al. (2005). Reduced Paneth cell alpha-defensins
in ileal Crohn's disease. Proc Natl Acad Sci U S A 102, 18129-18134.
Wilson, C.L., Ouellette, A.J., Satchell, D.P., Ayabe, T., Lopez-Boado, Y.S., Stratman, J.L.,
Hultgren, S.J., Matrisian, L.M., and Parks, W.C. (1999). Regulation of intestinal alpha-defensin
activation by the metalloproteinase matrilysin in innate host defense. Science 286, 113-117.
Wilson, K.J., Gilmore, J.L., Foley, J., Lemmon, M.A., and Riese, D.J., 2nd (2009). Functional
selectivity of EGF family peptide growth factors: implications for cancer. Pharmacol Ther 122,
1-8.
Winston, B.W., Lange-Carter, C.A., Gardner, A.M., Johnson, G.L., and Riches, D.W. (1995).
Tumor necrosis factor alpha rapidly activates the mitogen-activated protein kinase (MAPK)
cascade in a MAPK kinase kinase-dependent, c-Raf-1-independent fashion in mouse
macrophages. Proc Natl Acad Sci U S A 92, 1614-1618.
Wong, V.W., Stange, D.E., Page, M.E., Buczacki, S., Wabik, A., Itami, S., van de Wetering, M.,
Poulsom, R., Wright, N.A., Trotter, M.W., et al. (2012). Lrig1 controls intestinal stem-cell
homeostasis by negative regulation of ErbB signalling. Nat Cell Biol 14, 401-408.
Wu, F., Dassopoulos, T., Cope, L., Maitra, A., Brant, S.R., Harris, M.L., Bayless, T.M.,
Parmigiani, G., and Chakravarti, S. (2007). Genome-wide gene expression differences in
Crohn's disease and ulcerative colitis from endoscopic pinch biopsies: insights into distinctive
pathogenesis. Inflamm Bowel Dis 13, 807-821.
Yamaoka, T., Frey, M.R., Dise, R.S., Bernard, J.K., and Polk, D.B. (2011). Specific epidermal
growth factor receptor autophosphorylation sites promote mouse colon epithelial cell
chemotaxis and restitution. Am J Physiol Gastrointest Liver Physiol 301, G368-376.
88
Yang, Q., Bermingham, N.A., Finegold, M.J., and Zoghbi, H.Y. (2001). Requirement of Math1 for
secretory cell lineage commitment in the mouse intestine. Science 294, 2155-2158.
Yarden, Y., and Sliwkowski, M.X. (2001). Untangling the ErbB signalling network. Nat Rev Mol
Cell Biol 2, 127-137.
Zhang, C., Sherman, M.P., Prince, L.S., Bader, D., Weitkamp, J.H., Slaughter, J.C., and
McElroy, S.J. (2012a). Paneth cell ablation in the presence of Klebsiella pneumoniae induces
necrotizing enterocolitis (NEC)-like injury in the small intestine of immature mice. Dis Model
Mech 5, 522-532.
Zhang, Y., Dube, P.E., Washington, M.K., Yan, F., and Polk, D.B. (2012b). ErbB2 and ErbB3
regulate recovery from dextran sulfate sodium-induced colitis by promoting mouse colon
epithelial cell survival. Lab Invest 92, 437-450.
Zundler, S., and Neurath, M.F. (2016). Integrating Immunologic Signaling Networks: The JAK/
STAT Pathway in Colitis and Colitis-Associated Cancer. Vaccines (Basel) 4.
89

Asset Metadata

Creator Almohazey, Dana (author)

Core Title The role of neuregulin receptors in cell differentiation and the response to inflammatory cytokines in the intestinal epithelium

Contributor Electronically uploaded by the author (provenance)

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 10/21/2018

Defense Date 10/13/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Atoh1,ErbB receptor tyrosine kinases,intestinal epithelium,intestinal subepithelial myofibroblasts,JAK/STAT,MAPK,neuregulins,OAI-PMH Harvest,Paneth cells,PI3K,TNF

Format application/pdf (imt)

Language English

Advisor Frey, Mark R. (committee chair), Ouellette, Andre (committee member), Paine, Michael (committee member), Shi, Wei (committee member), Warburton, David (committee member)

Creator Email almohaze@usc.edu,dana.muhaizei@yahoo.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-315872

Unique identifier UC11213658

Identifier etd-AlmohazeyD-4890.pdf (filename),usctheses-c40-315872 (legacy record id)

Legacy Identifier etd-AlmohazeyD-4890.pdf

Dmrecord 315872

Document Type Dissertation

Format application/pdf (imt)

Rights Almohazey, Dana

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Abstract (if available)

Abstract Crohn’s disease (CD) and neonatal necrotizing enterocolitis are characterized by epithelial injury, defects in Paneth cells (PCs), and a disrupted subepithelial myofibroblast layer. Our lab is interested in investigating novel treatments for these diseases aimed at protecting the epithelium by studying the role of the neuregulin receptors, ErbB3 and ErbB4, and how they are regulated during inflammation. PCs support the intestinal stem cells (ISC) with growth factors and participate in innate immunity by releasing antimicrobial peptides, including lysozyme and defensins. The specific pathways regulating Paneth cell development and function are not fully understood. Here we tested the role of the neuregulin receptor ErbB3 in control of Paneth cell differentiation and the ISC niche. Intestinal epithelial ErbB3 knockout caused precocious appearance of Paneth cells as early as postnatal day 7, and substantially increased the number of mature Paneth cells in adult mouse ileum. ErbB3 loss had no effect on other secretory lineages, but increased expression of the ISC marker Lgr5. ErbB3-null intestines had elevated levels of the Atoh1 transcription factor, which is required for secretory fate determination, while Atoh1⁺ cells had reduced ErbB3, suggesting reciprocal negative regulation. ErbB3-null intestinal progenitor cells showed reduced activation of the PI3K-Akt and ERK MAPK pathways. Inhibiting these pathways in HT29 cells increased levels of ATOH1 and the Paneth cell marker LYZ. Conversely, ErbB3 activation suppressed LYZ and ATOH1 in a PI3K-dependent manner. Interestingly, expression of the neuregulin ligands for ErbB3 and ErbB4 in the intestinal subepithelial myofibroblasts (ISEMFs) is altered during inflammation. We previously showed that neuregulin 4 (NRG4- ErbB4 ligand) is downregulated in CD patients, but not neuregulin 1 (NRG1- ErbB3/4 ligand). Here, we show that TNF (tumor necrosis factor), an inflammatory cytokine, causes a reduction in NRG4 expression in ISEMFs. To understand the mechanism, we investigated pathways known to interact with TNF. Inhibiting the JAK/STAT pathway, but not JNK or MAPK, abrogated the reduction in NRG4. Our results show that TNF activation of the JAK/STAT pathway causes a downregulation of NRG4 expression in ISEMFs. Inflammatory cytokines result in a decrease in NRG4 with no change in NRG1, thus potentially favoring the activation of ErbB3 over ErbB4 during inflammation. The dominance of ErbB3 activation could lead to a reduction in PCs, thus compromising the epithelium. These projects are aimed at understanding the mechanism of two defects in CD with the hope of finding novel therapeutic options.