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Wnt/β-catenin/p300 induced transcription is critical for the differentiation and maintenance of Paneth cells
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Wnt/β-catenin/p300 induced transcription is critical for the differentiation and maintenance of Paneth cells
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Content
Wnt/ β-Catenin/p300 Induced Transcription is Critical for
the Differentiation and Maintenance of Paneth Cells.
by
Jia Wu
A Thesis Presented to the
FACULTY OF THE USC GRADUA TE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
May 2014
Copyright 2014 Jia Wu
i
ACKNOWLEDGMENTS
First and foremost I would like to express my deepest appreciation and thanks to my
mentor Professor Dr. Michael Kahn, who is a tremendous advisor for me. I appreciate for
the opportunity to do research in his lab and to work on this project. His intellectual
knowledge and enthusiasm for science has been such an inspire to me through my
research. His instructive and detailed requirements towards my project pushed me to be a
better researcher. The guidance he provided during my research and thesis writing has
been invaluable in enabling me to pursuit my degree. Besides my PI, I want to thank
Professor Dr. Andre Ouellette, who collaborates with Dr. Kahn to provide me this project.
I would like to express my sincere gratitude to him for the continuous support of my
research. His patience, motivation, passion and immense knowledge guide me and help
me to finish my project and to write this thesis. He has always been supportive to my
research. I also would like to thank my program advisor Dr. Zoltan Tokes, who is the
chair of my committee. His kindness, encouragement and support throughout these two
years has enabled me to grow in my pursuit of academic advancement. I want to thank
Tomoyo Sasaki, who trained me and supported me through my research and writing
thesis. I have gained much knowledge from her. I would like to express my gratefulness
to Cu Nguyen, our lab manager, who provided me so much help to finish all my
experiments.
Finally, I want to thank my parents and my friends, who share all the joy and tears
with me through these two years and support me to succeed in pursuit my master degree.
ii
TABLE OF CONTENTS
ACKNOWLEDGMENTS i
LIST OF FIGURES v
ABBREVIATIONS vii
ABSTRACT ix
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: MATERIALS AND METHODS 11
2.1 Animals 11
2.2 Expression Analysis 11
2.3 Real-Time RT-PCR 11
2.4 Antibodies for both Western-Blot and Immunohistochemistry 13
2.5 Protein Preparation 14
2.6 Western Blot Analysis 14
2.7 Histochemistry 15
2.8 Hematoxylin and eosin stain (H&E stain or HE stain) 15
2.9 Alcian blue stain 15
2.10 Phloxine Tartrazine Stain 15
2.11 Immunohistochemistry (IHC) 16
2.12 Bacteria DNA preparation 16
2.13 Antimicrobial Assays 17
2.14 Organoid culture from mouse small intestine 18
iii
2.15 Statistical Analyses 18
CHAPTER 3: RESULTS 19
3.1-Established small intestine crypt organoid culture system cannot maintain
regular growth. 19
3.2-Treatments by the β-catenin and p300 interaction blocker (IQ-1) induces weight
loss and sickness in C57BL mouse. 21
3.3-TheIQ-1 treatment significantly reduces both the Paneth cell specific gene
and protein expression in ileum, which indicates the loss of the Paneth cell
population 22
3.4-The IQ-1 treatment does not affect goblet cell gene expression in ileum or
goblet cell population. 26
3.5-The IQ-1 treatment maintains the small intestine stem cell gene expression
and population. 28
3.6- The IQ-1 treatment increases cell stress, reduces the length of the villus and
alters lineage markers. 29
3.7-The oral administration of IQ-1 specifically targets ileum compared to other
parts of small intestine. 31
3.8-Bacteria from the small intestine have been spread to the liver and spleen in
the IQ-1 treated mouse group and the species of bacteria from the small intestine
has been altered. 33
3.9-Anitimicrobial Assay-Radial Diffusion Assays of IQ-1. 36
iv
CHAPTER 4: DISCUSSION 38
REFERENCES 42
v
LIST OF FIGURES AND TABLES
Figure 1. Wnt/β-catenin coactivator switching model.
Figure 2. Model for the differentiation of the intestinal epithelial cell types.
Figure 3. Antimicrobial functions of secreted Paneth cell antimicrobial peptides.
Figure 4. Model of differential coactivator usage.
Figure 5. Model of organoid small intestine crypt culture.
Figure 6. Establishment of intestinal crypts culture system with DMSO and IQ-1
treatment for 7 days (1μM, every three days).
Figure 7. Body weights of control (DMSO) and IQ-1 were measured on Day 0 and
Day 14.
Figure 8. Mouse small intestine collected on Day 14.
Figure 9. Quantitative realtime PCR analysis at day 14 of transcripts from C57/BL mice
IQ-1or DMSO treatement.
Figure 10. IQ-1 decreases the expression of Sox 9.
Figure 11. Histology staining for Paneth cells.
Figure 12. IQ-1 treatment maintains the population or the expression of goblet cells.
Figure 13. qRT-PCR result for stem cell markers.
Figure 14. qRT-PCR result of Grp78.
Figure 15. qRT-PCR result of Vil, Sis and Alpi.
Figure 16. IQ-1 treatment specific affect ileum rather than rest of the small intestine.
Figure 17. IQ-1 treatment induce the impaired barrier function in the crypts of the small
vi
intestine and results in the leakage of small intestine lumenal bacteria into the circulation.
Figure 19. Radial Diffusion antibacteria assay.
Table 1. qRT-PCR primer
Table 2. 16S DNA group-specific and kingdom-specific oligonucleotide primers ofr
qPCR.
vii
ABREVIATIONS
APC: axin and adenomatous polyposis coli protein
Bact: Bacteroides
BrdU: 5-bromo-2-deoxyuridine
CBP: Creb-binding protein
CD: Crohn's disease
CKI: casein kinase I
Clept: C. leptum
DAB: diaminobenzidine
Defa5: α-defensin alpha 5
Defa22: α-defensin alpha 22
DMSO : Dimethyl sulfoxide
Ent: Enterobacteriaceae
Erec: E. Rectale/C. coccoides
ER: endoplasmic reticulum
Fz: Frizzled family protein
Grp78: glucose regulated protein 78
Grp94: glucose regulated protein 94
GSK3: glycogen synthase kinase 3
H&E stain or HE stain : Hematoxylin and eosin stain
IBD: intestinal bowl disease
viii
IHC: ImmunoHistoChemistry
IgG: Immunoglobulin G
Lact: Lactobacillus/Enterococcus group
Lgr5: Leucine-rich repeat-containing, G protein-coupled receptor 5
Lyz1: Lysosome 1
LRP6: low-density lipoprotein receptor-related protein 6
MIB: Mouse Intestinal Bacteroides
MMP7: matrix metalloproteinase-7
RBC: red blood cell count
SFB: Segmented Filamentous bacteria
Sis: Sucrase Isomaltase
TCF: Transcription Factor
Vil: Villin
WBC: white blood cell count
Wnt: Wingless/int
WT : Wild Type
ix
ABSTRACT
Our lab's previous studies have demonstrated that Wnt/β-catenin/p300 transcription
initiates cell differentiation, whereas Wnt/β-catenin/cyclic AMP-responsive element
binding protein-binding protein (CBP) transcription mediates cell
proliferation/maintenance of potency. Recent publications have shown that the Wnt
signaling pathway induces Paneth cell maturation and Sox9, a Wnt pathway target gene,
is required for Paneth cell differentiation. Paneth cells are terminally differentiated
epithelial cells in the small intestinal crypts, which contribute to the mucosal
antimicrobial barrier by generating and secreting antimicrobial peptides/proteins
including α-defensins through their abundant and large granules. We show that
β-catenin/p300 interaction is required for the expression of Sox9 and thereby Paneth
cell lineage commitment. Therefore the β-catenin/p300 interaction is critical for the
Paneth cell differentiation and maintenance, in contrast to the β-catenin/CBP interaction.
Our work shows that the blockage of β-catenin/p300 interaction dramatically reduces the
Paneth cell population, which is associated with impaired barrier function. In this way,
luminal bacteria can be disseminated through the circulation to the liver and spleen.
1
CHAPTER 1
INTRODUCTION
Wnt/β-catenin signaling pathway is known to play a pivotal role in tissue
homeostasis (Archbold, et al., 2012). Extracellular Wnts, which are glycosylated and
lipid modified proteins, bind to the Frizzled (Fz) transmembrane protein family and low
density lipoprotein receptor related protein 5 or 6 (LRP) on the cell surface to initiate
signaling in the pathway. Without Wnts, cytosolic β-catenin is constitutively inhibited by
a destruction complex composed by glycogen synthase kinase 3 (GSK3) and casein
kinase I (CKI), and the scaffolding proteins axin and adenomatous polyposis coli (APC)
protein. (Cadigan & Peifer 2009, Kennell & Cadigan 2009). When Wnt protein binds Fz
and LRP on the cell membrane, it recruits disheveled (DSH) subsequently inducing the
disruption of the destruction complex. This results in free cytosolic β-catenin
accumulating and entering the nucleus (Archibold, 2011). After β-catenin is translocated
into nucleus, it can bind to T-cell factor/lymphoid enhancer-binding factor (TCF), a DNA
binding transcription factor. This leads to a critical stage in the Wnt signaling pathway—
establishment of the interaction of β-catenin/TCF with their transcriptional coactivators in
the nucleus. By partnering with those coactivators, the same Wnt signaling network
integrates the various inputs in the same cell types to generate dramatically diverse
responses and therefore result in divergent gene expression. There are two major
coactivators that mediate the fate decisions of stem/progenitor cells, driving either
proliferation and maintenance of potency, or differentiation (Figure 1). These two
coactivators are Creb-binding protein (CBP) and its closely related homologue p300
(Emami, et al., 2004). Accumulating evidence demonstrates that utilization of either CBP
2
or p300 can guide stem cells to initiate one of these two processes respectively. The
CBP-β-catenin complex is essential for stem cell/progenitor cell maintenance and
proliferation, whereas the initiation of differentiation is mediated by the β-catenin/p300
interaction along with a decrease in cellular potency (Figure 1). Proper Wnt signaling is
associated with the requirement of a homeostatically living system. The Wnt signaling
pathway exerts profound influence on the small intestine development and its
homeostasis maintenance (Es, et, al., 2009).
Figure 1. Wnt/β-catenin coactivator switching model. β-catenin/CBP mediated
transcription is critical for stem/progenitor cell proliferation, whereas, coactivator
switching to β-catenin/p300 mediated transcription is critical to initiate a differentiation
program. (Adapted from Kahn, et, al., 2007)
3
The small intestine, part of the gastrointestinal tract, is a tube-like structure
composed of a proximal duodenum, mid jejunum, and distal ileum. The luminal wall of
this organ is covered by mucosa, which is in wrinkles or folds called plicae circulares
composed by monolayer of epithelial cells. From the plicae circulares, there are
microscopic finger-like pieces projecting into the lumen, called villi. The villi maintain a
high level of self-renewal, and cells in the villus, such as Paneth cells, goblets cells, and
intestinal epithelium cells, are continuously renewed from the intestinal stem cells at the
base of the crypts (Bevins, et, al., 2011). The life cycle for Paneth cells is approximate
70 days for murine animals (Clevers, et, al., 2013). Both plicae circulares and the villus
are evolved to increase the amount of the surface area, as this is where the most chemical
digestion takes place and where most of the nutrients from ingested food are absorbed. In
the small intestinal lumen, despite the digestive enzymes secreted by the pancreas and
others, the bacterial microbiota is also essential to fulfill this function. One of the key
benefits of the microbiota to host physiology is through the digestion and absorption of
nutrition. In fact, trillions of bacteria reside in the small intestine and build a
predominantly symbiotic relationship with their host. The composition of the small
intestinal microbiota varies between individuals, based upon a variety of factors including
diet(Costello, et, al., 2009). The Gram-positive Firmicutes and Gram-negative
Bacteroidetes are the two phyla, which dominate the intestinal microbiota in a healthy
living system (Bevins, et, al., 2011). Among the numerous species of bacteria existed in
the lumen, some are responsible from minor to serious bowel diseases. Therefore, this
requires the small intestine to take responsibility to maintain the balance of the bacterial
4
composition. Paneth cells contribute critically to the mucosal antimicrobial barrier
(Salzman, et, al., 2010).
Paneth cells, highly specialized epithelial cells, reside in small clusters at the
bottom of the crypts, and contain an apical clustering of secretary granules (Porter, et, al.,
2002). These abundant and large granules serve as histological hallmarks for Paneth cells
(Clevers, et, al., 2013). In the small intestine, each crypt/villus contains three to ten
Paneth cells, which are located adjacent to leucine-rich repeat-containing G protein-
coupled receptor 5( Lgr 5
+
) crypt base columnar cells or putative stem cells. Paneth cells,
along with enterocytes (absorptive cells), mucin-secreting goblet cells, and hormone-
secreting enteroendocrine cells derive from the Lgr5
+
stem cell population. Lgr5
+
stem
cells at the base of the crypt, are able to proliferate/maintain potency as well as to
differentiate into various cell types of the crypt/villus. Subsequently, enterocytes, goblet
cells and enteroendocrine cells migrate upwards from the crypts into the villus, whereas
the Paneth cells differentiate as they migrate towards the bottom of the crypts (Nori-
Akiyama, et, al., 2007 and Bastide, et, al., 2007) (Figure 2). In addition, β-catenin and
TCF transcription factor control the expression of ephrin B2 (Ephb2) cell-sorting
receptors, which allow the correct positioning of epithelial cells in a Wnt gradient
dependent fashion along the crypt-villus axis (Es, et, al., 2005). It has been previously
demonstrated that p300/β-catenin transcription is critical to regulate EphB2 expression
level in the small intestine (Kumar, et, al., 2009). The initial and terminal differentiation
of Paneth cells requires the Wnt signaling cascade through the accumulation and nuclear
translocation of β-catenin, which then interacts with the proper TCF to express the target
genes (Nori-Akiyama, et, al., 2007). One of the Wnt signaling pathway target genes in
5
the crypts is Sox9, which is a member of the SOX family of transcription factors. Recent
publication illustrate that Sox 9 is required for the differentiation of Paneth cells and the
development of mature Paneth cells (Nori-Akiyama, et, al., 2007) (Figure 2). However, it
is not clear which coactivor—either p300 and/or CBP—is able to control the Wnt
signaling pathway to initiate Paneth cell differentiation in the small intestine.
Figure 2. Model for the differentiation of the intestinal epithelial cell types. The
scheme on the left represents a crypt-villus unit in the adult mouse small intestine
epithelium. The critical feature of the left diagram is that Paneth cell is differentiated
from stem cell progenitors and driven by Sox9 expression (Gerbe, et, al., 2011,
permitted).
6
As mentioned above, in the small intestine, Paneth cells contribute to the
maintenance of a homeostatic relationship between the host and its microbiota. Paneth
cells are the primary producers of a diverse array of antimicrobial proteins/peptides,
including lysozyme (e.g. Lysozyme 1, Lyz1)and various defensins , which are an
essential part of innate immunity in the gut (Ouellette, 1997). Among the antimicrobial
defensins, the α-defensin-1to -5 are unique to Paneth cells (Es, Johan, et, al., 2005). The
α-defensins are released in response to a variety of stimuli, including acetylcholinergic
agonists, bacterial cell surface products and other Toll-like receptor (TLR) agonists
(Ouellette, et, al., 2010). Indeed, α-defensins are bactericidal for both Gram-negative and
Gram-positive bacteria. These antimicrobial products are delivered into and onto the
lumen to balance the composition of the small intestinal microbiota, thereby helping to
maintain the intestinal homeostasis (Figure 3). In mice the α-defensins are synthesized as
inactive precursors (pro-α-defensisns) that activated in the Paneth cells by the matrix
metalloproteinase 7(MMP7) (Ayabe, et, al., 2002) (Wilson, et, al., 1999). Therefore,
beside α-defensin 5 (Defa5), α-defensin 22 (Defa22), and other defensins, the expression
of MMP7, another Wnt target gene, also occurs in the Paneth cell population.
7
Figure 3. Antimicrobial functions of secreted Paneth cell antimicrobial peptides.
Proposed functions for Paneth cell antimicrobial peptides (AMPs). Paneth cells secrete a
potent combination of AMPs into lumen (Clevers, et, al., 2013 permitted).
Due to the high requirement for protein generation and secretion, it is important
for Paneth cells to maintain stress control, especially in the endoplasmic reticulum (ER)
to preserve intestinal homeostasis. An unstable homeostatic situation stimulates the
response from a family of ER heat shock proteins (glucose regulated protein) Grp (Grp78
and Grp94) to protect Paneth cells and other secretory cell types against various forms of
stress (Little, 1994). Grp 78, as an essential pro-survival chaperone, is critical to control
protein quality in the ER, especially by targeting misfolded protein for degradation.
Recent evidence demonstrates that this anti-apoptotic property is stress sensitive and
stress inducible (Li, Jianze; et, al., 2006). The other grp protein—Grp94— is able to
interact with LRP6, a coreceptor that binds Frizzled, which is exported from the ER to
the cell surface. Without Grp 94, the critical ER chaperone, the Wnt signaling pathway is
8
disturbed and in particularly results in the loss of the crypt-villus architecture and an
impaired barrier function (Liu, et, al., 2013). In this case, a leaky barrier function can
allow bacteria to translocate into the circulation, and thereby other internal organs,
including the liver and spleen.
Our lab has previously identified two compounds—ICG001 and IQ-1, which
modulate the Wnt pathway antagonize β-catenin co-activation (Emami, et, al., 2004).
ICG-001 is a specific inhibitor, which binds with high affinity to the coactivator CBP and
blocks the interaction between CBP and β-catenin (Emami, et. al., 2004). Since ICG-001
does not affect the p300/β-catenin interaction, it can be used as a tool to distinguish the
effects of the CBP/β-catenin interaction from the p300/β-catenin interaction (Figure 4
upper panel). On the other hand, IQ-1 is able to bind to regulatory subunits of the protein
phosphatase, PP2A, which indirectly plays a role in decreasing the phosphorylation of
p300 at serine 89 among many other things (Miyabayashi, et. al., 2007). Without this
Serine 89 phosphorylation modification, the affinity of p300 binding to β-catenin is
significantly reduced, thereby increasing the β-catenin bound to CBP (Miyabayashi, et,
al., 2007) (Figure 4 lower panel). Blocking this interaction with IQ-1 diminishes the β-
catenin/p300 interaction and thereby blocks differentiation. Therefore, we used IQ-1 as
the tool to study Paneth cell development.
9
Figure 4. Model of differential coactivator usage. (A) IQ-1 antagonizes the interaction
between p300 and catenin. (B)ICG-001 blocks the interaction between CBP and catenin
(adapted from Kahn, 2011).
In light of the previous work, we focused on the role of the β-catenin/p300
interaction in Paneth cell differentiation and maintenance. To do so, we use the small
molecule IQ-1 that specifically disrupts the β-catenin interaction with p300 and increases
its interaction with CBP. We hypothesized that the blockage of β-catenin/p300 would
diminish Paneth cell differentiation without adversely affecting β-catenin/CBP
coactivation maintenance of Lgr5
+
stem cells, or Wnt signaling pathway independent
cells. Since Paneth cells contribute to the formation of the crypt niche (Snippert, et, al.,
2010), the loss of the Paneth cell population might also cause impaired barrier function.
10
We also investigated the dissemination of the small intestinal bacteria leaking from the
small intestine lumen to peripheral organs.
11
CHAPTER 2
MATERIALS AND METHODS
2.1 Animal- C57BL/6 female mice, approximately 4 weeks old were purchased from
Jackson Laboratory. For analysis of IQ-1 effects on small intestine, the C57BL/6 mice
were randomized into two groups and housed and fed with regular diet. One group was
referred to as the control group, which was administered with DMSO, 15 µ l per day for
two weeks. The other group was IQ-1 treated group, and administered with 1 M IQ-1, 15
µ l per day for two weeks. Every 15 µ l, either DMSO or IQ-1 was mixed with about 1 g
of peanut butter. The body weight was measured before administering the drug as well as
at the end of the two-week administration period. Both groups were euthanized at the end
of the two-week DMSO/IQ-1 administration. Before euthanasia on Day 14, blood
samples were collected from each mouse and routine blood tests were run. The organs
were collected, and they included the small intestine, the liver, and the spleen.
2.2 Expression Analysis-The preparation of samples include ileal RNA, jejunal and
duodenal mixed RNA preparation, ileal protein preparation, and jejunal and duodenal
mixed protein preparation.
2.3 Real-Time RT-PCR- The total RNA of ileum was isolated by TRIzol® reagent
(Invitrogen) and reverse-transcribed using SuperScript III (Invitrogen). The same method
was used to prepare total RNA of mixed jejunum and duodenum. Real-time RT-PCR
(Sybr Green; BioRad, Hercules, CA) was performed using gene-specific primers, which
are markers for Paneth cells, small intestinal stem cells, and goblet cells. The primers
were designed using either Primer-Blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) or
followed the Primer Bank (http://pga.mgh.harvard.edu/primerbank/). The specificity of
12
each pair of primers was confirmed by a single peak of the melt curve after qRT-PCR as
well as running DNA products of RT-PCR on an agarose electrophoresis to demonstrate
only one band.
Oligo Sequence (5'--3')
Sox 9-F AGTACCCGCATCTGCACAAC
Sox 9-R ACGAAGGGTCTCTTCTCGCT
Defa5-F AGGCTGATCCTATCCACAAAACAG
Defa5-R TGAAGAGCAGACCCTTCTTGGC
Defa22-F ACCAGGCTGTGTCTGTCTCCTT
Defa22-R TGGCCTCAGAGCTGATGGTTGT
Defcr-rs1-F AGCAGCCATTGTGCGAAGAA
Defcr-rs1-R TGCTGTGTATTTGGAGCTTGG
Lyz1-F GAGACCGAAGCACCGACTATG
Lyz1-R CGGTTTTGACATTGTGTTCGC
Muc2-F ATGCCCACCTCCTCAAAGAC
Muc2-R GTAGTTTCCGTTGGAACAGTGAA
Lgr5-F CCTACTCGAAGACTTACCCAGT
Lgr5-R GCATTGGGGTGAATGATAGCA
EphB2-F GCCGTGGAAGAAACCCTGAT
EphB2-R GTTCATGTTCTCGTCGTAGCC
Grp94-F GTTCGTCAGAGCTGATGATGAA
Grp94-R GCGTTTAACCCATCCAACTGAAT
Grp78-F CATTGGTGGCCGTTAAGAATGAC
13
Grp78-R AGTATCGAGCGCGCCGTCGC
Vil1-F GGCAACGAGAGCGAGACTTT
Vil1-R GGAGTTTGTTTCTACGTGCTTCA
Sis-F GCTATCGCTCTTGTTGTGGTT
Sis-R TTCCAGGACTAGGGGTTGAAG
Alpi-F AGGACATCGCCACTCAACTC
Alpi-R GGTTCCAGACTGGTTACTGTCA
MMP7-F CTTACCTCGGATCGTAGTGGA
MMP7-R CCCCAACTAACCCTCTTGAAGT
CD44-F ACTTTGCCTCTTGCAGTTGAG
CD44-R TTTCTCCACATGGAATACACCTG
Ascl 2-F CCGTGAAGGTGCAAACGTC
Ascl 2-R CCCTGCTACGAGTTCTGGTG
Ephb2-F GAACACTATCCGTACCTACCAGG
Ephb2-R GGCTAAGTCAAAATCAGCCTCA
Table 1. qRT-PCR primers
2.4 Antibodies for Western-Blot and immunohistochemistry (IHC)- Primary
antibodies Anti-Sox 9 (Catalog # ab26414) and Anti-beta-Actin (β-Actin) (Catalog #
D2508 ) were purchased from Abcan (the catalog number of Anti-Sox 9 is ab26414). The
secondary antibodies used were goat anti-rabbit lgG-HRP (Catalog # J2811) and goat
anti-mouse lgG-HRP (Catalog # A2413) and were purchased from Santa Cruz
Biotechnology.
14
2.5 Protein Preparation- Total protein was extracted from mouse ileum using the T-
PER tissue protein extraction reagent (Thermo Scientific Inc.), following the
manufacturer’s protocol. The same reagent was used for the total protein extraction of
jejunum and duodenum mix. Protein concentration was measured using the Bio-Rad
protein assay (BioRad Laboratories Inc.). The samples were diluted with 1:40 ratio using
Millipore water. The Bio-Rad protein assay solution was diluted with 1:4 ratio with
Millipore water. The standard protein concentrations were 0 mg/ml, 0.125 mg/ml, 0.25
mg/ml, and 0.5 mg/ml. By using a 96-well plate, 10μl of either the sample or the
standards was pipetted into each well, and then added was 200 μl of a diluted Bio-Rad
protein assay solution. Sample protein as well as standard protein was duplicated when
measured at 595 nm absorbance on a Spectra Max spectrophotometer (Molecular
Devices).
2.6 Western Blot Analysis- 20 μg of sample protein was fractionated by electrophoresis
on a gradient (4-20%) polyacrylamide gel and the tris-glycine gel purchased from Bio-
Rad. Subsequently, the protein on the gel was transferred to a PVDF membrane via
overnight transfer. It was blocked with a 5% non-fat milk solution, which was made from
powder. There were two primary antibodies. The anti-Sox9 antibody was used in a
1:1000 ratio, and the anti-β-actin antibody was used in a 1:2000 ratio. After overnight
primary antibody incubation at 4˚C, the membrane was washed for 30 minutes at room
temperature, followed by a one-hour secondary antibody incubation. The protein was
detected by a ECL Select western blotting detection reagent (GE Healthcare) and was
imaged using the Bio-Rad ChemiDoc MP imaging System.
15
2.7 Histochemistry- The mouse ileum tissues were fixed by using 4% paraformaldehyde
in PBS. Subsequently, the fixed tissues were washed with PBS and dehydrated in graded
ethanol and xylene. Finally, the tissues were infiltrated with paraffin at 58 ˚C and cooled
down to room temperature. For histological slides, the paraffin-embedded tissues were
cut into 6-10um thickness sections, and the slides were dried on a 45 ˚C hot plate.
2.8 Hematoxylin and eosin stain (H&E stain or HE stain)- The paraffin sections were
deparaffinized and stained with hematoxylin and eosin (Sigma) according to the
manufacturer’s direction. Hematoxylin can color the cell nuclei into blue and eosin turns
eosinophilic structures into various shades from pink to red. The hematoxylin and eosin
were purchased from Sigma.
2.9 Alcian Blue stain- A 3% acetic acid solution was made with 3ml glacial acetic acid
(Sigma) and 97 ml distilled water. The alcian blue solution was made with 1g alcian blue
powder (8GX, Sigma) and 100ml acetic acid (3% solution). The pH of the alcian blue is
2.5 after adjusting with acetic acid and mixing well. The 0.1% nuclear fast read solution
was purchased from Electron Microscopy Sciences Cat. # 26356-01. Paraffin sections
were deparaffinized and incubated in the Alcian blue solution which stains goblet cells
blue. Then, the sections were counterstained with Nuclear fast red solution.
2.10 Phloxine Tartrazine stain- The phloxine solution was made with 0.5g phloxine
(Sigma), 0.5g calcium chloride (Sigma), and 100ml distilled water. Tartrazine saturated
cellosolve was purchased from Electron Microscopy Sciences. First, the nuclei was
stained in haematoxylin (blue), and after, it was washed in running tap water. The
phloxine solution stained the Paneth cells to bright red. Tatrazine solution was used to
washed away the unspecific phloxine staining.
16
2.11 Immunohistochemistry (IHC)- The primary antibody was anti-Sox9 antibody
(Abcam) with a 1:500 ratio. The secondary antibody was goat-anti-rabbit HRP with a
1:1000 ratio. The diaminobenzidine (DAB) kit was used as the developing color for Sox9
in the Paneth cells. When the dark brown color was stable, the slides were washed with
distilled water to stop the imaging reaction. Finally, the slides were counterstained with
methyl green for the nuclei.
2.12 Bacteria DNA preparation- Total DNA from liver and spleen were extracted by
using the QIAamp DNA Stool mini kit (QIAGEN). Total amount of commensal bacteria
in each liver or spleen sample was determined by using the conserved 16S rRNA-specific
Eubacteria primer pair amplified by qRT-PCR (Salzman, et, al., 2010).
A gradient/dilution series of Eubacteria plasmid (10
8
copy, 10
7
copy, 10
6
copy, 10
5
copy,
10
4
copy, 10
3
copy) was used to obtain the standard curve by qRT-PCR. Both the sample
and the standard plasmid were duplicated in the same qRT-PCR plate. Seven group-
specific 16S rRNA gene primers were tested in order to determine the abundance of the
small intestinal bacterial group, which might exist in the liver and spleen samples
(Barman, et, al., 2008). Each set of primer had its own graded plasmid standard curve as
the Eubacteria plasmid set.
Group Reference strain Primers
Eubacteria
Ruminococcus
productus
(ATCC27340)
UniF334:
ACTCCTACGGGAGGCAGCAGT
UniR514
ATTACCGCGGCTGCTGGC
Bacteroides (Bact) Bacteroides fragilis
(ATCC 25285D)
BactF:
GGTTCTGAGAGGAGGTCCC
UniR349: CTGCCTCCCGTAGGAGT
Mouse Intestinal
Bacteroides (MIB)
Plasmid DNA
(CT11-6)
UniF516:
CCAGCAGCCGCGGTAATA
MIBR: CGCATTCCGCATACTTCTC
17
E. RECTALE/C.
coccoides group (Erec)
Ruminococcus
productus (ATCC
27340D)
UniF338:
ACTCCTACGGGAGGCAGC
CcocR:
GCTTCTTAGTCAGGTACCGTCAT
C. leptum (Clept) Plasmid DNA
(Mmp7+/+-3)
ClepF1123:
GTTGACAAAACGGAGGAAGG
ClepR1367:
GACGGGCGGTGTGTACAA
Segmented filamentous
bacteria (SFB)
Plasmid DNA
(CT25-6)
SFBF:
GACGCTGAGGCATGAGAGCAT
SFBR:
GACGGCACGGATTGTTATTCA
Enterobacteriaceae
(Ent)
Escherichia coli
(ATCC 10798D)
Uni515F:
GTGCCAGCMGCCGCGGTAA
Ent826R:
GCCTCAAGGGCACAACCTCCAAG
Table 2. 16S DNA group-specific and kingdom-specific oligonucleotide primers of
qPCR. (Salzman, et, al., 2010 supplementary material)
2.13 Antimicrobial Assay-Radial Diffusion Assays- The bottom layer of agarose media
was made with 1% w/v agarose (Sigma A-6013), 10mM sodium phosphate buffer (pH
7.4), and 0.3 mg/ml trypticase soy broth powder. The top layer of agarose media was
made with 60mg/ml trypticase soy broth powder in 1% w/v agarose and 10mM sodium
phosphate buffer (pH7.4). There were four bacteria tested, which were E coli, Listeria,
Staphylococcus aureus, and Pseudomonas. In each test plate, 15 ml bottom layer media
was mixed with 10ul bacteria culture (overnight incubation with selected single colony).
After 30 minutes, the bottom layer was solidified, and a 1000ul pipet was used to make a
nice round hole. Then, 2μl IQ-1 (5μM, 10μM, 20μM and 25μM) was added to each hole.
The plate was incubated in a 37 ˚C incubator for three hours. Then 15 ml top layer media
was poured onto the bottom layer. After it solidified, the plate was stored in the 37 ˚C
18
incubator. The E. coli test plate was incubated overnight, and the other three kinds of
bacteria test plates were incubated for eight hours (Tincu et al., 2003).
2.14 Mouse crypts organoid culture- The small intestine crypts were collected from
fresh euthanized C57/BL mice and cultured in a 37 ˚C incubator with 5% CO
2
according
to Sato's protocol (Sato, et, al., 2009). The culture was randomly split into two groups: a
DMSO control group and a IQ-1 tested group. The media was replaced every three days
and along with it a new dosage was added at 10 μM. Both DMSO and IQ-1 were 10 μM.
2.15 Statistical Analyses- Statistical Analyses were obtained by using unpaired Student's
t test, where p<0.05 was set as statistically significantly different.
19
CHAPTER 3
RESULTS
3.1-Established small intestine crypt organoid culture cannot maintain regular
growth in the presence of IQ-1. To investigate the role of the β-catenin/p300 interaction
in small intestinal development, we first set up an in-vitro model, which utilizes mouse
crypt organoid culture. We treated crypt organoid cultures, derived from C57BL/6 mice
small intestine, with the β-catenin/p300 interaction blocker—IQ-1and monitored the
influence on growth/differentiation. Fresh mouse small intestinal crypts were collected
from C57BL/6 WT mice and incubated at 37 ˚C, with media containing essential growth
factors according to the published protocol (Sato, et, al., 2009). The main composition of
the crypts are mixed intestinal stem cells and Paneth cells. Paneth cells are terminally
differentiated cells, therefore, only the mixed stem cells/progenitor cells are able to
generate new cells. After the organoid culture was seeded and established, the media was
changed every three days, along with a fresh dose of DMSO 10 µ M) or IQ-1(10 µ M) in
DMSO (Figure 5). In DMSO treated organoid culture, crypt protrusion was first seen on
Day 3 and massive expansions were observed by Day 7, which represent normal growth
with significant branching and regular differentiation. Whereas in IQ-1 treated organoid
cultures, the crypt protrusion first appeared on Day 7, and even at that time, the cultures
displayed poor growth with little expansion (Figure 6). This indicates that majority of the
intestinal stem cells/progenitor cells in the crypts experienced delayed differentiation or
none at all. These results suggested that the β-catenin/p300 interaction is critical in
inducing differentiation in vitro.
20
Figure 5. Model of organoid small intestine crypt culture. In vitro, 10μM IQ-1 was
replaced along with the crypt organoid culture media every three days. (Kanmura, and
Ouellette, unpublished)
A. DMSO
B. IQ-1
Day 1 Day 3 Day 7
Day 1
Day 3 Day 7
21
Figure 6. Establishment of intestinal crypts culture system with DMSO and IQ-1
treatment for 7 days (1μM, every three days). A. The schema reveals DMSO group
time course of an isolated single crypt growth. Start day 3, crypt-like domain formed.
B. The IQ-1 treated time course organoid. Crypt-like domain formed until day 7.
3.2- IQ-1 treatment induces weight loss and sickness in WT C57BL/6 mice. In order
to explore the role of β-catenin /p300 interaction in vivo, we orally administered IQ-1 or
DMSO in peanut butter to C57BL/6 mice and analyzed the small intestine especially the
ileum, because among the three parts of mouse small intestine— duodenum, jejunum,
and ileum, the majority of Paneth cells reside in the ileum. The mice (approximately four
weeks old, female) were assigned to two groups according to their body weights, four in
each group, to ensure the average body weights of these two groups were similar at Day 0.
After 14-day treatment with either DMSO (control) or IQ-1, we measured the body
weight of each mouse as we did on the Day 0. The IQ-1 treated mouse group showed
approximately 20% body weight loss. In addition to the body weight loss, the IQ-1
treated mice also had oily hair, and cowered in the corner with slow movement and
decreased activity. These are likely signs that IQ-1 treatment is making the mice sick.
Whereas, the DMSO treated mouse group gained 20% body weight as expected (Figure
7). The growth pathology of the mouse small intestine showed that the IQ-1 treated mice
had gas-bloated small intestines, whereas the DMSO control mice didn't experience this
and appeared normal (Figure 8).
22
Figure 7. IQ-1 [270mg/kg/day IQ-1 group (n=4)] or DMSO [Control group (n=4)] was
administered orally for 14 days. Body weights of control (DMSO) and IQ-1 were
measured on Day 0 and Day 14. The bar graph represents body weight change (%) in
each group compared to the body weight of that group measured on Day 0.
Figure 8. Mouse small intestine collected on Day 14. A. Small intestine sample from
DMSO control group. B. small intestine sample from IQ-1 treated group, which is filled
with gas and floating on the PBS.
3.3- IQ-1 treatment significantly reduces Paneth cell markers, which indicates the
loss of Paneth cell population. The Wnt signaling pathway directs Paneth cell
P<0.05
*
DMSO
IQ-1
A B
23
differentiation and induces maturation of Paneth cells (Es, et, al., 2005). Sox 9 is a of the
Wnt pathway target gene that is required for Paneth cell differentiation (Nori-Akiyama, et,
al., 2007). Sox9 expression level can be used as a biomarker to evaluate the generation of
new Paneth cells. Paneth cells function to generate and secrete bactericidal
proteins/peptides. Therefore, in order to explore the effect of IQ-1 on Paneth cells, we
analyzed a panel of genes at the RNA messenger level including Sox 9, matrix
metalloproteinase-7 (MMP7), α-defensin 5 (Defa 5), α-defensin 22 (Defa 22), general
Paneth cell defensin (Pan-Defcr), lysozyme 1(Lyz 1), and heat shock protein (glucose
regulated protein) Grp94 by quantitative RT-PCR (qRT-PCR). We collected the ileal
samples from each mouse in both the DMSO and IQ-1 treated groups, as mentioned in
the previous section. All eight mice (four mice per group) were euthanized according to
an approved IACUC animal protocol on Day 14 and RNA was isolated from the ileums.
The expression level of each gene from IQ-1 treated mice was less than 50% of the level
detected in the DMSO group(p<0.001) (Figure 9). The decrease of the Wnt target gene—
Sox 9 indicates that blocking the β-catenin/p300 interaction with IQ-1 decreased the
message level of Sox9. Reduction of Sox9 at the protein level was consistent with qRT-
PCR result by Western Blot (Figure 10 A). IHC anti-Sox 9 staining was performed to
confirm this protein level reduction (Figure 10 B). The significantly decreased Sox9
message RNA and protein level demonstrates that Paneth cell differentiation was
defective during the 14-day treatment. In order to verify the Paneth cell population
decrease, we used H and E staining and phloxine and tartrazine staining to stain the apical
clustering of secretary granules in Paneth cells. The staining indicates that with
24
diminished β-catenin/p300 interaction through the use of IQ-1, a decrease in the Paneth
cell population is evident (Figure 11A,B).
Figure 9. Quantitative realtime PCR analysis at day 14 of transcripts from C57/BL
mice IQ-1or DMSO treatement.
A.
P<0.001
25
B.
Figure 10. IQ-1 decreases the expression of Sox9 in the ileum. A. Two samples on the
left of the membrane are from the DMSO control group, and three samples on the right
are from the IQ-1 treated group. The Sox9 antibody was purchased from Abcam. B. IHC
staining for Sox9.
A.
DMSO
IQ-1
DMSO Group
IQ-1 group
26
B.
Figure 11. Histology staining for Paneth cells. A. H and E staining. Paneth cells are
stained in pink left panael DMSO sectioning, due to the large granules. IQ-1 treated
ileum section lack stainable Paneth cells. B. Phloxine and tartrazine stains Paneth cell
(large granules) in a bright red, shown in the DMSO section as pointed. The IQ-1
sections lack stainable Paneth cell.
3.4-IQ-1 treatment does not affect goblet cell density. Next we questioned whether the
blockage of the β-catenin/p300 interaction could affect goblet cells, which is another cell
linage of intetine. The goblet cell is derived from the same progenitor cell as the Paneth
cell, however, the differentiation of goblet cells does not require Wnt target gene. To
answer this question, we used Muc2 as a specific goblet cell marker. Muc2 mRNA level
was tested from the same samples obtained previously from the IQ-1 and DMSO treated
mice by qRT-PCR. The results indicate there is no significant change in goblet cell gene
expression level (Figure 12 A). Alcian blue staining also confirms that there is no
noticable change of goblet cell numbers per villus in IQ-1 treated samples compared to
the DMSO group (Figure 12 B). However, the distribution of goblet cells is different
DMSO
IQ-1
27
between the two treatments. In the DMSO control group, the goblet cells are at the
bottom half of the villus, whereas in the IQ-1 treated group, the goblet cells are widely
disprersed all over the villus, especially at the top of the villus (Figure 12 B). This result
suggests that goblet cell differentiation process is not affected by disruption of β-
catenin/p300 interaction, however epithelial migration and organization appear to be
affected.
A.
B.
DMSO
IQ-1
28
Figure 12. IQ-1 treatment maintains the population or the expression of goblet cells.
A. qRT-PCR result for Muc2 indicates there is no significant change between DMSO and
IQ-1 treatment. B. Alcian blue staining stains goblet cells in blue. The density of the
goblet cells does not change, however the distribution of goblet cells is different. In the
DMSO control group, goblet cells are on the lower part of the villus, whereas in the IQ-1
treated group, goblet cells have migrated toward the top of the villus.
3.5-IQ-1 treatment does not affect the small intestinal stem cell population. In the
small intestine, both the Paneth cells and the goblet cells are differentiated from the same
stem/progenitor cells at the bottom of crypts. Therefore, we questioned whether IQ-1
could affect small intestinal crypts stem cells by enhancing β-catenin/CBP interaction.
We examined the mRNA level of Lgr5, CD44, Ascl2, and Ephb2, Wnt target genes
selected as the potential stem cell markers. RNA from the ileum of DMSO and IQ-1
treated groups was isolated and quantified by qRT-PCR. The expression of Lgr5, CD44,
and Ascl2 were not significantly reduced by IQ-1 treatment (Figure 13). However, Ephb2,
which is involved in numerous developmental processes (NCBI gene), is the only
intestinal stem cell marker ate the ones we examied, that was significantly decreased by
IQ-1 treatment (Figure 13). A recent publication from our group demonstrates that
EphB2 expression requires β-catenin-p300-complex, in contrast to the β-catenin-CBP-
complex, which represses EphB2 (Kumar, et, al., 2009). IQ-1 is able to block the β-
catenin/p300 intreaction, therefore the decreased message RNA level of EphB2 is
consistent with the effects of IQ-1. The result represents that the stem cell population in
the ileum isn't affected by IQ-1treatment, however, the differentiation pathway target
29
gene—Ephb2 is significantly reduced by IQ-1. The decrease of Ephb2 also affects
epithelial architecture—maybe it is the reason that why goblet cells are repositioned.
Figure 13. qRT-PCR result for stem cell markers. Lgr5, CD44, and ASCL2 are not
affected by IQ-1 treatment, however, Ephb2 is reduced significally, which may suggest
the reason that goblet cells have been repositioned.
3.6 IQ-1 treatment increases ER stress, and reduces the villus length. The Paneth cell
population was significantly reduced by IQ-1 treatment, which significantly decreased
the expression of genes involved in bactericidal peptide/protein production (Figure 9).
This could create an unhealthy and stressful envrionment in the IQ-1 treated mouse small
intestine, which is consistent with the IQ-1 treated mice becoming sick and their small
intestine being bloated (Figure 8). The Wnt signaling pathway is also related to stress
response in the samll intestine. Therefore we examined if cellular stress was induced
increased. We first analyzed the stress marker Grp78. Grp78, a luminal stress marker of
the endoplasmic reticulum, was analyzed by qRT-PCR in the same ileal sample. The
results indicate that Grp78 expression was elevated 250% compared to the DMSO control
group (Figure 14). This suggested that IQ-1 treatment increases cellular stress in the
small intestine. Histochemical staining (Figure 12,14) illustrates that the villus of the IQ-
30
1 treated mouse group is shorterned and is not able to maintain its normal shape, in
contrast to the DMSO control group, where the villus is long and compact. This is
consistent with the increased Grp78 expression in the IQ-1 treatment. Another panel of
lineage markers, which includesVillin (Vil), Sucrase-isomaltase (Sis), and intestinal
Alkaline phosphatase (Alpi) also was analyzed by qRT-PCR (Figure 15). The
significanty diminished Vil expression is consistent with the decreased villus length. The
expression of Alpi also showed significant change. Indeed, since IQ-1 is a blocker of
proetin phosphatase PP2A, it could possess a broader effect rather than limited in Wnt
signaling pathway.
Figure 14. qRT-PCR result of Grp78. Grp78 is a stress induced protein, and its
significant increase in mRNA expression indicates the increased stress in the ileum due to
IQ-1 treatment.
31
Figure 15. qRT-PCR result of Vil, Sis and Alpi. Since IQ-1 can interfere with
phosphotase, Alpi as a phosphatase could be competed out by IQ-1 treatment, and result
in the significant decrease of mRNA expression.
3.7 Oral administration of IQ-1 specifically targets the ileum compared to other
parts of the small intestine. The same markers of the Paneth cells, gobelts cells, stem
cells and other lineage cells were evaluated by qRT-PCR. The RNA sample from
jejunum and duodenum were collected from the same IQ-1/DMSO treated groups. The
results indicate that there is no stastically significant reduction in those genes, which
suggests that the orally administrated IQ-1 more specifically targets on the ileum, where
the Paneth cells are most abundant, compared to the rest of the small intestine (Figure 16
A, B, C). There is only one exception—Grp78 expression was dramtically increased in all
areas of the small intestine. This suggests that the IQ-1 treatment induces general stress
in cells in the small intestine.
32
A.
B.
C.
Figure 16. IQ-1 treatment specifically affects the ileum rather than rest of the small
intestine. A. qRT-PCR of Paneth cell markers were tested from the jejunum and
33
duodenum. There is no significant change in the markers analyzed. B. qRT-PCR of stem
cell markers were also tested from the jejunum and duodenum, and showed no significant
change as well. C. qRT-PCR of goblet cell markers and other markers, except for Grp78,
showed no noticeable change.
3.8 Bacteria from the small intestine are found in the liver and spleen in the IQ-1
treated mouse group. Paneth cells contribute to the formation of the crypt niche, and
loss of the Paneth cell population can cause impaired barrier function (Bevins, et, al.,). In
the histological stained ileum, barrier function appears compromised (Figure 12,13).
Therefore, if the space is large enough, the bacteria in the small intestinal lumen may
pass through the barrier and enter the circulation. In this case, the liver and the spleen
would be the primary organs that the bacteria can migrate toward colonize. Due to the
inherent complexity of the intestinal microbiota, often-neglected experimental variables,
such as husbandry and environmental influences, must be tightly controlled to minimize
differences. We kept the two groups under the exact same environment and fed them with
the same diet except for the treatment. We analyzed the bacterial composition using total
genomic DNA from both IQ-1 and DMSO treated mouse livers (fresh frozen) and spleens
(fresh frozen) by using the QIAamp DNA Stool mini kit. Full-length 16S rDNA
sequences were amplified from these samples using Eubacterial primers. In this way, we
generated a pool of PCR products, which reflect the entire complex mixture of bacteria
(Salzman, et, al., 2010). When we lysed the liver, the IQ-1 treated samples appeared
crimson, whereas the DMSO sample was bright red (Figure 17 A). In the IQ-1 treated
spleen sample, total bacterial 16S DNA levels were more than three times higher
34
compared to the control (Figure 17 B). In the IQ-1 treated liver sample, this number is
more than five times higher compared to the control (Figure 18 C). This suggests that
there is a significant increased bacterial population in the IQ-1 treated mouse liver and
spleen compared with the samples from the DMSO group. In order to estimate microbial
diversity, we selected seven common bacterial primer sets (Table 2) to perform the PCR.
In the spleen lysate, the proportion of Mouse Intestinal Bacteroides (MIB) and C. leptum
(Clept) in the IQ-1 treatment group are elevated compared to the DMSO group (Figure 17
D). In the liver lysate, the proportion of undefined microbiota is severely increased in the
IQ-1 treated group. Meanwhile, the percentages of Enterobacteriaceae (Ent) and
Bacteroides (Bact) are evidently reduced by IQ-1 treatment. (Figure 17 E). This indicates
that the IQ-1 treatment results in the translocation of the bacteria from the small intestine
to the circulation and represents change in the microbiota population in the small
intestine lumen.
A.
IQ-1
DMSO
35
B. C.
D. E.
Figure 17. IQ-1 treatment induces the impaired barrier function in the crypts of the
small intestine and result in the leakage of small intestine lumenal bacteria into the
circulation. Total DNA were extracted from whole liver and whole spleen lysate. A.
Whole liver lysate from IQ-1 and DMSO treatment. The three tubes on the left side of the
picture contain IQ-1 treated whole liver lysate, the three tubes on the right side of the
picture contain the whole liver lysate from the DMSO control group. B and C. relative
total bacteria 16S rDNA copy amount from whole liver and whole spleen lysate. IQ-1
treated samples indicate significant increase. D and E. Distribution of bacterial species in
36
the liver and spleen from DMSO and IQ-1 treated group as judged by PCR using seven
common bacterial primer sets (Table 2).
3.9 Antimicrobial assay (radial diffusion assays) of IQ-1 indicates that IQ-1 is not
bactericidal. IQ-1 treatment affects the Paneth cell population, which leads to an
alteration in the bacterial composition in the small intestinal lumen. However, we wanted
to exclude that IQ-1 itself might have an antimicrobial effect on small intestinal bacteria.
There are four common small intestinal bacteria that we tested, including gram positive
and gram negative species, which are E coli (gram negative), Listeria (gram positive),
Staphylococcus aureus (gram positive), and Pseudomonas (gram negative). The
antimicrobial peptide Crp4, was used as the positive control, Crp4 treatment results in the
formation of a clear, circular no-growth zone. The test plates showed that IQ-1 does not
affect the growth of these four bacterial species similar to the DMSO control (Figure 18A,
B,C,D).
A. E. coli Test Plate B. Listeria Test Plate
37
C. Staphylococcus aureus Test Plate D. Pseudomonas Test Plate
Figure 18. Radial Diffusion anti-bacteria assay. Crp4 is an anti-bacterial peptide, that
inhibits the growth of E coli, Listeria, Staphylococcus aureus, and Pseudomonas. Crp4
was used as a positive control and formed a clear circle zone. A.B and C. E coli Test
Plate, Listeria Test Plate, Staphylococcus aureus Test Plate, and Pseudomonas Test Plate
for IQ-1 and DMSO. The data indicate that IQ-1 and DMSO both have no effect on these
four bacterial species, representing both the gram negative and gram positive bacterial
spectrum.
38
CHAPTER 4
DISCUSSION
The maturation of Paneth cells requires the Wnt signaling cascade (Es, et, al.,
2005). Sox9, a Wnt pathway target gene, is required for Paneth cell differentiation
(Mori-akiyama, et, al., 2007). We have previously described that the β-catenin/p300
interaction initiates stem cell differentiation. Therefore, we hypothesized that blockage of
the β-catenin/p300 complex using the small molecule IQ-1would diminish the Paneth cell
differentiation without adversely affecting the β-catenin/CBP interaction (Miyabayashi,
et, al., 2009). The observed decrease in both Sox9 protein level (Figure 10, western blot
and IHC) and message RNA level (Figure 9, qRT-PCR) confirms that Sox9 is a β-
catenin/p300 complex target gene in the Wnt signaling pathway. The diminished Sox9
levels suggest a potential decrease in the Paneth cell population. To verify this, we
analyzed specific Paneth cell marker levels of expression (Figure 9). Our data shows that
in the IQ-1 treated mouse ileum, at the mRNA level by qRT-PCR all Paneth cell
expression was reduced by more than 50% compared to the DMSO group. However in
the reminder of the small intestine the effects were apparently much less significant,
except Grp 78 (Figure 9 and Figure 16 A). The majority of Paneth cells reside in the
ileum, this result illustrates that the β-catenin/p300 interaction is particularly critical for
maintaining the Paneth cell population. This was also confirmed by our histological
staining (Figure 11). The Paneth cell stainable granules were significantly reduced as
judged by H and E staining and phloxine and tartrazine staining. This is consistent with
the decreased Paneth cell population. We conclude that the β-catenin/p300 interaction is
critical in the induction of Paneth cell differentiation and maintenance of Paneth cell
39
population. The 14-day mouse experiment demonstrated that there was approximately
80%-90% decrease in the Paneth cell population. Since the life cycle of Paneth cell is
approximately 70 days, this decrease suggests that the IQ-1 treated mice not only lack
newly differentiated Paneth cells, but are also losing at least part of the existing Paneth
cell population. In future studies, an apoptosis detection assay (by using anti-caspase 3
IHC staining and TUNEL staining kit) could be applied to confirm whether the existing
Paneth cells undergo apoptosis upon IQ-1 treatment.
Since IQ-1 does not affect the β-catenin/CBP interaction, which plays an essential
role in the proliferation/maintenance of stem cells (Miyabayashi, et, al., 2007), we
hypothesized that the intestinal stem cell population should not be affected by IQ-1. The
mRNA expression level of the intestinal (i.e. Lgr5, CD44, Ascl2) stem cell markers
indicates only minor changes in the stem cell population (Figure 13). The only exception
was Ephb2, which was reduced significantly. Work from out laboratory previously
demonstrated that Ephb2 is Wnt/β-catenin/p300 target gene (Kumar, et, al., 2009). IQ-1
treatment affects a significant decrease in EphB2 expression, consistent with our previous
results. Also, our histological staining shows that the villus in the ileum is shortened and
abnormally shaped. Since Ephb2 mediates epithelial patterning along the crypt-villus axis
in the small intestine (Jubb, et, al., 2005), down regulated expression Ephb2 is consistent
with the abnormal villus growth in the IQ-1 treatment. Beyond the small intestinal stem
cells, there is another cell lineage that is closely related to the Paneth cell, the goblet cell.
Goblet cells and Paneth cells are derived from the same progenitor cells (Paulus, et, al.,
1993), however, goblet cell differentiation does not require activation of Wnt pathway
target genes. We hypothesized that the disruption of the β-catenin/p300 interaction by IQ-
40
1 would not affect the goblet cell population. By analyzing Muc2 message RNA
expression levels, there was no significant difference between the IQ-1 treated group and
the DMSO group (Figure 10 A). The slight decrease in Muc2 gene expression can be
explained by the decrease in total cell population associated with the shortened and
loosened villus. Also, alcian blue staining confirmed that the density of the goblet cells
was not affected (Figure 10 B). Since IQ-1 is an indirect inhibitor of β-catenin-p300
complex, it might act in a broader manner. In conclusion, the β-catenin/p300 interaction
specifically exerts a profound influence on the Paneth cell, however does not or only
minimally affect the small intestinal stem cells and goblet cell population.
IQ-1 treatment reduces the Paneth cell population, and consequently results in
impaired barrier function. Paneth cells also contribute to the niche formation in the small
intestine (Sato, et, al., 2011). In addition, decreased the Paneth cell population causes
diminished anti-bacterial protein/peptide production. Therefore, it is not surprising to see
a change in the composition of luminal bacteria (Figure 17 D and E). This might be
associated with the IQ-1 treated mice occurring sick. We speculated that this would also
allow the luminal bacteria to pass through the weakened intestinal barrier and enter the
circulation. In this case, the liver and spleen would be the primary organs for these
bacteria to migrate to and inhabit. After amplifying full length 16S DNA by PCR, the
bacteria in the liver and spleen from the IQ-1 treated mice were significantly elevated
compared to the DMSO group (Figure 17 B and C). Therefore, we conclude that
disruption of the β-catenin/p300 interaction induces impaired barrier function potentially
due to the decreased Paneth cell population, as well as aberrant epithelial architecture
(EphB2). This contributes to the leakage of small intestinal bacteria and dissemination to
41
the liver and the spleen. Since there are many undefined bacterial species from the liver
and spleen lysate (Figure 17 D and E), in future study, the bacterial 16S DNA sequence
could be analyzed to identify the precise bacterial species. Furthermore, a fresh liver and
spleen from the IQ-1 treated mouse could be cultured in a bacteria dish. This could aid in
the determination of the viability of the bacteria found in the liver and spleen.
In conclusion, the β-catenin/p300 interaction in the Wnt signaling pathway is
particularly critical for Paneth cell differentiation and maintenance. Blockade of this
interaction can cause impaired barrier function in the small intestine. Accumulating
studies reveal that in diseases of small intestine and appendix, there is offer observed a
decrease in the amount of Paneth cells that are normally present (Ellis, 2013). Crohn's
disease (CD), one type of the inflammatory bowel disease (IBD), is associated with
decreased Paneth cell products—α-defensins (Wehkamp, et, al., 2005). Intestinal
microbiota are believed to trigger disease in genetically susceptible individuals (Bonen, et,
al., 2003). According to our study, one of the consequences by blocking β-catenin/p300
interaction is dramatically reduced defensin production (Figure 9). This might be a future
direction to explore, to rescue the Paneth cell population to restore by enhancing the β-
catenin/p300 interaction through the use of the specific CBP/β-catenin antagonized ICG-
001.
42
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Abstract (if available)
Abstract
Our lab's previous studies have demonstrated that Wnt/β‐catenin/p300 transcription initiates cell differentiation, whereas Wnt/β‐catenin/cyclic AMP‐responsive element binding protein‐binding protein (CBP) transcription mediates cell proliferation/maintenance of potency. Recent publications have shown that the Wnt signaling pathway induces Paneth cell maturation and Sox9, a Wnt pathway target gene, is required for Paneth cell differentiation. Paneth cells are terminally differentiated epithelial cells in the small intestinal crypts, which contribute to the mucosal antimicrobial barrier by generating and secreting antimicrobial peptides/proteins including α-defensins through their abundant and large granules. We show that β‐catenin/p300 interaction is required for the expression of Sox9 and thereby Paneth cell lineage commitment. Therefore the β‐catenin/p300 interaction is critical for the Paneth cell differentiation and maintenance, in contrast to the β‐catenin/CBP interaction. Our work shows that the blockage of β‐catenin/p300 interaction dramatically reduces the Paneth cell population, which is associated with impaired barrier function. In this way, luminal bacteria can be disseminated through the circulation to the liver and spleen.
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Wu, Jia
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Wnt/β-catenin/p300 induced transcription is critical for the differentiation and maintenance of Paneth cells
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Keck School of Medicine
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Master of Science
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Biochemistry and Molecular Biology
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04/29/2014
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