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Investigation of butyrate’s effects on colonic stem cell development
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Investigation of butyrate’s effects on colonic stem cell development
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Content
Investigation of Butyrate’s Effects on Colonic Stem Cell Development
by
Canran Deng
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)
August 2022
Copyright 2022 Canran Deng
ii
Acknowledgments
I would like to thank my mentor, Dr. Mark Frey who was always kind and helpful. Thanks
for being patient and listening to all my questions and giving me feedback in time. You
broke my stereotype of a professor. You were approachable all the time which made me
feel like getting along with you just like friends. In addition to the personality I admired,
you were also professional in academics. I could not image how struggling and stumbling
I would be during the thesis project without your guidance in designing every detail of the
experiment.
I would also express my gratitude to my thesis committee members, Dr. Pragna Patel and
Dr. Ching-Ling Lien. Thanks for being supportive of my thesis project and writing
recommendation letters for me on PhD applications. Without your support, I could not
have completed my thesis.
I am very grateful to the “Frey Lab Family Members”, especially Edie Bucar, Jonathan
Hsieh, and Mike Schumacher. Thanks for teaching me so many wet lab techniques and
answering a lot of the stupid questions I came up with. During the year I spent in Dr. Frey’s
lab, I felt I have been taken good care of by Mark and all the lab members.
Last but not least, I would like to say thank you to my parents. Thanks for supporting my
decision to study at the University of Southern California. Without your mental and
financial support, I could not achieve a master’s degree in the U.S. You not only gave me
opportunities to explore the diversity of the world, but also allowed me to meet different
people coming from all over the world.
iii
Table of Contents:
Acknowledgments ............................................................................................................ ii
Abbreviations .................................................................................................................. vi
Abstract ........................................................................................................................... vii
Chapter 1: Introduction ..................................................................................................... 1
1. Background ......................................................................................................... 1
1.1 Anatomy and histology of the colon .................................................................. 1
1.2 Colon epithelial cells ......................................................................................... 2
1.3 Dysfunction and disease .................................................................................. 3
2. Colonic stem cells and secretory cells ................................................................. 4
2.1 Colonic stem cells ............................................................................................. 4
2.2 Secretory cells .................................................................................................. 6
3. Microbiota and butyrate ....................................................................................... 7
3.1 Gut microbiota .................................................................................................. 7
3.2 Short chain fatty acids and butyrate ................................................................. 8
4. Pathways involved in intestinal differentiation ..................................................... 9
4.1 Nrg1 and PI3K signaling pathway .................................................................... 9
4.2 Wnt ................................................................................................................. 10
Chapter 2: Methods ........................................................................................................ 12
iv
Chapter 3: Results ......................................................................................................... 17
Chapter 4: Discussion .................................................................................................... 24
References ..................................................................................................................... 26
v
List of Figures:
Figure 1. H&E staining of the murine colon ...................................................................... 2
Figure 2. Schematic of the colonic epithelial crypt unit .................................................... 6
Figure 3. Progression of 3D organoid growth in Matrigel. .............................................. 13
Figure 4. mRNA levels of Muc2 and Dclk1 were altered by butyrate treatment ............. 18
Figure 5. Butyrate promotes the expression of colonic stem cell markers at the mRNA
level ................................................................................................................................ 20
Figure 6. Lgr5 and Lrig1 protein levels did not change after 24h butyrate treatment ..... 22
Figure 7. The effects of PI3K inhibition on butyrate stimulation of Lgr5 ......................... 23
vi
Abbreviations
SCFA short chain fatty acid
CSC colonic stem cell
YAMC young adult mouse colonic epithelial cell
Nrg1 neuregulin-1
GI tract gastrointestinal tract
Lgr5 leucine-rich repeat-containing G protein-coupled Receptor 5
7TM G-protein-coupled 7-transmembrane
Lrig1 leucine rich repeats and ImmunoGlobulin like domains 1
Ascl2 achaete-scute family BHLH transcription factor 2
IBD inflammatory bowel disease
EGF epidermal growth factor
PI3K phosphatidylinositol 3-kinase
IFN-γ interferon γ
RT-qPCR reverse transcription quantitative polymerase chain reaction
vii
Abstract
The colonic microbiome plays critical roles in health. Short chain fatty acids (SCFAs)
produced by bacteria drive key colonic epithelial processes, including proliferation and
maintenance of barrier integrity. Literature suggests that the SCFA butyrate has a
functional influence on colon cancer cells, but its effects on non-transformed cells are
unknown. Understanding how butyrate interacts with colonocytes, especially colonic
stem cells (CSC), is of great value for diseases that involve epithelial dysfunction. In this
study, I treated mouse colon epithelial cells (YAMCs) (2D) and colonic organoids (3D)
with 5 mM butyrate for 24 hours. Subsequent qPCR analysis determined that butyrate
stimulated expression of secretory cell markers (Muc2 and Dclk1) and stem cell
markers (Lgr5 and Lrig1) in YAMCs. Organoid data also supported positive regulation of
CSC markers by butyrate. However, western analysis did not detect increases in the
protein level of CSC markers after treatment for 24h. Interestingly, inhibition of PI3K (a
key driver in cell proliferation and CSC function) did not interrupt the effect of butyrate,
suggesting PI3K signaling is not likely involved in this regulation, though it did block
enhancement of the effect by the growth factor neuregulin-1 (Nrg1). Future studies that
include RNAseq could help uncover the underlying mechanisms of the effect of butyrate
and guide the identification of relevant signaling pathways. By showing that butyrate
exposure supports CSCs, my studies have identified a possible mechanism through
which the microbiome promotes gut health.
1
Chapter 1: Introduction
1. Background
1.1 Anatomy and histology of the colon
The large intestine is a part of the gastrointestinal (GI) tract. It begins just after the
terminal segment of the small intestine, the ileum, and continues on to the terminal
segment of the entire digestive tract, the rectum. The large intestine is also colloquially
termed the colon, but that is actually one specific portion of the large intestine. Overall,
the large intestine consists of three major parts: cecum, colon, and rectum. The colon is
in the middle segment of large intestine and is where the majority of water and
electrolyte absorption occurs, resulting in the production of fecal pellets (Karam, 1999;
Newton, 1987).
Microscopically (Figure 1), the colonic structures in order from the lumen (the inside of
the tube) to the outer (body-facing) layer are the mucosa, submucosa, and muscularis
externa. The colonic mucosa is made up a single, folded layer of specialized epithelial
cells overlaying connective tissue called the lamina propria. This tissue provides a
critical barrier separating the organism from its gut contents (Pangtey et al., 2017).
2
Figure 1. H&E staining of the murine colon. Colonic tissue from a C57Bl/6 mouse
was stained by H&E to visualize structures. From the outer surface inward, the colonic
mucosa is made up of three main layers: muscularis externa, thin submucosa and
mucosa. The mucosa includes epithelial cells, interepithelial stromal cells, and
supporting lamina propria.
1.2 Colon epithelial cells
The colonic mucosa is a critical barrier between the body and the intestinal contents,
including the trillions of microbes that live in the lumen (Vancamelbeke & Vermeire,
2017). The colonic epithelium is a key part of this barrier. It is a simple columnar
epithelium made up of multiple secretory and absorptive lineages (Noah et al., 2011), all
derived from common adult stem cells. Unlike in the small intestine, villi are absent from
the colonic epithelium (Kong et al., 2018; Sato et al., 2011). Instead, the tissue folds into
3
simple “crypts” (Figure 2) with stem cells anchored at the base (Yip et al., 2018). Lgr5
is
recognized as a common marker for active CSCs (Barker et al., 2007). These CSCs
proliferate continuously, and daughter cells move upward while differentiating into
absorptive enterocytes or secretory cells—tuft cells, enteroendocrine cells and goblet
cells (Altmann, 1983; Sasaki et al., 2016). Unlike the small intestine secretory cell
lineages, the colon does not have Paneth cells (Lueschow & McElroy, 2020).
The colonic epithelium constantly and rapidly renews, turning over in 5-7 days in
humans (Karam, 1999; Potten & Loeffler, 1990; Umar, 2010) or 3-4 days in mice
(Darwich et al., 2014). This rapid turnover is necessary to maintain gut epithelial
homeostasis in the face of continual challenge from luminal contents (Okumura &
Takeda, 2017). However, sometimes this rapid turnover rate is also linked with diseases
like colorectal cancer (Cao et al., 2015).
1.3 Dysfunction and disease
A number of important clinical problems involve impaired colonic function. If the colon
does not have enough time to absorb water from the fecal stream (due to rapid transit)
or water reabsorption is biochemically dysfunctional (Sandle, 1998), watery stool
results, which is also called diarrhea. In contrast, it can also be a problem when the
stool stays in the colon for too long or excessive absorption occurs (constipation).
Dysfunction of the colonic epithelium can lead to multiple diseases (O'Connor et al.,
2010; Seidelin, 2004). Colon cancer typically initiates from over-proliferation of
transformed stem cells (Boman et al., 2001; Zeki et al., 2011). In contrast, deficient stem
4
cell activity can exacerbate inflammatory conditions such as Crohn’s disease and
ulcerative colitis (Duran & Hommes, 2016; Gillespie et al., 2011; Lanzoni et al., 2008;
O'Connor et al., 2010) by decreasing barrier function and making the gut “leaky.”
2. Colonic stem cells and secretory cells
2.1 Colonic stem cells
The colonic crypts house the Lgr5+ CSCs at the crypt base (Barker et al., 2007; Zeki et
al., 2011). These cells constantly renew, routinely replacing the multiple cell types of the
epithelium (Karam, 1999), including absorptive colonocytes, mucus-secreting goblet
cells (Muc2+) (Karam, 1999; Tytgat et al., 1994), tuft cells (Dclk1+) (Gerbe et al., 2012),
and enteroendocrine cells (Chga+) (Forster et al., 2014). The continuous renewal of
these populations is necessary to keep a functional balance of cell types despite chronic
damage leading to necrosis or apoptosis in the gut, so that the colon maintains a
dynamic equilibrium of this complexity (Creamer et al., 1961). Thus, dysfunction arises
when CSCs proliferate either too fast or too slow (Gillespie et al., 2011; O'Connor et al.,
2010; Zeki et al., 2011). Additionally, altered differentiation programs that re-balance the
number of stem cells versus secretory cells versus absorptive cells can be an important
mechanism for the tissue to respond to a challenge. With their individual cell markers, it
is possible to visualize all of these cell populations in the epithelium and study their
behavior and differentiation.
A robust set of markers has been defined that can be used to indicate or localize CSCs,
5
including Lgr5 (Barker et al., 2007), Lrig1 (Jensen et al., 2009; Jensen & Watt, 2006),
and Ascl2 (Schuijers et al., 2015). Leucine-rich repeat-containing G protein-coupled
Receptor 5 (Lgr5), also known as Gpr49 (McClanahan et al., 2006; McDonald et al.,
1998), is one the most distinct and specific stem cell markers in both small and large
intestine. Lgr5 mRNA marks CSCs definitively (Beumer & Clevers, 2021). Furthermore,
Lgr5 is a Wnt target gene (Carmon et al., 2011; Glinka et al., 2011), consistent with the
role of Wnt signaling in maintaining stem cells. However, because Lgr5 protein shares
high structural similarity with G-protein-coupled 7-transmembrane (7TM) family proteins
including Lgr4 and Lgr6 (Carmon et al., 2011; de Lau et al., 2011), available antibodies
targeting Lgr5 are not as specific as RNA detection through methods such as in situ
hybridization.
Other than Lgr5, there are also some other markers that can be used to visualize CSCs
or support the evidence of their presence. For example, Leucine Rich repeats and
ImmunoGlobulin like domains 1 (Lrig1) is highly expressed in CSCs. However, though
lineage mapping of Lrig1+ and Lgr5
+
cells is very similar, some non-CSC progenitor
cells are also labeled by Lrig1 detection, making it less specific (Powell et al., 2012).
Achaete-Scute Family BHLH Transcription Factor 2 (Ascl2) is another gene that is
enriched by R-spondin/Lgr5 mediated Wnt signaling in intestinal stem cells. Ascl2+ cells
can function as stem cells in mouse (Schuijers et al., 2015). However, this molecule has
been less well-studied than Lgr5 and its potential as a definitive marker is unclear.
6
Figure 2. Schematic of the colonic epithelial crypt unit. This tissue consists of
colonic stem cells (CSC) in the base of the crypt, absorptive cells, and secretory cells
(including tuft cells and goblet cells), and mucus covering the single cell layer containing
microbiota and their secretome. Figure created in BioRender.
2.2 Secretory cells
The colonic epithelium contains two main secretory cell lines—tuft and goblet cells.
Enteroendocrine cells are also present, but in very few numbers. Paneth cells are also
common gut secretory cells but these are specific to the small intestine.
Secretions from these lineages contribute to the gut barrier protection by providing the
overlying mucus layer with signals that modify immune and enteric nervous activity, and
7
anti-microbial peptides (Noah et al., 2011). Tuft cells can be visualized by the Dclk1
+
marker (Gerbe et al., 2012), which labels a small (0.4%) but important percentage of the
epithelial cells. These cells have chemosensory receptors, giving them the ability to
sample the microenvironment and release immunomodulatory peptides. Similarly, goblet
cells produce mucus and multiple immune-active molecules to protect the gut epithelial
integrity as well as create an environment for commensal microbiota (Gerbe et al.,
2012; Knoop & Newberry, 2018). They can be localized by Muc2
+
expression (Tytgat et
al., 1994).
3. Microbiota and butyrate
3.1 Gut microbiota
Microbes living in the intestine, though technically not “self”, are an indispensable part of
the whole organism. These micro-organisms live in the mucus, swim in the lumen, or
adhere to the colon epithelial cells (Bull & Plummer, 2014). By sheer numbers, this
population is mostly bacteria (Bäckhed et al., 2005) but also includes some viruses (Gill
et al., 2006) and fungi. The microbiome contains approximately the same number of
bacteria as there are cells in the mammalian body, It actually represents the largest
proportion of the genome in the body with ~3 million encoding genes (Valdes et al.,
2018), while the human genome is much smaller with about 23,000 coding genes (Gill
et al., 2006). This huge microbial population is engaged in and responsible for many
metabolic processes, ultimately exerting profound influence on the health and fitness of
8
the host (Rath & Dorrestein, 2012).
Studies have shown that the composition of the gut microbiota is highly responsive to
environmental factors (Goodrich et al., 2014; Rothschild et al., 2018) such as, for
instance, diet and drugs (Hasan & Yang, 2019). Dietary fiber has been reported to be
good for health because of the generation of short chain fatty acids (Silva et al., 2020).
Fiber is poorly digestible in the absence of bacteria (Valdes et al., 2018; Wong et al.,
2006). However, with the help of gut bacteria, fiber can be metabolized into SCFAs
through fermentation (Wong et al., 2006). SCFAs can be directly used by colonocytes
as fuel, and can also be absorbed from the large intestine into the blood, allowing them
to function throughout the body (Cummings et al., 1987).
3.2 Short chain fatty acids and butyrate
As the main metabolites of fiber fermentation by gut bacteria, SCFAs play an important
role in normal immune function and in maintaining colon homeostasis (Topping &
Clifton, 2001). One of the key effects of SCFAs is suppression of inflammation (Rutting
et al., 2019), which may explain the correlation between fiber in diet with low risks of
colon cancer (McIntyre et al., 1993; Weisburger, 1991). SCFAs and a high-fiber diet
have been recognized as potential treatments for gastrointestinal dysbacteriosis and
other colonic diseases by manipulating gut microbiota (Sommer & Bäckhed, 2013; Xu et
al., 2020). Previous studies also found that there is a decrease in SCFAs in
inflammatory bowel disease (IBD) patients (Parada Venegas et al., 2019).
Butyrate is one of the SCFAs produced by colonic bacteria. It not only provides an
9
energy source for colonocytes (Donohoe et al., 2011) but also modulates several
epithelial processes including inflammation, proliferation, and barrier integrity (Sturm &
Dignass, 2008). Studies have shown that a reduced level of butyrate in the gut has a
harmful impact on Crohn’s disease patients (Geirnaert et al., 2017), and that IBD
patients have lower butyrate concentrations than healthy controls (Panasevich et al.,
2015). Moreover, although butyrate treatment helps to ameliorate gut dysbiosis in mice
with intestinal tumors (Chen et al., 2020), the relationship between butyrate and
colonocyte development/differentiation is not well-understood.
4. Pathways involved in intestinal differentiation
4.1 Nrg1 and PI3K signaling pathway
Neuregulin 1 (Nrg1) is a member of the epidermal growth factor (EGF)-like family of
ligands, part of the neuregulin subgroup that also includes Nrg2, Nrg3, and Nrg4. The
Nrg1 protein has been found mostly in epithelial tissues (Peles et al., 1992; Zhang et al.,
1997). It participates in various cellular responses by activating the receptor tyrosine
kinase activity of ErbB-family receptors (Falls, 2003). They are not only capable of
enhancing cellular proliferation and differentiation (Jardé et al., 2020), but they play an
essential role in protecting the mucosal layer of GI tract and maintaining the
homeostasis of epithelial cells (Jacenik et al., 2019).
The phosphatidylinositol 3-kinase (PI3K) pathway is one of the most important
pathways downstream of ErbBs. Since Nrg1 can stimulate PI3K signaling during
10
proliferative and survival responses (Jardé et al., 2020), the PI3K inhibitor LY294002
inhibits a number of Nrg1 signaling pathway effects. This signaling cascade has also
been reported to have high relevance to colorectal cancers (Koveitypour et al., 2019;
Malinowsky et al., 2014).
4.2 Wnt
Wnt signaling is critical for developing stem cells (Farin et al., 2012; Sato et al., 2004;
Willert et al., 2003) and progenitor cell expansion (Chenn & Walsh, 2002). Previous
studies have suggested that Wnt promotes colonic epithelial cell proliferation (Flanagan
et al., 2018). In contrast, if there is a reduction in Wnt signaling, then epithelial
regeneration is impaired (Barker et al., 2013). Because this study focuses on stem cell
regulation, Wnt is a good target to evaluate. Lgr5 and Ascl2 will be used as CSC
markers, as they are Wnt target genes responsive to Wnt3a/R-spondin mediated Wnt
signaling (Schuijers et al., 2015). In addition, butyrate is able to regulate the Wnt
signaling pathway in some settings (Chen et al., 2020). Therefore, we hypothesize that
the regulation between butyrate and stem cells involves Wnt.
The gut microbiome plays critical roles in colonic health and disease. Much of this is
thought to be through the action of bacterial metabolites such as butyrate. Evidence in
the literature suggests that butyrate influences the function of colon cancer cells, but its
regulation of non-transformed colonic epithelial cells remains unclear. The main purpose
of this study is to reveal the relationship between the gut microbial metabolite butyrate
11
and CSC development. The findings in this study will be of potential benefit to future
therapy development for inflammatory bowel disease or other GI disorders.
12
Chapter 2: Methods
Cell culture:
Young Adult Mouse Colon (YAMC) epithelial cells were genetically engineered to switch
on and off an immortalized state to allow for routine culture use. In order to turn on the
immortalized state and rapidly expand cultures, 0.5 µL/mL interferon (IFN)-γ was added
to activate production of a mutant SV40 Large T antigen (a viral oncoprotein) driven by
the IFN-γ promoter. This mutant T antigen is temperature-sensitive, so YAMC cells need
to be incubated at 33
o
C for the Large T antigen to be stable. To de-immortalize cells in
preparation for experiments, the IFN-γ is removed and the cells placed in a 37
o
C
incubator for 24 hours. ~600000 cells were plated in each well of a 6-well plate in 2 ml
complete growth medium [RPMI 1640 (Gibco) with 5% FBS, 1% Penicillin and
Streptomycin, and insulin/transferrin/selenium (ITS, Gibco) additive plus IFN-γ as
above] and incubated for 24 hours to ensure that each well contained a confluent
monolayer of cells. Before butyrate treatment, medium was replaced with low-serum
starve medium (RPMI 1640 with 0.5% FBS and 1% Penicillin and Streptomycin only) for
24 hours. Cells were then treated with 5 mM sodium butyrate, 10 ng/ml Nrg1, or 10 µM
PI3K inhibitor LY294002 for 2, 6, or 24 hours, and harvested 24 hours after treatment.
13
Organoid culture:
Organoids were used for a 3D culture model containing all mature colonic epithelial cell
types. C57BL/6J mice were sacrificed and colonic crypts were liberated by calcium
chelation with 5 mM EDTA and shaking as previously performed (Almohazey et al., 2017),
then embedded in Matrigel with 20 ng/ml EGF (PeproTech), 100 ng/ml Noggin (R&D),
and 500 ng/ml R-Spondin-1 (R&D). The mix of Matrigel and crypts was incubated at 37°C
for 15 minutes to allow them solidify, and overlaid with IntestiCult organoid culture medium
(Stemcell Technologies).
Figure 3. Progression of 3D organoid growth in Matrigel. Images show the daily
change in morphology during growth of the organoid starting with crypts isolated from
mouse colon. Single crypts turned into spheroids, and finally started budding in all
14
directions. At day 5, debris from ongoing cell turnover can be seen within the organoid.
Images were obtained using an iPhone 11 built-in camera.
mRNA extraction and cDNA synthesis:
Cells or organoids were collected after treatment and RNA isolated by using a column-
based extraction kit (Omega Bio-Tek) following the manufacturer’s instructions. mRNAs
were either stored in a -80°C freezer for long-term storage, or kept on ice for immediate
use. RNA concentration (by A260, absorbance at 260 nm) and quality (A260/A280 and
A260/A230 ratios) were measured on a NanoDrop spectrophotometer. One microgram
of RNA was used with Applied Biosystems High Capacity Reverse Transcriptase cDNA
Synthesis Kit to synthesize the cDNA, following the manufacturer’s instructions.
RT-qPCR:
To quantify gene expression in samples, cDNA was used for quantitative PCR (qPCR)
using probes and primers from predesigned TaqMan Gene Expression Assays (see
Assay IDs in Table 1 below). qPCR assays were performed using a StepOne real-time
PCR system (ABI) following the manufacturer’s instructions. Relative expression was
determined by the 2
−∆∆Ct
method, using mouse Hprt as the reference gene.
15
Table 1. Taqman Predesigned Gene Expression Assays
Gene Symbol Assay ID
Lgr5 Mm00438890_m1
Lrig1 Mm00456116_m1
Ascl2 Mm01268891_g1
Muc2 Mm01276696_m1
Dclk1 Mm00444950_m1
Western blot analysis:
Proteins were extracted from YAMCs by scraping them into RIPA buffer (50 mM Tris, pH
7.4, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) plus protease and
phosphatase inhibitors (Halt Protease Inhibitor Cocktail and Phosphatase Inhibitor
Cocktails I and II, Sigma) on ice to avoid protein degradation. Protein concentration was
determined using the BCA Protein Assay Kit (Pierce). 30 µg of protein from each sample
was loaded on to Bolt 4-12% Bis-Tris Plus gels (Invitrogen) for separation at 200V
followed by transfer to a nitrocellulose membrane. The membrane was blocked in 5%
milk in Tris-buffered saline (TBS) for 1 hour and then incubated in TBS with primary
antibodies against β-actin (Sigma A228, 1:2000), Lgr5 (Abcam ab75850, 1:2000), and
Lrig1 (Cell Signaling #12752, 1:2000) overnight at 4°C. Membranes were washed three
times in in TBS plus 0.5% Tween-20. Lgr5 signal was detected by incubation for 1 hour
with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling). After
16
three TBS washes, signal was detected by incubation with SuperSignal enhanced
chemiluminescence reagent (Pierce) and detection on a C-Digit scanner (Li-Cor Inc).
Lrig1 signal was detected by incubation for 1 hour with IR-dye tagged secondary
antibodies (Li-Cor Inc.). After three TBS washes, signal was detected by scanning on a
Li-Cor Odyssey imager.
17
Chapter 3: Results
Butyrate induces expression of colonic secretory cell markers in YAMCs but not
in colonoids
I first hypothesized that butyrate would promote development of secretory cells and that
markers for these lineages would be upregulated after exposure. The treatments used
to test this in both cell and organoid culture were 24 h exposure to 5mM sodium
butyrate (NaB), 10ng/ml Nrg1, and the co-treatment (NaB+Nrg1). The reason why I
additionally added Nrg1 is that ErbB signaling has been shown to play a crucial role in
gut development, and developmental disruption of this signaling pathway leads to
altered epithelial differentiation in the small intestine (Almohazey et al., 2017). Thus, a
secondary hypothesis was that Nrg1 signaling would modify butyrate effects.
Representative markers for goblet cells and tuft cells are Muc2 and Dclk1, respectively. I
first tested Muc2 and Dclk1 expression in YAMCs (Figure 4A&B) using qPCR. Both
secretory cell markers Muc2 and Dclk1 were remarkably increased in the presence of
NaB compared to control. However, Nrg1 in YAMCs did not modify this response.
Interestingly, the results in colonoids were quite different from what we learned in the
cell model. Secretory cell markers were reduced in the colonoid NaB treatment group
(Figure 4C&D). Again, Nrg1 also did not affect the Muc2 and Dclk1 expression.
18
Figure 4. mRNA levels of Muc2 and Dclk1 were altered by butyrate treatment.
qPCR analysis for goblet cell marker Muc2 (A) and tuft cell marker Dclk1 (B) in YAMCs
treated with 5 mM NaB +/- 10 ng/ml Nrg1 for 24 h compared to controls. Hprt was used
as a reference gene. Dashed line represents baseline expression in controls. *P<0.05.
n=3 for each group. (C) and (D) shows the changes of relative expression in Muc2 and
Dclk1 in colonoids. ***P<0.001. n=5 for each group.
19
Butyrate induces colonic stem cell markers at the mRNA level
After determining how butyrate regulates gut secretory cells, I tested the butyrate effect
on CSCs. I first performed a time-course with the same treatments as in Figure 4 and
assessed Lgr5 expression in YAMC cells. Lgr5 expression was induced with NaB over
time, with a maximum observed effect at 24 h (Figure 5A). Therefore, I set my treatment
time at 24h in the experiments that followed. In both cell (Figure 5B) and organoid
(Figure 5C) cultures, NaB strongly induced Lgr5, with Nrg1 co-treatment trending
towards enhancing this induction. Nrg1 alone had no apparent effect on CSC marker
expression. I also quantified Lrig1 (Figure 5D) which is another marker for CSCs and
Ascl2 (Figure 5E) which is a Wnt target gene that is specific to R-spondin/Lgr5
regulation. These increased nearly four times and 40 times, respectively, in the NaB
group compared with control.
20
Figure 5. Butyrate promotes the expression of colonic stem cell markers at the
mRNA level. (A) qPCR analysis of stem cell marker Lgr5 in YAMC cells after treatment
21
for 2h, 6h, or 24h with NaB, Nrg1, or both. (B) qPCR of colonic stem cell marker Lgr5 in
YAMC cells treated with 5 mM NaB +/- 10 ng/ml Nrg1 for 24 h compared to controls.
n=4 for each group. (C) qPCR of organoids treated with 5 mM NaB +/- 10 ng/ml Nrg1
for 24 h compared to controls. n=4 for each group. (D) qPCR analysis of Lrig1 in
organoids. n=5 for each group. (E) qPCR analysis of Ascl2 in organoids. n=5 for each
group. Hprt was used as the reference gene. Dashed line represents baseline
expression in controls. ***P<0.001; *P<0.05; ns, not significant.
24h NaB treatment of YAMCs did not show clear effects on stem cell markers at
the protein level
To test the butyrate effects at the protein level, I performed western blot analysis of
YAMC cells treated with 5 mM butyrate for 24 h. Although I obtained robust evidence
showing butyrate upregulates the stem cell markers at the mRNA level in both 2D and
3D models, the western blots demonstrated no noticeable differences at the 24 h time
point. Ongoing experiments will include testing protein expression over a broader time
course, testing organoid expression in case YAMCs are unable to translate or sustain
protein expression of these stem cell markers, and further testing the antibodies for
specificity since there is a possibility of cross-reactivity with other family members.
22
Figure 6. Lgr5 and Lrig1 protein levels did not change after 24h butyrate
treatment. Western blot analysis on Lgr5 (A) and Lrig1 (B), using β-actin as loading
control. YAMCs were treated (+) or untreated (-) with 5 mM NaB. MW, molecular weight.
Butyrate effects on stem cell regulation are not directly through PI3K signaling.
The PI3K signaling pathway is critical for cell proliferation, survival and differentiation.
PI3K is also a key downstream pathway of NRG/ErbB signaling. Therefore, I
hypothesized that this pathway might participate in mediating the effect of butyrate. In
order to study whether NaB-stem cell regulation or the enhancement with Nrg1 is
dependent on PI3K, LY294002, a PI3K inhibitor, was used for blocking PI3K signaling,.
As shown in Figure 6, there was no distinct difference on Lrg5 mRNA level between
NaB treatment alone and the co-treatment (NaB + PI3K inhibitor). On the other hand,
enhancement of Lgr5 expression caused by Nrg1 co-treatment was ablated by
LY294002. Thus, the baseline butyrate effect on stem cell regulation is unlikely through
PI3K signaling pathway directly, but Nrg1 activation of PI3K can enhance the response.
23
Figure 7. The effects of PI3K inhibition on butyrate stimulation of Lgr5. qPCR
analysis on Lgr5 in YAMCs. Every treatment lasted 24h. NaB: 5 mM; Nrg1: 10 ng/ml;
LY294002: 10 µM. ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; ns, P>0.05.
24
Chapter 4: Discussion
Studying colonic epithelial cell proliferation and differentiation is necessary for us to
understand how to keep gut integrity and health intact. This single cell layer to us is like
a shield separating “ourself” from “non-self”, for instance, micro-organisms. Though it is
not generated by our own body, the microbiome is in turn indispensable to normal
health and plays a significant role in digesting as well as promoting gut health (Sommer
& Bäckhed, 2013). However, the mechanisms underlying microbe-epithelial
communication have not been well-studied. This topic has been brought to more
researchers’ interests and attention recently. Early this year, a report was published
discussing the diet effect on mitigating colitis, in which the investigators also linked this
dietary intervention with the microbiome (Ahmed et al., 2021). Their studies shared
some similarity with mine, but I was more focused on the interaction between butyrate
and stem cell development. To investigate this regulatory relationship, I tested both
mRNA and protein levels for stem cell and differentiation markers in both a 2D model
(YAMC cells) and a 3D model (colonic organoids).
Due to the limited time available for research, several important questions remain to be
addressed. First of all, the data showed that butyrate effects on secretory cell markers
were opposite in cell culture (stimulated) versus organoid culture (suppressed). The
underlying mechanisms are unclear, but it is possible that positional cues from the 3D
organoids make the difference. Additionally, the presence of all mature cell types in the
organoids may influence discrete cell type-specific responses. On the other hand,
25
glutamine (Gln) is a key source for intestine epithelial cell proliferation and survival,
which is required for organoid expansion, and the lack Gln in starve medium could
subsequently decrease epithelial cell proliferation and reduce the secretory cell
differentiation after 24 hours’ starvation process (Moore et al., 2015). To resolve this
dilemma, we could either add Gln to starve medium or minimize the starvation time.
Secondly, Lgr5 is a specific marker to distinguish stem cells, but measuring its protein
level is difficult. Since Lgr5 is highly similar in structure to other members of G-protein-
coupled 7-transmembrane (7TM) family, there is no highly specific antibody available to
detect Lgr5 protein. The western blots in Figure 6 likely show a sum of Lgr5 with Lgr4
and Lgr6. I also looked at Lrig1 and Ascl2, in order to visualize the stem cell in multiple
ways. If we have to compare the changes in Lgr5, we can use organoids from a
genetically modified mouse with its Lgr5 fused to a GFP tag which will allow Lgr5 to be
specifically detectable by western blot. Thirdly, an increase in stem cell markers at the
RNA level but not the protein level could be a timing issue. For example, treatment with
butyrate for 24 hours altered stem cell marker transcription but may not yet result in
accumulation of protein because of the possible low translation rate and short protein
half-life (Liu et al., 2016). Last but not least, these findings need to be confirmed in an in
vivo animal model.
26
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Abstract (if available)
Abstract
The colonic microbiome plays critical roles in health. Short chain fatty acids (SCFAs) produced by bacteria drive key colonic epithelial processes, including proliferation and maintenance of barrier integrity. Literature suggests that the SCFA butyrate has a functional influence on colon cancer cells, but its effects on non-transformed cells are unknown. Understanding how butyrate interacts with colonocytes, especially colonic stem cells (CSC), is of great value for diseases that involve epithelial dysfunction. In this study, I treated mouse colon epithelial cells (YAMCs) (2D) and colonic organoids (3D) with 5 mM butyrate for 24 hours. Subsequent qPCR analysis determined that butyrate stimulated expression of secretory cell markers (Muc2 and Dclk1) and stem cell markers (Lgr5 and Lrig1) in YAMCs. Organoid data also supported positive regulation of CSC markers by butyrate. However, western analysis did not detect increases in the protein level of CSC markers after treatment for 24h. Interestingly, inhibition of PI3K (a key driver in cell proliferation and CSC function) did not interrupt the effect of butyrate, suggesting PI3K signaling is not likely involved in this regulation, though it did block enhancement of the effect by the growth factor neuregulin-1 (Nrg1). Future studies that include RNAseq could help uncover the underlying mechanisms of the effect of butyrate and guide the identification of relevant signaling pathways. By showing that butyrate exposure supports CSCs, my studies have identified a possible mechanism through which the microbiome promotes gut health.
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Creator
Deng, Canran
(author)
Core Title
Investigation of butyrate’s effects on colonic stem cell development
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Medicine
Degree Conferral Date
2022-08
Publication Date
07/22/2022
Defense Date
06/15/2022
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University of Southern California
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butyrate,colonic stem cell,OAI-PMH Harvest,secretory cell
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Frey, Mark (
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), Lien, Ching-Ling (
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), Patel, Pragna (
committee member
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canrande@usc.edu,canrandeng98@gmail.com
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Tags
butyrate
colonic stem cell
secretory cell