Interactions Between EGF and the IGF-1 Receptor in Mediating Hgly2-GLP-2-Induced Proliferation in the Murine Small Intestine

Interactions Between EGF and the IGF-1 Receptor in Mediating hGly2-GLP-2-Induced Proliferation in the Murine Small Intestine

Zivit Fesler

A thesis submitted in conformity with the requirements for the degree of Master’s of Science Department of Physiology University of Toronto

Interactions Between EGF and the IGF-1 Receptor in Mediating hGly2-GLP-2-Induced Proliferation in the Murine Small Intestine

Zivit Fesler

Master’s of Science

Department of Physiology University of Toronto

2019 Abstract

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone that promotes intestinal proliferation through downstream mediators. Two known mediators are insulin-like growth factor-1 (IGF-1) and epidermal growth factor (EGF), both essential for the proliferative effect of

GLP-2. I hypothesized that pre- and/or co-EGF enhances IGF-1-mediated GLP-2-induced proliferation in the murine model. Chronic combination treatment of EGF and GLP-2 increased intestinal growth in C57Bl/6 mice compared to vehicle, GLP-2 and respective EGF treatments.

Treatment of pre-EGF+co-EGF+GLP-2 in intestinal epithelial IGF-1 receptor (IGF-1R) knockout mice restored intestinal growth despite the lack of a receptor essential for GLP-2 signaling. Finally, IGF-1 treatment in organoids did not have an additive effect to EGF on proliferation, but managed to replace EGF as an essential growth factor in the media to maintain normal proliferation. Together, these data suggest that exogenous EGF is an essential mediator for GLP-2 that can supersede the missing signaling pathway of IGF-1R.

Acknowledgments

Graduate school has been an important learning and growing environment for me. It was both joyful and challenging. I met many people who became mentors and good friends and honed my research and critical thinking skills which I will carry on in my future endeavors. There are many people I would like to thank for their support and belief in me as I took this important step.

First, I would like to thank my supervisor, Dr. Patricia Brubaker, for her unconditional support and patience as I learned and grew as a researcher. Her continuous mentorship and guidance allowed me to develop invaluable skills that I will carry wherever I go. I would also like to thank my committee member Dr. Dan Drucker for challenging me during committee meetings and providing me with essential tools to better myself as a scientist and a critical thinker.

I would like to thank Jennifer Chalmers and Melanie Markovic from the Brubaker lab for their valuable teachings and continuous support of me and my project. Jennifer taught me many of the methods I have mentioned through this thesis, as well as provided me with support through problem-solving, critical thinking, and teaching me how to be an ethical scientist. I want to thank

Melanie for providing me with friendship and mentorship throughout my two-year degree, and for teaching me how to be critical of my data, how to analyze it, and how to be a better scientist. I would like to thank Alexandre Hardy from the CFI lab for providing me with much needed training, as well as continuous support during my extensive use of the facility.

I would also like to thank Dr. Katie Rowland, who has established the IE-IGF-1R knockout model in our lab, as well as Dr. Robine, Dr. Holzenberger and Dr. Kulkarni for initially providing the transgenic animals. I would like to thank Dr. O’Brien from the University of Toronto and Dr.

Kuo from Stanford University for providing me with HEK293 cells for the organoid experiments.

iii

I would also like to thank the Department of Physiology and the administration, particularly

Colleen, Rosalie, and Eva for all their assistance.

Finally, I would like to thank my mother, Zila Fesler, and my two close friends, Adrian

Esser and Christine Qian for supporting me and continuously pushing me to better myself. I would have not been able to achieve this degree without your emotional support and listening ear.

In memory of Bertie Fesler.

Table of Contents

Acknowledgments ...... iii

Table of Contents ...... v

List of Tables ...... viii

List of Figures ...... ix

List of Abbreviations ...... x

Introduction ...... 1

1.1 Rationale ...... 1

1.2 Intestinal Architecture and Proliferation ...... 2 1.2.1 Types of cells in the intestine ...... 2 1.2.2 Lgr5 and Quiescent Stem Cells ...... 3 1.3 Glucagon Like Peptide 2 ...... 5 1.3.1 Structure, Expression and Secretion ...... 5 1.3.2 Clearance ...... 6 1.3.3 Effects on the Gastrointestinal Tract ...... 6 1.4 GLP-2 Receptor ...... 8 1.4.1 Discovery and Location...... 8 1.4.2 Signaling ...... 10 1.5 Intestinal EGF ...... 11 1.5.1 Structure, Expression and Secretion ...... 11 1.5.2 Effects of EGF on the Gut ...... 11 1.5.3 EGF Receptors and Signaling ...... 13 1.5.4 EGF and GLP-2 ...... 14 1.6 Intestinal IGF-1 ...... 15

1.6.1 Structure, Expression and Secretion ...... 15 1.6.2 Effects of IGF-1 on the Gut ...... 16 1.6.3 IGF-1 receptors and signaling ...... 17 1.6.4 IGF-1 and GLP-2...... 17 1.7 The Known Interactions Between EGF and IGF-1 ...... 19

1.8 Hypothesis and Specific Aims...... 20

Materials and Methods ...... 21

2.1 In Vivo Studies ...... 21

2.2 In Vitro Studies ...... 22

2.3 RNA Extraction and Analysis ...... 24

2.4 Microscopy ...... 25

2.5 Proliferation Analysis ...... 26

2.6 Statistics ...... 27

Results ...... 28

3.1 Determining the Optimal Timing for EGF-GLP-2 Treatment ...... 28 3.1.1 A Combination Treatment with EGF and GLP-2 Increases Small Intestinal Weight...... 29 3.1.2 All Combination Treatments with EGF and GLP-2 Increase Crypt-Villus Height ...... 31 3.1.3 GLP-2 Increases Proliferation while EGF Decreases Apoptotic Markers ...... 33 3.1.4 Transcripts of Ligands and Their Receptors Did Not Change Between Groups ...... 35 3.2 Investigating the Relationship Between GLP-2, EGF and IGF-1R Using IE-IGF-1R-KO Mice ...... 37 3.2.1 Validation of the IE-IGF-1R KO in Mice ...... 38 3.2.2 IE-IGF-1R KO Mice and Control Mice Respond Similarly to Pre-EGF+Co-EGF+GLP-2 Combination Treatment ...... 39 3.3 Organoids ...... 43 3.3.1 qRT-PCR of Organoids Confirms the Absence of GLP-2R and the Presence of EGFR and IGF-1R 43 vi

3.3.2 Determination of Optimal Timepoint and EGF Withdrawal Treatments to Detect Proliferation ...... 45 3.3.3 EGF and IGF-1 Stimulate Proliferation Independently, But Do Not Have an Additive Effect on Organoid Proliferation ...... 48 Discussion ...... 52

4.1 General Discussion ...... 52

4.2 Limitations ...... 56

4.3 Future Directions ...... 57

4.4 Conclusion ...... 60 References ...... 61

vii

List of Tables

Table 2.1: List of primers used for experiment...... 25

Table 3.1: Treatment groups designed to determine the optimal timing for EGF-GLP-2 treatment

……………………………………………………………………………………………28

Table 3.2: Determination of Optimal Timepoint and EGF Withdrawal Treatments ...... 47

Table 3.3: EGF and co-IGF-1 Treatment of Organoids ...... 50

viii

List of Figures

Figure 3.1.1: Combination Treatment with EGF and GLP-2 Increases Small Intestinal Weight 31

Figure 3.1.2: All Combination Treatments with EGF and GLP-2 Increase Crypt-Villus Height 32

Figure 3.1.3: GLP-2 Increases Proliferation while EGF Decreases Apoptosis ...... 35

Figure 3.1.4: Transcripts of Ligands and Their Receptors Did Not Change Between Groups .... 37

Figure 3.2.1: Validation of the IE-IGF-1R KO in Mice ...... 39

Figure 3.2.2: IE-IGF-1R KO Mice Respond Similarly to EGF-GLP-2 Combination Treatment as Control Animals ...... 43

Figure 3.3.1: qRT-PCR of Organoids Confirms the Absence of GLP-2R and the Presence of EGFR and IGF-1R ...... 44

Figure 3.3.2: Determination of Optimal Timepoint and EGF Withdrawal Treatments ...... 47

Figure 3.3.3: EGF and IGF-1 Stimulate Proliferation Independently, But Do Not Have an Additive Effect on Organoid Proliferation ...... 51

List of Abbreviations

ANOVA Analysis of variance

AUC Area under the curve bp Base pair cAMP Cyclic adenosine monophosphate

Cre Cyclization recombination

DAPI 4'-6-diamidino-2-phenylindole

DPP-4 Dipeptidyl peptidase-4

EDTA Ethylenediaminetetraacetic acid

EdU 5-ethynyl-2’-deoxyuridine

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

ERK Extracellular signal-regulated kinase

FBS Fetal bovine serum

Fl Flox

GH Growth hormone

GLP-2 Glucagon-like peptide-2

GLP-2R Glucagon-like peptide-2 receptor

HB-EGF Heparin-binding EGF

IE Intestinal epithelial

IEC Intestinal epithelial cells

IGF Insulin-like growth factor

IGF-1R Insulin-like growth factor-1 receptor

IGFBP Insulin-like growth factor binding protein ip Intraperitoneal

KO Knockout

MEK MAPK/ERK kinase mRNA Messenger ribonucleic acid

NEC Necrotizing enterocolitis

Lgr5 Leucine-rich repeat-containing G protein-coupled receptor 5

PBS Phosphate-buffered saline

PC Prohormone convertase

PCR Polymerase chain reaction

PI3K Phosphoinositide-3-kinase

PKA Protein kinase A

PN Parenteral nutrition qRT Quantitative reverse transcription

SGLT-1 Sodium-dependent glucose cotransporter-1

SH2 SRC-homology 2

SI Small intestine

TA Transit amplifying

TNFα Tumour necrosis factor alpha

TPN Total parenteral nutrition

Symbols and units

% Percent

°C_ _ Degrees Celsius

µg micrograms g Grams hr Hours kg kilograms mM Millimolar mL Milliliters p Statistical p-value

SEM Standard error of the mean

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Introduction 1.1 Rationale

Glucagon-like peptide-2 (GLP-2) is an intestinotrophic hormone that increases gut growth in response to nutrient absorption. It promotes growth by increasing proliferation in the intestinal crypt, particularly in the jejunum.1–3 The GLP-2 receptor (GLP-2R) is found in three cell types in the intestine: subepithelial myofibroblasts, enteric neurons and enteroendocrine cells.4–7

However, no GLP-2R is found on the crypt cells of the intestine, where the proliferative action of

GLP-2 is observed.4–7 Therefore, several intermediaries are at play, among them are epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) and both have been separately shown to be essential for GLP-2-mediated induction of intestinal proliferation.2,3,8 Furthermore, it has previously been shown that while IGF-1 causes a reduction of IGF-1 receptor (IGF-1R) in vitro, EGF maintains IGF-1R levels in the presence of IGF-1.9 Moreover, incubation of intestinal epithelial cells with IGF-1 after EGF treatment increases proliferation synergistically compared to incubation with IGF-1 alone.10 However, a clear relationship between all three growth factors,

GLP-2, EGF and IGF-1, has not been established as of yet. Therefore, the goal of this study was to investigate the relationship between GLP-2, EGF and IGF-1R to modulate proliferation in the mouse jejunum.

1 2

1.2 Intestinal Architecture and Proliferation

1.2.1 Types of cells in the intestine

The intestinal epithelium is a monolayer of cells that lines the small and large intestine and that serves as a selective barrier. It functions as both an absorptive organ for beneficial nutrients, as well as a protective barrier against harmful nutrients and microorganisms. Although the monolayer is composed of columnar cells, these cells serve different functions within the gut.

There are four main types of cells: enterocytes, which serve to absorb nutrients from the gut lumen and are most abundant type, enteroendocrine cells which secrete various hormones, as well as goblet cells which secrete mucous and Paneth cells which secrete antimicrobial substances to protect the gut from harmful bacteria.11 There are also two types of stem cells: actively proliferating stem cells that actively renew the intestinal epithelium, which has to be replaced every 4-5 days, and the +4 quiescent stem cells which replace the actively proliferating stem cells in response to injury.12 While goblet cells and enteroendocrine cells tend to be present throughout the crypt and villus, Paneth cells reside at the base of the intestinal crypt, among leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) stem cells, while protecting the sensitive environment of the crypt.13 Enteroendocrine cells are particularly rare in the gut and compose approximately 1% of the entire epithelium. They secrete hormones such as GLP-1 and

GLP-2 (L cells) and glucose-dependent insulinotrophic polypeptide (K cells) and cholecystokinin (I cells).14

1.2.2 Lgr5 and Quiescent Stem Cells

Actively proliferating stem cells in the crypt of the intestine were first reported in 1974 by Cheng et al.15 This was achieved by injecting mice with 3H-thymidine, into mice. The authors sacrificed mice at 1 hour, 6 hours and 12 hours. At 1 hour, the labeled cells were confined to the base of the crypt, while by 6 hours the labeled cells reached the top of the crypt. By 12 hours labeled cells were found along the villus epithelial cells, suggesting migration of cells from the bottom of the crypt to the top of the villus. The first discovery of Lgr5, a member of the wingless

(Wnt) signaling pathway, whose ligand is R-spondin 1, as an active stem cell marker was by

Barker et al. in 2007.16 The research team looked for specific Wnt target genes and found Lgr5 to be a prominent marker specifically located at the base of the crypt. Of importance to this project, Lgr5 stem cells were also found to express IGF-1R and EGF receptor (EGFR/ErbB1), two downstream intermediaries of GLP-2, as described in section 1.3.17–19

Quiescent stem cells, also known as +4 stem cells due to their position in the intestinal crypt at the +4 position, have first been identified by Potten in 1977.20 Potten irradiated mice and found that the crypt architecture remained despite the cells at the base of the crypt going through apoptosis, whereas the surviving cells resided directly above these cells, at the +4 position. These

+4 cells are dormant stem cells that can be activated upon injury to the intestine. Once injury occurs, these stem cells will begin to divide to replace the missing or non-functioning Lgr5 cells.

The cells will drop down to the base of the crypt and will then begin to proliferate to replace damaged epithelial cells.21,22 These cells are marked by HopX, Tert, and Sox9. Both have been shown to have limited overlap with Lgr5 and were found to mark slowly cycling cells.22–25

Organoids are mini organ systems that can be grown in vitro and contain structurally accurate architecture of the organ from which they are derived. They have a heterogeneous

4 population of cells that mirrors the organ and can secrete and function normally. Intestinal organoids are composed of the intestinal epithelium alone, without its supportive tissue, and are normally derived from the Lgr5 stem cells and are grown in several growth factors, among them

R-spondin-1 and EGF. Intestinal organoids typically contain all types of intestinal epithelial cells that are found in a normal intestine, including stem cells, transit amplifying (TA) cells, enterocytes, goblet, enteroendocrine and Paneth cells. Organoids have been used as an in vitro model to study gut proliferation and cell maturation.26 A study by Van Landeghem et al.27 has used organoids measure intestinal growth in response to various growth factors. The research group reported that while IGF-1 does not have impact on the growth on organoids derived from isolated Lgr5 stem cells, it promotes the growth of organoids derived from stem cells marked with high Sox9, a marker of quiescent stem cells. However, this was not true in vivo, where IGF-

1 promoted the proliferation of the active stem cell population that are marked by low Sox9 expression, thus showing that IGF-1 has a differential pathway to activating proliferation in different stem cell pools. The Clevers group has also used organoids to investigate the effects of

EGF in proliferation of the gut. They have shown that abolition of EGFR signaling stops proliferation and organoids enter a quiescent period and can be awakened by reintroduction of

EGF into the media.28 Organoids have proven to be an effective in vitro tool to investigate cellular and signaling mechanisms in a heterogeneous cellular environment.

1.3 Glucagon Like Peptide 2

1.3.1 Structure, Expression and Secretion

GLP-2 is a peptide hormone 33-amino acids in length that is secreted from the intestinal

L cell in response to nutrient intake in order to maximize absorption.29 GLP-2 is synthesized from the proglucagon gene, a highly conserved gene across species with homology of 88% between humans and rats.30 It is transcribed in the α cells of the pancreas and the L cell of the intestine, and to a lower degree in the brain.31–33 It is found on chromosome 2 in the human genome and contains 6 exons, with exon 5 encoding for GLP-2.30,34 In the L cell, the proglucagon gene is transcribed and then translated before being modified by prohormone convertase 1/3, which cleaves the protein into GLP-1, GLP-2, oxyntomodulin and glicentin. In contrast, in the α cells proglucagon is modified by the prohormone convertase 2 to produce glucagon, major proglucagon fragment and glicentin-related pancreatic peptide in the pancreas.35,36

GLP-2 is co-secreted with GLP-1 in a biphasic manner from the L cell, with an acute increase at 30-45 minutes for carbohydrates and 45-60 minutes for fat, and a prolonged increase at 90-120 minutes for carbohydrates and 150 minutes for fat.37,38 The first peak in GLP-2 secretion occurs in response to signaling from the vagus nerve during nutrient intake,39,40 while the second peak occurs in response to direct stimulation of the L cell by the ingested nutrients.41–

1.3.2 Clearance

GLP-2 has a half-life of approximately 7 minutes under normal physiological conditions.

It is rapidly cleared by the kidneys as well as cleaved by dipeptidyl peptidase-4 (DPP-4) at alanine at position 2 of the peptide on the N-terminal side, producing GLP-2(3-33).45,46 DPP-4 is both free-circulating in the blood as well as membrane-bound in endothelial cells, including in the kidney. GLP-2(3-33) is a partial agonist that can activate the GLP-2R at high concentrations.45,47 The partial agonist has been shown to block regeneration in the intestinal epithelium in refed mice, reducing proliferation, and increasing apoptosis.47 DPP-4 is more abundant in rats versus mice, rendering native GLP-2 less potent in rats. Thus, DPP-4 deficient rats had a much higher intestinal response to GLP-2.46

Due to its short half-life, a long-acting human analog has been produced, where glycine replaces alanine at position 2, rendering DPP-4 inactive. This analog, hGly2-GLP-2, is currently used in research and as a therapeutic for patients with short bowel syndrome.49

1.3.3 Effects on the Gastrointestinal Tract

The effect of an unknown proglucagon-derived peptide on the intestine was first reported by Gleeson et al and Stevens et al.50,51 Patients were reported to present with L cell derived tumours expressing glucagon, with accompanying symptoms of enlarged intestine and villi, among others. However, the symptoms were not attributed to GLP-2 until much later, in a paper published by Drucker et al. in 1996.1 It was reported in the paper that mice with engrafted proglucagon-producing tumours had larger intestines compared to the sham controls. The authors

7 then injected the various products of proglucagon into mice to see which peptide induced the observed intestinal growth and found GLP-2 to produce the most significant growth effect. In subsequent years, GLP-2 has been shown to increase proliferation, decrease apoptosis, increase blood flow to the intestine, and increase tight junctions and nutrient absorption.2,3,6,8,29,52–57 The

Brubaker lab has previously shown that GLP-2 increases proliferation in the intestinal crypt, particularly in the TA zone.2,3,58 Furthermore, the Drucker lab has shown an increase in proliferation in the stem cell zone in mice treated acutely with GLP-2,8 and Jeppessen showed proliferation occurring in the jejunum of patients with short bowel.49 GLP-2 has also been shown to decrease intestinal epithelial cell apoptosis in control mice59, total parenteral nutrition fed mice60 and piglets61.

GLP-2 also affects blood flow to the intestine via the nitric oxide pathway. It has been shown in the porcine model that acute administration of GLP-2 increases superior mesenteric arterial blood flow, which supplies the intestine with fresh blood.6 Increased blood flow is also observed in rats and humans in response to GLP-2 administration.53

It has previously been reported by our lab as well as others that GLP-2 increases tight junctions in the intestine in mice. Treatment with GLP-2 decreases membrane permeability to both ions and macromolecules62, as well as decreases in permeability to bacteria-derived toxins63. Treatment with GLP-2 was also reported to increase the expression of claudin-3 and -7, two proteins that are important components of the tight junction complex.52 Finally, nutrient absorption also reportedly increases in mice, rats, and hamsters with GLP-2. Rodents treated with GLP-2 have increased leucine, glycine, and galactose absorption.29,54 Increased glucose transport in response to GLP-2, but not GLP-1, treatment in rats was also reported56 , and this increase was later attributed to an increase in sodium-dependent glucose cotransporter-1 (SGLT-

1), particularly after 60 to 120 minutes of GLP-2 infusion.55 It has also been shown in hamsters that GLP-2 increases the levels of apolipoprotein B48 in the blood, a component of a lipid- carrying particle, thus confirming the increase in fat absorption.29,57 Finally, It has been shown in patients with short-bowel syndrome that GLP-2 administration increased carbohydrate absorption.64

Physiologically, GLP-2 has no significant role in intestinal health.65 However, it has been shown that it is important for intestinal adaptation following fasting as shown in mice treated with GLP-2(3-33). GLP-2 receptor (GLP-2R) knockout mice are also not able to recover their atrophied intestine the same way as control mice following refeeding after 24 hour fast47,66

1.4 GLP-2 Receptor

1.4.1 Discovery and Location

The GLP-2 receptor (GLP-2R) was first been identified in 1999 by Munroe et al.67 The authors cloned the receptor from a library of targets taken from rat hypothalamus and duodenum/jejunum. The GLP-2R sequence was revealed to have a 7-transmembrane structure typical of the class B G protein-coupled receptor family. The receptor was found to be highly conserved among species, with over 81% homology between human and rat, and did not demonstrate any alternative splicing. Munroe et al.67 found it to be located along the gastrointestinal tract, including the stomach, duodenum, jejunum, ileum and colon. The group also found low expression of the receptor in the kidney, lung, muscle, liver and spleen. Later studies by Yusta et al.4,68 has disputed the localization of the receptor in most tissues except the

9 digestive tract. Interestingly, GLP-2R was found to be expressed by HeLa cells, which are derived from a cervical tumour.69

Later studies attempted to localize the receptor to specific intestinal cell types. GLP-2R is not expressed on the majority of epithelial cells of the intestine, where the actions of GLP-2 are exerted. Moreover, it is expressed at extremely low levels in the intestine.4,67 GLP-2R however, is found on three cell types in the intestine, the enteroendocrine cells, enteric neurons, and subepithelial myofibrolasts. First, GLP-2R was also found to be expressed by enteroendocrine cells by Yusta et al.4, where it is expressed more highly in the jejunum in human intestinal sections using antisera was developed to detect the receptor. The GLP-2R staining was co- localized with staining for chromogranin, a marker for enteroendocrine cells. The localization of the receptor in the enteric neurons was first reported by in situ hybridization of subepithelial tissue of the intestine. The location of the receptor in the neuronal cells was confirmed by triple labelling of the receptor and the neuronal marker β-tubulin III.5 Another study confirmed the presence of the receptor in the myenteric plexus in the porcine model.6 Lastly, the location of

GLP-2R in the subepithelial myofibroblasts in the intestinal subepithelial myofibroblast was first discovered by immunohistochemistry and in situ hybridization of the intestine of rat, mouse, human and marmoset. The staining was found directly below the enterocytes, in cells that were identified to be the subepithelial myofibrolasts. The most intense staining was found to be in the proximal intestine.7

1.4.2 Signaling

G protein-coupled receptors signal through one main signaling pathway, cyclic-adenosine monophosphate (cAMP). GLP-2R signaling begins when GLP-2 binds to the receptor on the N- terminal side, which is required for the signaling action.70 The receptor then activates its GαS subunit, which dissociates from the β and ɣ subunits, which goes on to activate adenylyl cyclase, protein kinase A (PKA), Rap/Raf, and the MEK1/2 Erk1/2 pathway to increase cAMP levels in the cell.71 The evidence for downstream GLP-2R signaling pathways varies between cell types and perhaps also depends on whether the receptor is native to that cell type. When the GLP-2 receptor was first cloned and transfected into COS cells, which are fibroblasts derived from monkey kidney tissue, treatment of the cells with GLP-2 markedly increased cAMP levels in these cells.67 Both Yusta et al.72 and Shin et al.47 also confirmed the increase in cAMP levels in baby hamster kidney cells transfected with the rat and mouse GLP-2R when treated with GLP-2.

However, they noted that it was not coupled to proliferation. Three studies by the Brubaker lab have confirmed the increase in cAMP in response to GLP-2 binding in natively GLP-2R- expressing cultures. Anini et al.73 has shown that cAMP levels increase in muscle strips containing native GLP-2R treated with GLP-2, an increase that was not seen in muscle strips lacking functional PI3-Kɣ. Furthermore, in fetal rat intestinal cultures and isolated rat epithelial cells in which GLP-2R is also natively expressed, GLP-2 treatment also elicited an increase in cAMP production.74,75

There, however, have been other reports that GLP-2 can signal through other pathways.

In HeLa cells expressing native GLP-2R there was no discernible increase in cAMP, but pERK1/2 levels did increase.69 Furthermore, in intestinal subepithelial myofibroblasts and enteric neurons, GLP-2 binding increases pAKT/tAKT, but not cAMP, levels in the cultures. In

11 the subepithelial myofibroblasts this signaling pathway leads to increased expression and release of IGF-1 from in cultured cells.76–78

1.5 Intestinal EGF

1.5.1 Structure, Expression and Secretion

Epidermal growth factor (EGF) is a growth hormone that affects many tissues in the body, promoting proliferation and differentiation of stem cells and reducing apoptosis. 79,80 EGF was first discovered by Stanley Cohen in 1962 when he injected newborn mice with an extract from submaxillary glands. He recorded an accelerated development in these newborn mice. The active ingredient in the extract was then isolated and then reinjected into mice to verify it was the correct compound.81 The peptide, EGF, was later confirmed to have 53 amino acids, to be a single peptide chain, and to have six cysteine residues that create three disulfide bonds.82

EGF is encoded by EGF gene, which located on chromosome 4 of the human genome. It is most highly expressed in the kidney, followed by the pancreas83, as well as mainly secretory glands, such as mammary salivary glands.84 EGF is then secreted into fluids such as milk and saliva, and travels throughout the body to bind to its receptor. EGF is also secreted from Paneth cells, goblet cells and Brunner’s duodenal glands.13,85,86

1.5.2 Effects of EGF on the Gut

The importance of EGF to the gut was first reported by Miettinen et al.87 when they observed that epidermal growth factor receptor (EGFR) knockout animals had compromised intestines. Further investigation by Duh et al.88 has shown that by treating organ cultures with

EGF, small intestinal growth increases and intestinal lining thickens. It is now recognized that inhibition of EGF signaling can render the intestine vulnerable. For example, treatment with

EGFR inhibitors such as gefitinib can cause gastrointestinal side effects which include diarrhea.89 Conversely, studies by the Polk lab have shown that EGF has a protective effect on the damaged intestine. EGF promotes cell migration in vitro in colonic cells following wounding.90 Furthermore, a recent study has shown that EGF is an effective treatment for colitis.

Mice were induced with colitis using dextrate sulfate sodium and were treated with either EGF or an EGFR inhibitor. Mice treated with EGF were able to recover their intestinal architecture while mice receiving the inhibitor demonstrated exasperated damage to the intestine.91 EGF treatment also reduces the severity of necrotizing enterocolitis (NEC) in rat pups. Newborn rats were removed from their mothers and stressed to induce NEC and were then given milk supplemented with EGF. Rats given EGF along with their milk had a much better disease outcome.92 These protective effects are a result of increased proliferation in the intestine demonstrated using both in vivo and in vitro models. Yusta et al.8 have shown that an acute treatment with EGF to mice markedly increases proliferation in the stem cell zone of the jejunum, and others have shown it to promote proliferation in intestinal organoids28 and intestinal epithelial cells in culture, where EGF was a more effective proliferative agent than IGF-19. EGF also enhances IGF-1-mediated proliferation as will further be discussed in section 1.7.

EGF also reduces apoptosis in damaged intestine as reported by Feng and Teitelbaum. In their paper they showed that EGF reduces apoptosis induced by tumour necrosis factor (TNF)-α in mice on total parenteral nutrition (TPN), which normally leads to degeneration of the intestine.93 Additionally, EGF prevents epithelial cell shedding in intestinal organoids as reported by Miguel et al.94 Organoids were treated with EGF and were observed to have lower cell shedding compared to their vehicle control. It was also observed in the intestinal epithelial cell

(IEC)-6 cell line that the reduction in shedding in response to EGF treatment is due to activation of the MEK-ERK pathway.

1.5.3 EGF Receptors and Signaling

EGF has one main receptor it binds to: EGFR, also known as ErbB1. EGFR is a tyrosine kinase transmembrane receptor with an intracellular and extracellular domain.95,96 The receptor is a monomer that dimerizes to another EGFR unit or an ErbB2 unit upon ligand binding on the extracellular domain.97 ErbB2 is not known to have any ligands of its own.98 Binding leads to autophosphorylation of tyrosine residues in the intracellular domain,99 and eventually leads to cell proliferation, survival, and differentiation.

Although EGFR signal transduction involves a network of signaling molecules, there are two main pathways: Akt and Erk1/2. EGFR signaling cascade starts at the phosphorylation of the tyrosine residues, which in turn phosphorylate SRC-homology 2 (SH2) subunit, on which PI3-K docks. PI3-K is made of two subunits: the inhibitory subunit p85 and the catalytic subunit p110.

P85 is released from p110, which then converts PIP2 to PIP3. PIP3 then phosphorylates AKT, which then goes on to affect many proteins, including activation of mTOR, and subsequently

S6K, leading to proliferation.100 Erk1/2 signaling starts by receptor phosphorylation of SHC, which then phosphorylates in sequence GRB2, SOS, and RAS. RAS then activates RAF which goes on to activate MEK1/2 which activates ERK1/2. ERK1/2 translocates to the nucleus to induce cell proliferation survival.101–103

1.5.4 EGF and GLP-2

EGF has long been associated with GLP-2 activity in the gut. The Drucker group has shown GLP-2 dependence on EGF signaling. In a study published in 2009, Yusta et al.8 reported that a single acute treatment with GLP-2 increased ErbB ligand transcripts, such as epiregulin, amphiregulin and HB-EGF, but not EGF. This effect was non-existent or diminished in GLP-2R knockout and EGFR mutant mice. Furthermore, chronic treatment with GLP-2 in the presence of an EGFR inhibitor blocked crypt-villus height growth in these mice. Bahrami et al.66 have also shown that GLP-2R knockout mice were not able to properly rescue their gut from atrophy following refeeding, but EGF administration in these mice restored the intestine. They also reported that refed wild-type mice that were given an EGFR inhibitor had reduced proliferation in the gut.

The interaction between EGF and GLP-2 was also investigated in mice receiving TPN. In these mice, GLP-2 treatment increased EGF mRNA levels in the intestinal mucosa, while EGF treatment increased GLP-2R levels in the mucosa. EGF and GLP-2 alone were also able to increase proliferation and decrease apoptosis in these TPN mice. Treatment with either of these growth factors with the inhibitor of the other (that is, co-treatment with EGF and GLP-2 (3-33) or treatment with GLP-2 in combination with an EGFR inhibitor) blocked the growth effects seen in response to EGF and GLP-2. Respectively, this was confirmed in intestinal epithelial

EGFR knockout mice receiving GLP-2, wherein no growth effect was seen in the intestine.60 A porcine model of bowel resection, mimicking neonatal short-bowel syndrome, was also used to investigate the effects of EGF and GLP-2. While EGF treatment alone had little effect on intestinal weight, GLP-2 treatment with and without EGF did have a significant effect on intestinal weight. Crypt-villus height in the jejunum and ileum also increased in response to both

GLP-2 and combination treatment.104 A paper by Kitchen et al.105 has reported the effects of combination treatment with EGF and GLP-2 in parenteral nutrition (PN). They found that while

GLP-2 treatment increased small intestinal weight, a combination treatment with EGF and GLP-

2 was more effective than GLP-2 alone. Crypt-villus height and proliferation were also more significant in the combination treatment compared to GLP-2 alone in the duodenum, suggesting that the combined effect of GLP-2 and EGF together was more effective in promoting gut growth.

Finally, a study investigating the EGFR inhibitors gefitinib and erlotinib, which are used in cancer treatment was also investigated in mice. These inhibitors are known to worsen intestinal outcomes, so mice were treated with these inhibitors along with GLP-2. Mice treated with gefitinib or erlotinib had decreased intestinal weight, but treatment with GLP-2 along with these inhibitors partially rescued intestinal weight, albeit not to the same level as control mice receiving GLP-2. This suggests that EGFR is perhaps not required, or not essential, for GLP-2 signaling.106,107

1.6 Intestinal IGF-1

1.6.1 Structure, Expression and Secretion

IGF-1 is a single-chain peptide 70 amino acids in length and is structurally similar to insulin on the A and B domains, but not C domain, while also having an additional D domain.108

IGF-1 is also structurally similar to IGF-2. The liver is the main source for IGF-1 in the circulation, however, IGF-1 is produced by other tissues as well, including the intestine. Hepatic

IGF-1 is secreted in response to growth hormone (GH) release from the pituitary109 and is age-dependent, with higher expression in the fetal stage.110 In the intestine, GLP-2 stimulates the release of IGF-1 from subepithelial myofibroblasts.76,78 Circulating IGF-1 is bound by IGF-1 binding proteins (IGFBPs) to increase its half-life. The most highly circulating IGFBP is IGFBP-

3 and it is mostly bound to IGF-1111, but in the intestine IGF-1 is mostly bound to IGFBP-3/5112 and IGFBP-410,113. When IGF-1 is not bound to IGFBP, it will be cleared by the kidneys.114

1.6.2 Effects of IGF-1 on the Gut

IGF-1 has an important role in promoting growth in the intestine. Normal mice treated with IGF-1 show modest intestinal growth in a study published by Dubé et al.2 Rowland et al.3 showed that IGF-1 administration to control mice increased proliferation specifically in the TA zone. The Lund group has also shown that IGF-1 is able to increase crypt-villus height, as well as the number of crypts, and proliferation in mice.27 Similarly, mice were reported to respond similarly to GLP-2 and IGF-1 treatments by increase in jejunal mass, protein content, and proliferation. Crypt-villus height was dramatically higher in these two treatment groups compared to the control group.112 Interestingly, in vitro, particularly in IEC-6 cells, IGF-1 has a reduced proliferative effect compared to EGF.9 However, the Brubaker lab reported that increasing concentrations of IGF-1 added to these cells increases their proliferation in a dose- dependent manner.10

IGF-1 has been suggested as a management therapy for short bowel syndrome. A study by Gillingham et al.115 reported that rats that went through bowel resection and were on TPN were able to transition to enteral nutrition after treatment with IGF-1. Furthermore, rats that went through intestinal resection and were receiving IGF-1 had larger jejunal sections. Another paper

17 reported that resected mice overexpressing IGF-1 in the intestinal smooth muscle experienced increased small intestinal weight and crypt-villus height, as well as proliferation.116

1.6.3 IGF-1 receptors and signaling

IGF-1 binds to the IGF-1 receptor (IGF-1R), with some low affinity binding to the insulin receptor. IGF-1R is a tyrosine kinase receptor that is composed of two α and two β chains held together by disulfide bonds. The β chains contain a transmembrane sequence and have both intra- and extracellular domains, while the α chain is found on the cell surface.117–120 The receptor has several signaling pathways, the two main ones, similar to EGFR, being Akt and

Erk1/2. Signaling cascade starts by IGF-1 binding to the extracellular α chains. The receptor is then autophosphorylated, which then in turn phosphorylates the IRS1/IRS2 complex to which

PI3-K is docked. Alternatively, SHC phosphorylates GRB and SOS. From then, the activation cascade is similar to that of the EGFR to activate Akt and Erk1/2 respectively.121 These processes lead to cell proliferation, survival, and differentiation.

1.6.4 IGF-1 and GLP-2

The relationship between IGF-1 and GLP-2 has long been established by the Brubaker lab. Dubé et al2. have shown that IGF-1 is an important component in the GLP-2 signaling pathway in the intestine. The first paper utilized IGF-1 knockout mice to assess the ability of

GLP-2 to induce intestinal growth in vivo in the absence of its downstream growth factor. It was found that IGF-1 knockout mice did not respond to GLP-2 treatment, albeit there was some response when treated with a higher dose and when intestinal weight was normalized to body

18 weight. IGF-1 knockout mice were able to increase intestinal growth when given exogenous

IGF-1 or Rspondin-1, another gut growth factor. Subsequently, both knockout and control mice were treated acutely with either vehicle, IGF-1 or GLP-2 acutely to look at β-catenin signaling, which is linked to increased proliferation.122 Both IGF-1 and GLP-2 treatments increased nuclear

β-catenin localization in the crypt cells of control mice. However, when IGF-1R was inhibited, or when IGF-1 knockout mice were treated with GLP-2, β-catenin levels in the nucleus were not increased, thus suggesting that IGF-1 is imperative for GLP-2 signaling.75

The importance of IGF-1 was also studied in IGF-1R knockout mice. Rowland et al.3 used intestinal epithelial specific (IE) IGF-1R knockout mice to assess whether GLP-2-mediated intestinal growth was dependent on IE-IGF-1R signaling. Mice were treated acutely with GLP-2 and as seen with the IGF-1 knockout mice, nuclear β-catenin levels in the intestine were not increased in the IE-IGF-1R knockout mice. Chronic treatment with GLP-2 increased crypt-villus height and crypt cell proliferation in both control and knockout mice. However, knockout mice experienced a reduced response to GLP-2. Furthermore, IE-IGF-1R knockout mice that were fasted for 24 hours and then refed did not have the same intestinal regeneration as controls.

The connection between GLP-2 treatment and an increase in the production of IGF-1 was established in two studies done in vitro in intestinal subepithelial myofibroblast cultures. Cells were treated with GLP-2 in a dose- and time-dependent manner. It was found that 2-hour treatment with GLP-2 had the highest increase of IGF-1 levels in culture. Inhibition of PI3-K in these cells prevented an increase in IGF-1 mRNA in response to GLP-2, thus proving that GLP-

2-dependent IGF-1 production is mediated through the Akt pathway.76 Shawe-Taylor et al.78 have shown that GLP-2 promotes the degradation of IGFBPs in media of myofibroblasts, particularly IGFBP-4, suggesting an increase in released IGF-1 in the media. IGF-1 is also

involved in the increase of tight junctions in response to GLP-2 treatment. Dong et al.52 used IE-

IGF-1R knockout mice to show that there was a decrease in jejunal resistance and in tight junction protein expression. Furthermore, knockout animals with induced enteritis that were treated with GLP-2 did not experience the same improvement in barrier function.

Austin et al.113 also showed the importance of IGFBP-4 in the function of GLP-2.

IGFBP-4 knockout mice had a reduced growth response to GLP-2, where control mice responded to GLP-2 treatment by increased intestinal growth. Furthermore, both crypt-villus height and proliferation did not significantly increase in the knockout animals in response to

GLP-2 treatment. Finally, Murali et al.112 have shown that IGFBP-3/-5 knockout does not affect the growth response to GLP-2 treatment. Interestingly, treatment with GLP-2 decreased jejunal

IGF-1 mRNA levels in both wild-type and knockout mice, while others have either not seen any change in IGF-1 mRNA8, or found it was increased2.

1.7 The Known Interactions Between EGF and IGF-1

The interaction between EGF and IGF-1 has been noted in several, non-intestinal, cell lines. It has been shown in COS-7 cells, cells derived from monkey kidney tissue, that IGF-1R stimulates the transphosphorylation of EGFR, thus increasing the activity of EGFR in these cells.

It has been suggested that this is done through IGF-1R activating heparin binding (HB)-EGF, which then goes on to bind EGFR.123 In esophageal epithelial cells, co-administration of EGF and IGF-1 increases cell proliferation in a synergistic manner.124 Moreover, a combination treatment of EGF with IGF-1 or IGF-2 has an additive effect on proliferation in fetal adrenal cortical cells.125

EGF-IGF-1 interactions were also observed in intestinal cells by both the Brubaker and

Lund labs. Austin et al.10 have shown that pre-treatment of IEC-6 cells with EGF before a treatment with IGF-1, increases proliferation synergistically. Simmons et al.9 have shown in the same cell line that proliferation increases dramatically following a combination treatment with both EGF and IGF-1 together. Furthermore, they have shown that while IGF-1 treatment decreases IGF-1R transcript levels, EGF maintains them at basal level in the presence of IGF-1 in the media. These set of data create a clear relationship between EGF and IGF-1 that can be investigated further to elucidate their potential interaction with GLP-2.

1.8 Hypothesis and Specific Aims

Both EGF and IGF-1 have been shown to be an important part of GLP-2-mediated intestinal growth. Clear interactions between EGF and GLP-2 have been established by the Drucker lab while the Brubaker, Lund and Ney groups have established a relationship between IGF-1 and

GLP-2. Several research groups have also established a relationship between EGF and IGF-1 in vitro. However, it has yet been shown whether GLP-2, EGF, and IGF-1 interplay to produce their effects on intestinal proliferation. Therefore, my hypothesis was that pre- and/or co- treatment with EGF enhances the IGF-1R dependent intestinal proliferative effects of GLP-2 in murine models. The aims of this hypothesis were to: (a) determine the optimal timepoint and combination of EGF and GLP-2 treatments in vivo, (b) use intestinal epithelial IGF-1R knockout mice to determine the interactions between EGF, IGF-1R and GLP-2, and (c) use an in vitro organoid model to determine a direct interaction between IGF-1 and EGF, all with respect to crypt cell proliferation in the jejunum.

Materials and Methods 2.1 In Vivo Studies

All animal protocols were approved by the University of Toronto Animal Care

Committee. Animals were housed in a 14/10-hour light/dark cycle room at the University of

Toronto. For the EGF and GLP-2 combination optimization study 6-8-week-old male and female

C57Bl/6 mice (Charles River, St. Constant QC) were divided into eight treatment groups, consisting of 8-10 animals each. Mice were treated daily for 10 days with a subcutaneous injection of phosphate-buffered-saline (PBS), 0.2µg/kg h(Gly2)GLP-2 (American Peptide company; Sunnyvale, CA) and/or 0.4 µg/kg recombinant mEGF (per dose) (R&D Systems,

Minneapolis, MN). Pre-EGF or vehicle were given at -3 hours, co-EGF and/or GLP-2 or vehicle were given at 0 hours as previously described.8,58 On day 11, mice received their final treatments

(as above) plus an EdU (100mg/kg ip, Invitrogen, Eugene, OR) injection 2 hour later and were sacrificed 1 hour after that by anesthetization with isoflurane followed by sacrifice with a heart puncture. The intestine was removed and cleared of luminal content to measure small and large intestinal weight, and small and large intestinal length under constant tension. Body weight was measured prior to sacrifice. Two cm sections of the jejunum were fixed in 10% neutral-buffered formalin at room temperature before being stored in ethanol at 4 °C. Samples were then embedded paraffin and sectioned (Toronto General Hospital pathology lab). 2cm Sections of the jejunum were collected for mRNA, histological and proliferation analyses. Sections of the duodenum, ileum and colon were also collected for storage.

For IGF-1R knockout studies, age and sex-matched IE-IGF-1R KO mice with C57BL/6J background were generated by crossing villin-cre ERT2/+ (gift of Dr. S Robine) with Igf1rfl/fl mice

(gift of Dr. M. Holzenberger via Dr. R. N. Kulkarni). Mice were genotyped as previously described3. Briefly, Igf1rfl/fl mice were identified by the floxed allele while the KO mice were identified by an additional Cre allele using oligonucleotides 5’-

ATCTTGGAGTGGTTGGGTCTGTTT-3’ and 5’-ATGAATGCTGGTGAGGGTTGTCTT-3’, which produced a 327-bp fragment of the floxed allele, and the primers 5’-

CCTGGAAAATGCTTCTGTCCG-3’ and 5’-CAGGGTGTTATAAGCAATCCCC-3’ which produced a 390-bp fragment from the Cre coding region.

To induce the knockout at exon 3 (the ligand binding domain) of IGF-1R, mice were either treated with tamoxifen (100µl ip, 10mg/mL) in oil (vehicle) for 5 days.3 Nuclear translocation of Cre recombinase was induced by the tamoxifen treatment. Under the villin promoter, cre is known to be expressed in all cells of the intestinal epithelium,126 including the the actively proliferating intestinal stem cells.17–19 Additional controls were used: fl/fl and cre mice with tamoxifen or vehicle, as well as cre/fl mice with vehicle. Mice were then treated chronically with either vehicle, 0.2µg/kg h(Gly2)GLP-2, and/or 0.4 µg/kg recombinant mEGF per dose bid followed by a final treatment and an EdU injection 1 hour before sacrifice at day 11 by anesthetization with isoflurane followed by sacrifice with a heart puncture. Measurements and intestinal samples were then collected as indicated above.

2.2 In Vitro Studies

Organoids were produced from the jejunum of 5-8-week-old mice as previously described.127 Briefly, the small intestine was extracted and 10cm of jejunum was collected and placed in cold PBS. The jejunum was then cleaned of fat and cut longitudinally to dispose of feces and villi mechanically. The intestine was then cut into 2-4mm pieces and was further

23 cleaned mechanically by pipetting up and down in cold PBS. The intestinal pieces were incubated in 2mM EDTA (BioShop, Burlington, ON, Canada) in cold PBS for 30 minutes while shaking at a 1.5 speed on a platform shaker to extract the crypts. Crypts were then mechanically extracted by pipetting up and down in cold PBS. The isolated crypts were then spun down in

40ml PBS and 4ml fetal bovine serum (FBS) (Gibco, Billings, MT) and were embedded in 50µL

Matrigel basement membrane (Corning, Corning, NY).

Advanced DMEM/F12 media was supplemented with GlutaMAX 100x, Hepes 1M and

Pen Strep as well as B27 50x, N2 100x (all from Gibco, Billings, MT), 10% R-spondin (from

HEK293 cells donated by Dr. Catherine O’Brien), noggin (100 ng/µl) and EGF (50 ng/µl, both from PeproTech, Montreal, QCanada) growth factors. Media was also supplemented with 10mM

Y-27632 dihydrocloride and 500nMN-acetyl-L-cysteine (both from Sigma-Aldrich, St. Louis,

MO). Media was replaced every 3-4 days and organoids were passaged every 7 days.

To optimize the time window for EdU treatment, organoids were replated into 8-chamber slides and treated with EdU for 20, 40, 60 and 120 minutes. The organoids were then washed with PBS and fixed with 4% PFA. For the pilot EGF withdrawal study, organoids from a 6 week- old male fl/fl mouse were replated into 8-well chamber slides (Falcon, Corning, NY) and EGF was withdrawn a day later for 4 days and then restored for either 1 or 2 days.28 As control, one group of organoids did not experience EGF withdrawal and one group of organoids had EGF withdrawal without restoration. For the third experiment, one female and male IGF-1R KO mouse were used along with vehicle treated female villin-cre ERT2/+ Igf1rfl/fl, male and female

Igf1rfl/fl mice for controls. Cre mouse-derived organoids were not used due to inability for them to survive passaging. Organoids were divided into 6 treatment groups: no EGF withdrawal with and without IGF-1 (100ng/mL10, Abcam, Cambridge, UK), EGF withdrawal and restoration with

24 and without IGF-1, and EGF withdrawal without restoration with and without IGF-1. IGF-1 was given at the same time as the restored EGF and organoids were sacrificed one day later. For all organoid experiments following the EdU optimization experiment, organoids were treated with

EdU for 20 minutes before Collection. Collection was done by removing the media and washing the organoids once with room temperature PBS. Organoids were then fixed in 4% PFA at room temperature for 30 minutes before continuing with the EdU staining protocol.

2.3 RNA Extraction and Analysis

RNA was extracted from mouse jejunal mucosal scrapes. RNA extracted from organoids was done by harvesting organoids after first or second passage. Organoids were harvested by dissolving the Matrigel and spinning down to extract the organoids from the media. RNA was then isolated using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). RNA was then converted to cDNA using 5x All-in-One RT Mastermix (Applied Biological Materials Inc., Richmond, BC,

Canada). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed for RNA analysis using the Taqman Gene Expression Assay (Thermo Fisher,

Weltham, MA). Primers for proglucagon, GLP-2R, IGF-1, IGF-1R, EGF, ErbB1, ErbB2, Lgr5,

Ki67, Bax, and Bcl-2 with the internal control, 18S2 for the in vivo experiments and H3a for the in vitro experiments, were used in a 384-well plate (primers list provided in Table 2.1, Thermo-

Fisher, Weltham, MA)

Primer ID Number

18S Hs99999901_s1

H3a Mm01612808_g1

Gcg Mm00801712_m1

GLP-2R Mm_01329477_m1

IGF-1 Mm00439559_m1

IGF-1R Mm_00802837_m1

EGF Mm00438696_m1

EGFR Mm01187858_m1

ErbB2 Mm00658541_m1

Bax Mm00432051_m1

Bcl-2 Mm00477631_m1

Lgr5 Mm01251805_m1

Ki67 Mm01278617_m1

Table 2.1: List of primers used for experiment

2.4 Microscopy

Crypt depth and villus height were measured on hematoxylin and eosin-stained slides prepared by the University Health Network Pathology Department (Toronto, Canada) using the

AxioVision software by Zeiss. At least 20 crypts and 20 villi were measured for each mouse in a blinded fashion.

For in vivo EdU measurements, AxioVision software by Zeiss was used. For organoid imaging, 90 Nikon Eclipse T Swept Field microscope was used and imaging processing was done with the NISA software.

2.5 Proliferation Analysis

The Click-iT EdU Alexa Fluor 488 Imaging Kit (Invitrogen, Eugene, OR) was used to stain for EdU, using Vectashied Antifade Mounting Medium with DAPI (Vector Laboratories) for detection of nuclei, and DAPI alone used for the negative control. Briefly, sections were washed with 3% bovine serum albumin (BSA) in PBS and then were permeablized with 0.5%

Triton X-100 (both from Sigma-Aldrich, St. Louis, MO) in PBS for 20 minutes. Samples were then washed in 3% BSA with PBS and were incubated for 20 minutes with the reaction mixture.

Samples were washed again with 3% BSA in PBS. Mounting medium was then put on and the slides coverslipped. 20 crypts were then selected per mouse and 20 cells were counted on the right side of the crypt, starting at position 1 in the crypt base. Measurements were done in a blinded fashion.

To measure proliferation in the organoids, the Click-iT EdU Alexa Fluor 488 Imaging Kit was used to stain for EdU. To stain for nuclei, stock concentration of 10mg/mL of DAPI (Roche,

Basel, Switzerland) was diluted to 1:1000 for use and was incubated with the organoids for 20 minutes. Organoids were then washed three times with 3% BSA in PBS and were coverslipped with Vectashied Antifade Mounting Medium without DAPI. Proliferation was then measured n a blinded fashion as percent EdU cells in each crypt. One to four crypts were measured from at least 10 organoids per treatment group.

2.6 Statistics

GraphPad Prism was used for the statistical tests. Significance was determined by 1-way

ANOVA followed by post hoc Sidak’s multiple comparisons test for all in vivo studies and all in vitro studies except for the IGF-1-EGF experiment, which was analyzed by 2-way ANOVA. * indicates significance vs. vehicle or as indicated otherwise, # indicates significance compared to

GLP-2 for the mouse studies. For the organoid withdrawal, * indicates significance vs. the EGF withdrawal group. For the EGF-IGF-1 organoid study, * indicates significance vs. EGF withdrawal group within genotype groups, # indicates significance vs. IGF-1 alone group within genotype groups, $ indicates significance between genotypes.

Results

3.1 Determining the Optimal Timing for EGF-GLP-2 Treatment

To determine the optimal timing for GLP-2 and EGF treatments and elucidate their combined

effect on intestinal proliferation, I treated with one of eight treatments: vehicle, GLP-2 at 0 hours

timepoint, EGF at -3 hours, EGF at 0 hours, EGF at both timepoints, or a combination of GLP-2

with one of the above EGF treatments (Table 3.1).

Treatment Group Group Group Group Group Group Group Group 1 2 3 4 5 6 7 8 Pre- - - + + - - + + Treatment with EGF @ t=-3 hr Co- - - - - + + + + Treatment with EGF @ t=0hr Treatment - + - + - + - + with GLP2 @ t=0hr

Table 3.1: Treatment groups designed to determine the optimal timing for EGF-GLP-2 treatment

6-8-week-old male and female C57Bl/6 mice treated with vehicle, 0.2µg/g GLP-2, pre- and/or co-EGF at 0.4µg/g per dose or a combination of EGF and GLP-2 doses for 11 days.

3.1.1 A Combination Treatment with EGF and GLP-2 Increases Small Intestinal Weight

Body weight and small intestinal length of all mice were generally uniform across all treatment groups. (Figures 3.1.1A and 3.1.1B) Small intestinal weight increased significantly in the pre-

EGF+co-EGF+GLP-2 group, and was higher than vehicle, GLP-2 alone and its respective EGF treatment alone. Co-EGF+GLP-2 had also a significant effect on small intestinal weight compared to vehicle, but not compared to GLP-2 or its respective EGF treatments. (Figure

3.1.1C) When small intestinal weight was normalized to small intestinal length, the significance for the pre-EGF+co-EGF+GLP-2 treatment group remained (Figure 3.1.1D). However, although the pre-EGF+co-EGF+GLP-2 group remained significant compared to vehicle when small intestinal weight was normalized to body weight, it did not remain significant compared to GLP-

2 alone. (Figure 3.1.1E) Large intestinal weight of both the pre-EGF+co-EGF group and the pre-

EGF+co-EGF+GLP-2 group increased significantly compared to vehicle, while the pre-

EGF+GLP-2 group had a significant increase compared to its EGF control group. (Figure

3.1.1F) Large intestinal length did not change between the groups. (Figure 3.1.1G) When large intestinal weight was normalized to large intestinal length, the pre-EGF+co-EGF+GLP-2 group also became significantly higher compared to the GLP-2 group. (Figure 3.1.1H) Large intestinal weight normalized to body weight did not change in any of the groups except for the pre-

EGF+co-EGF that had a significant growth compared to vehicle. (Figure 3.1.1I)

A Body Weight B Small Intestinal Length C Small Intestinal Weight

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Figure 3.1.1: Combination Treatment with EGF and GLP-2 Increases Small Intestinal Weight

Body, small and large intestinal weights and lengths for 6-8-week-old male and female C57Bl/6 mice treated with vehicle, 0.2µg/g GLP-2, pre- and/or co-EGF at 0.4µg/g per dose or a combination of EGF and GLP-2 doses for 10 days and sacrificed on day 11 as summarized in

Table 3.1. Small intestinal and colonic measurements were taken following clearing of luminal content. (A) body weight, (B) small intestinal length, (C) small intestinal weight, (D) small intestinal weight normalized to small intestinal length, (E) small intestinal weight normalized to body weight, (F) large intestinal weight, (G) large intestinal length, (H) large intestinal weight normalized to large intestinal length, (L) Large intestinal weight normalized to large intestinal length (*p<0.05 **p<0.01 ***p<0.001 as indicated, ## p<0.01 ### p<0.001 compared to GLP-2 treatment group, n=8-10) Data analyzed by 1-way ANOVA.

3.1.2 All Combination Treatments with EGF and GLP-2 Increase Crypt-Villus Height

Crypt-villus height measurements were taken to further elucidate the effect that the treatments had on intestinal growth. GLP-2 and pre-EGF+co-EGF treatment groups increased in crypt depth and villus height compared to vehicle. Interestingly, all combination groups had a significant increase in villus height compared to vehicle, GLP-2 and their respective EGF treatments. Crypt depth increase was most profound in the co-EGF+GLP-2 group, differently from vehicle, GLP-2, and co-EGF alone. (Figure 3.1.2)

Crypt-Villus Height

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C ic P G P G P G P h L -E L -E L -E L e G e G o G o G V r + C + C + P F F + F G G F G -E -E G -E e o -E o r C e C P r + P F G -E re P

Figure 3.1.2: All Combination Treatments with EGF and GLP-2 Increase Crypt-Villus Height

Jejunal crypt depth and villus height measurements for 6-8-week-old male and female C57Bl/6 mice treated with vehicle, 0.2µg/g GLP-2, pre- and/or co-EGF at 0.4µg/g per dose or a combination of EGF and GLP-2 doses for 10 days and sacrificed on day 11 as summarized in

Table 3.1. (*p<0.05 **p<0.01 ***p<0.001, # p<0.05 ## p<0.01 compared to GLP-2 treatment group, n=8-10). Data analyzed by 1-way ANOVA.

3.1.3 GLP-2 Increases Proliferation while EGF Decreases Apoptotic Markers

Proliferation was measured using the EdU assay. Areas under the curve were then quantified and divided into three zones: position 1-3 for the stem cell zone, position 4-7 for the quiescent stem cell zone, and position 8-16 for the TA zone.58 The active and quiescent stem zones did not show a significant increase in proliferation in any of the groups. (Figures 3.1.3A and 3.1.3B)

However, in the TA zone there was a significant increase in proliferation in all the groups receiving GLP-2. (Figure 3.1.3C) However, EGF did not seem to have a proliferative effect as none of the combination groups had a significantly higher proliferation than vehicle alone. As this did not translate into the observed results in the crypt-villus height data, apoptotic markers were looked at using qRT-PCR. The ratio between Bax, an apoptotic marker, and Bcl-2, a counterapoptotic marker, was used to measure apoptosis. It was found that all groups containing co-EGF treatment had a significantly reduced apoptosis compared to GLP-2 treatment alone.

(Figure 3.1.3D)

A Proliferation AUC Position 1-3 B Proliferation AUC Position 4-7 0.3 1.5

0.2 1.0

C C

U U

A A 0.1 0.5

0.0 0.0 le -2 F -2 F -2 F -2 le -2 F -2 F -2 F -2 ic P G P G P G P ic P G P G P G P h L -E L -E L -E L h L -E L -E L -E L e G e G o G o G e G e G o G o G V r + C + C + V r + C + C + P F F + F P F F + F G G F G G G F G -E -E G -E -E -E G -E e o -E o e o -E o r C e C r C e C P r + P r + P F P F G G -E -E re re P P

Proliferation AUC Position 8-16 Bax/Bcl-2 qRT-PCR C *** D *** 2.0 5 *** *

*** **

2 1.5 -

4 l

3 C 1.0 # #

C /

x ###

a A

B ### 2

T 0.5

C D 1 0.0 0 le -2 F -2 F -2 F -2 c P G P G P G P i E E E le -2 F -2 F -2 F -2 h L - L - L - L c G G G e G e G o G o G i P E P E P E P V r + C + C + h L - L - L - L P F F + F e G e G o G o G G G F G V r + C + C + P F F + F -E -E G -E G G F G e o -E o E E E r C e C - - G - P r + e o -E o P F r C e C P r + G P F -E G re -E P re P

Figure 3.1.3: GLP-2 Increases Proliferation while EGF Decreases Apoptosis

Proliferation measurements and qRT-PCR data for jejunal mucosa from 6-8 week old male and female C57Bl/6 mice treated with vehicle, 0.2µg/g GLP-2, pre- and/or co-EGF at 0.4µg/g per dose or a combination of EGF and GLP-2 doses for 10 days and sacrificed on day 11 as summarized in Table 3.1. (A) area under the curve proliferation for positions 1-3, the Lgr5 stem cell zone, (B) area under the curve proliferation for positions 4-7, the +4 stem cell zone, (C) area under the curve proliferation for positions 8-16, the TA zone, (D) Ratio of the ΔΔCt measurements between Bax and Bcl-2 as a measure for apoptosis (*p<0.05 **p<0.01

***p<0.001, # compared to GLP-2 treatment group, n=8-10). Data analyzed by 1-way ANOVA.

3.1.4 Transcripts of Ligands and Their Receptors Did Not Change Between Groups

Transcripts for proglucagon, GLP-2R, IGF-1, IGF-1R, EGF, EGFR and ErbB2 were analyzed via qRT-PCR from the mucosa of the jejunum. Transcripts were normalized to the house- keeping gene 18S. None of the transcripts changed significantly based on treatment. (Figure 3.4)

Gcg Glp2r

6 6

d 4 4

r o

2 N 2

L G

0 G 0 le -2 F -2 F -2 F -2 le -2 F -2 F -2 F -2 ic P G P G P G P ic P G P G P G P h L -E L -E L -E L h L -E L -E L -E L e G e G o G o G e G e G o G o G V r + C + C + V r + C + C + P F F + F P F F + F G G F G G G F G -E -E G -E -E -E G -E e o -E o e o -E o r C e C r C e C P r + P r + P F P F G G -E -E re re P P Igf1 Igf1r

6 6

d d

4 e 4

o o

2 N 2

I G 0 I 0 le -2 F -2 F -2 F -2 le -2 F -2 F -2 F -2 ic P G P G P G P ic P G P G P G P h L -E L -E L -E L h L -E L -E L -E L e G e G o G o G e G e G o G o G V r + C + C + V r + C + C + P F F + F P F F + F G G F G G G F G -E -E G -E -E -E G -E e o -E o e o -E o r C e C r C e C P r + P r + P F P F G G -E -E re re P P Egf Egfr

6 6

d 4 4

r o

o 2 2

r E 0 E 0 le -2 F -2 F -2 F -2 le -2 F -2 F -2 F -2 ic P G P G P G P ic P G P G P G P h L -E L -E L -E L h L -E L -E L -E L e G e G o G o G e G e G o G o G V r + C + C + V r + C + C + P F F + F P F F + F G G F G G G F G -E -E G -E -E -E G -E e o -E o e o -E o r C e C r C e C P r + P r + P F P F G G -E -E re re P P Erbb2

d 4

m r

o 2

b r E 0 le -2 F -2 F -2 F -2 ic P G P G P G P h L -E L -E L -E L e G e G o G o G V r + C + C + P F F + F G G F G -E -E G -E e o -E o r C e C P r + P F G -E re P

Figure 3.1.4: Transcripts of Ligands and Their Receptors Did Not Change Between Groups

qRT-PCR data for jejunal mucosa from 6-8 week old male and female C57Bl/6 mice treated with vehicle, 0.2µg/g GLP-2, pre- and/or co-EGF at 0.4µg/g per dose or a combination of EGF and GLP-2 doses for 10 days and sacrificed on day 11 as summarized in Table 3.1. (n=8-10)

Transcripts are normalized to 18S and measured by ΔΔCt. Data analyzed by 1-way ANOVA.

3.2 Investigating the Relationship Between GLP-2, EGF and IGF-

1R Using IE-IGF-1R-KO Mice

IE-IGF-1R-KO mice were utilized to determine the interactions of EGF, IGF-1R and GLP-2 and their effect on intestinal growth and proliferation. Four groups out of the eight used above were chosen for this experiment: vehicle treatment, GLP-2 treatment at 0 hours timepoint, EGF at both timepoints, and a combination of GLP-2 with the above EGF treatments. These treatment groups were chosen based on the normal mouse data, particularly the data on small intestinal weight both not normalized, and normalized to small intestinal length, which showed that pre-EGF+co-

EGF+GLP-2 provided the largest growth effect in the small intestine.

3.2.1 Validation of the IE-IGF-1R KO in Mice

Cre/flox and flox pups were identified by genotyping for the flox and cre fragments. Flox pups had one band while the cre/flox pups had two bands for cre and flox. (Figure 3.2.1A) Cre/flox mice were treated with tamoxifen for 5 days intraperitonially to induce the knockout. The knockout was then verified by qRT-PCR for exons 2-3 of the IGF-1R in jejunal mucosa. (Figure

3.2.1B)

Cre/Fl Fl

Cre Band (390bp) Flox Band (327bp)

Igf1r

1.5

d 1.0

i l

a *

m r

o 0.5

)

(

C D D 0.0 l t o u r o t k n c o o C n K Genotype

Figure 3.2.1: Validation of the IE-IGF-1R KO in Mice

6-10-week-old mice were genotyped and then treated with vehicle, 0.2µg/g GLP-2, pre- and co-

EGF at 0.4µg/g per dose, or a combination of EGF and GLP-2. (A) Sample gel electrophoresis of a genotyping. One band represents a flox mouse, two bands represent a cre/fl mouse, band at

390bp represents the Cre band and band at 327bp represents the flox band, (B) qRT-PCR for the

Igf1r in whole jejunal mucosa.

3.2.2 IE-IGF-1R KO Mice and Control Mice Respond Similarly to Pre-EGF+Co-

EGF+GLP-2 Combination Treatment

Body weight did not change in the control group, but the knockout group receiving EGF-GLP-2 had a significant increase compared to its vehicle. (Figure 3.2.2A) Small intestinal weight

40 increased significantly in the control mice in response to the EGF-GLP-2 treatment group. This effect was also observed in the knockout group. (Figure 3.2.2B) Small intestinal length did not change for any of the groups. (Figure 3.2.2C) When normalized to small intestinal length, small intestinal weight increased significantly in the EGF-GLP-2 group compared to vehicle treatment in both the control and the knockout mice. (Figure 3.2.2.D) This same effect remained when small intestinal weight was normalized to body weight. (Figure 3.2.2.E) Crypt-villus height was also measured in the control and knockout treated animals. Control mice responded to GLP-2 and EGF-GLP-2 treatment as expected. There was a significant increase in villus height with

GLP-2, an effect that was retained in the EGF-GLP-2 treatment group. THE EGF-GLP-2 group had also significantly longer villi compared to its EGF control group. Crypt depth was significantly higher in the EGF-GLP-2 group. The knockout group had an increased response to

GLP-2 treatment, but the delta between the control and knockout groups indicated that the response to GLP-2 was less profound, confirming a functional knockout. The knockout group receiving EGF alone also had significantly longer villi compared to vehicle. This effect was not seen in the control group. Interestingly, the knockout group responded to the EGF-GLP-2 treatment similarly to the control group with both longer villi and deeper crypts, suggesting that exogenous EGF is sufficient to bypass the lack of IGF-1R. (Figure 3.2.2F) Proliferation measurements in the transit amplifying zone in control and knockout mice showed that there was a trend towards increased proliferation in the control GLP-2 treatment group, and to a lesser extent in the knockout group. No significance was achieved for any treatment. (Figure 3.2.2G)

A Body Weight B Small Intestinal Weight

1.5 2.0 -EGF -EGF * *** ** -GLP-2 -GLP-2 1.5 1.0 -EGF -EGF +GLP-2 +GLP-2 1.0 +EGF +EGF 0.5 -GLP-2 -GLP-2 +EGF 0.5 +EGF +GLP-2 +GLP-2 0.0 0.0 Control Knockout Control Knockout Genotype

C Small Intestinal Length D Small Intestinal Weight Normalized to Small Intestinal Length 1.5 -EGF 2.0 -GLP-2 * -EGF ** -GLP-2 1.0 -EGF 1.5 +GLP-2 -EGF +GLP-2 +EGF 1.0 +EGF 0.5 -GLP-2 -GLP-2 +EGF 0.5 +GLP-2 +EGF +GLP-2 0.0 Control Knockout 0.0 Control Knockout Genotype Genotype

Small Intestinal Weight Normalized E to Body Weight 2.0 * * -EGF -GLP-2 1.5 -EGF +GLP-2 1.0 +EGF -GLP-2 0.5 +EGF +GLP-2 0.0 Control Knockout Genotype

G Proliferation Positions 8-16

1.5 -EGF -GLP-2

1.0 -EGF

+GLP-2

C U

A +EGF 0.5 -GLP-2 +EGF +GLP-2 0.0 Control Knockout Genotype

Figure 3.2.2: IE-IGF-1R KO Mice Respond Similarly to EGF-GLP-2 Combination Treatment as Control Animals

Body weight and small intestinal weight and length and crypt-villus height measurements of 6-

10 week old male and female control and IE-IGF-1R-KO mice treated with vehicle, 0.2µg/g

GLP-2, pre- and co- EGF at 0.4µg/g per dose, or a combination of EGF and GLP-2 for 10 days.

Small intestinal and colonic measurements were taken following clearing of luminal content. (A)

Body weight, (B) small intestinal length, (C) small intestinal weight, (D) Small intestinal weight normalized to small intestinal length, (E) Small intestinal weight normalized to body weight, (F) crypt-villus height (G) Area under the curve of proliferation in positions 8-16 (*p<0.05 **p<0.01

***p<0.001 as indicated, #p<0.05 compared GLP-2 between genotypes, n=6-8). Data analyzed by 1-way ANOVA.

3.3 Organoids

Organoid cultures from jejunum of both control and IE-IGF-1R KO mice were used to determine the possible interactions between the effects of EGF and IGF-1 on proliferation in vitro.

3.3.1 qRT-PCR of Organoids Confirms the Absence of GLP-2R and the Presence of

EGFR and IGF-1R

Organoids from a flox/flox female 8-week-old mouse were pooled and used for qRT-PCR data confirmed the absence of the GLP-2R and the presence of the EGFR, ErbB2 and IGF-1R.

Furthermore, there was presence of Ki67, a marker for proliferation at all active stages of cell cycle, LGR5, and proglucagon, but no IGF-1 transcripts were detected. (Figure 3.3.1)

Organoids

0.20

o t

0.15

i l

a 0.10

) 0.05

(

C D D 0.00

G R -1 R F R 2 7 5 C -2 F -1 G F B i6 R G P G F E G rb K G L I G E E L G I

Figure 3.3.1: qRT-PCR of Organoids Confirms the Absence of GLP-2R and the Presence of EGFR and IGF-1R qRT-PCR data for organoids derived from a fl/fl 6-week-old female mouse. Organoids were harvested 24 hours after the first passage. (n=6 wells)

3.3.2 Determination of Optimal Timepoint and EGF Withdrawal Treatments to Detect

Proliferation

Organoids were produced from a male C57Bl/6 mouse to determine optimal EdU treatment timepoint. Organoids were treated with EdU for 20, 40, 60 and 120 minutes before harvest.

Proliferation was measured by calculating percent of EdU positive cells in each crypt. A linear relationship between time and percent proliferation was determined (Figure 3.3.2A) and the 20- minute timepoint was chosen to allow for any increases in proliferation in response to treatments.

Conversely, the effect of Y-inhibitor, a component of the organoid media that allows for maintenance of organoids in culture, on proliferation was analyzed due to a previous report suggesting it dampens proliferation.128 Measurements showed there was a 15% decrease in proliferation in the presence of the Y-inhibitor, thus confirming that a decrease in proliferation can possibly be observed in our treatments. (Figure 3.3.2B) Y-inhibitor was excluded from future proliferation experiments. Finally, to determine the optimal EGF withdrawal protocol for the organoid experiments, three treatment groups were designed (Table 3.2). EGF was either given throughout the 5-day experiment, was withdrawn throughout the experiment, or was withdrawn for 4 days and then restored for 1 day. Proliferation counting revealed that complete withdrawal of EGF to the media for 1 day has restored proliferation restoration completely to control levels, while dropped proliferation by 32%. (Figure 3.3.2C)

A EdU Organoid Timepoints 100

80 R = 0.99

t a

r 60

l o

r 40

% 20

0 0 20 40 60 80 100 120 140 EdU Treatment Duration (Minutes)

B Organoid Proliferation With 80 and Without Y-Inhibitor 70

s l

l *

e 50

U 40

d E

% 20 10 0 r r o o it it ib ib h h n n -i -I Y Y t u th o i h W it W

1 Day EGF C

80 *** ***

e C

U 40

% 20

0 + - + F F F G G E G E - -E + F F F G G E G E E

Figure 3.3.2: Determination of Optimal Timepoint and EGF Withdrawal Treatments

Organoids were derived from control mice. (A) EdU treatment timepoints at 20, 40, 60 and 120

minutes, (B) Organoids proliferation and the presence and absence of Y-27632, excluded at 24

hours prior to EdU treatment. (* p<0.05 *** p<0.001. n=1 muse per group, n=10-20 organoids

per mouse, n=1-4 crypts per organoid). Data analyzed by linear regression in the first experiment

and t-test in the second experiment.

Group EGF 4d Prior EGF 1d

EGF Present (+ve Ctrl)

EGF Withdrawal (-ve Ctrl) Ⅹ Ⅹ

EGF Restoration Ⅹ

Table 3.2: Determination of Optimal Timepoint and EGF Withdrawal Treatments

All three organoid treatment groups. Organoids were derived from control mice and treated as

follows: EGF was either present in the media for 5 days, withdrawn for 5 days, or withdrawn for

4 days and then restored for 1 day.

1 Day EGF C

80 *** ***

U 40

% 20

0 l) l) n tr tr o ti C C a e e r v -v to (+ ( s t l e n a R e w s a F e r G r d E P th F i G W E F G E

Figure 3.3.2: Determination of Optimal Timepoint and EGF Withdrawal Treatments

Organoids were derived from control mice. (c) Pilot EGF withdrawal experiment. As treated per Table 3.2, EGF was either left in the media for 5 days, withdrawn for 5 days, or withdrawn for 4 days and then restored for 1 day. (p<0.001. n=1 mouse per group, n=10-20 organoids per mouse, n=1-4 crypts per organoid). Data analyzed by 1-way ANOVA.

3.3.3 EGF and IGF-1 Stimulate Proliferation Independently, But Do Not Have an

Additive Effect on Organoid Proliferation

Organoids from both control and IE-IGF-1R knockout mice were generated and then divided into six treatment groups: EGF positive control with and without co-IGF-1 on day 5, EGF withdrawal with and without co-IGF-1 on day 5, and EGF Restoration with and without co-IGF-

1 on day 5. (Table 3.3) The EGF control group in both genotypes produced 58% proliferation,

49 while EGF withdrawal reduced proliferation to 43% in the control group and 41% in the knockout group. EGF Restoration restored proliferation to positive control levels in both control and knockout organoid groups. The addition of IGF-1 to any of the control treatment groups (ie.

EGF present) did not yield any change in proliferation compared to the positive control. As expected, it did not have an additive effect in the knockout group either. Interestingly, the addition of IGF-1 to the EGF Withdrawal group on the fifth day of the experiment restored proliferation to normal levels, suggesting a compensatory mechanism taking place. That change in proliferation in response to IGF-1 alone was significant compared to the EGF Withdrawal groups in both the control and knockout organoids, and comparable to the EGF Restoration and

EGF present groups. Overall, these data suggest that IGF-1 does not have an additive effect on jejunal organoid proliferation in the presence of EGF but can promote normal proliferation in the absence of EGF in the media. (Figure 3.3.3)

Group EGF 4d Prior EGF 1d Post IGF-1 1d Post

EGF Present (+ve Ctrl) Ⅹ

EGF Present + IGF-1

EGF Withdrawal (-ve Ctrl) Ⅹ Ⅹ Ⅹ

EGF Withdrawal + IGF-1 Ⅹ Ⅹ

EGF Restoration Ⅹ Ⅹ

EGF Restoration + IGF-1 Ⅹ

Table 3.3: EGF and co-IGF-1 Treatment of Organoids

All six organoid treatment groups. Organoids were derived from control and knockout mice and

treated as follows: EGF was either present in the media for 5 days with and without IGF-1 on the

last day, withdrawn for 5 days with and without IGF-1 on the last day, or withdrawn for 4 days

and then restored for 1 day with and without IGF-1 on the last day.

0.8 $$$ ### ### ### EGF Present (+ve Ctrl) ### *** *** *** *** *** *** *** *** *** EGF Present + IGF-1

0.6

s l

l EGF Withdrawal (-ve Ctrl)

e C EGF Withdrawal + IGF-1

U 0.4

d E

EGF Restoration % 0.2 EGF Restoration + IGF-1

0.0 Control Knockout Genotype

Figure 3.3.3: EGF and IGF-1 Stimulate Proliferation Independently, But Do Not Have an Additive Effect on Organoid Proliferation

Organoids from both control and IR-IGF-1R-KO mice treated according to Table 3.3, EGF was either left in the media for 5 days with and without IGF-1 on the last day, withdrawn for 5 days with and without IGF-1 on the last day, or withdrawn for 4 days and then restored for 1 day with and without IGF-1 on the last day. (*** p<0.001 vs EGF withdrawal group in respective genotype groups. ### p<0.001 indicates significance vs. EGF withdrawal + IGF-1 group in respective genotype groups. $$$ p<0.001 indicates significance between genotypes. n=2-3 mice per group, n=10-20 organoids per mouse, n=1-4 crypts per organoid) Data analyzed by 2-way

ANOVA.

Discussion 4.1 General Discussion

GLP-2 promotes proliferation in the intestine in response to nutrient intake to prime the intestine for increased absorption and it does so through signaling cascade that involves EGF and

IGF-1. Due to the lack of GLP-2 in the intestinal crypt, it is still unclear how GLP-2R acts to promote the proliferative effect. These set of data attempt to shed a light and consolidate some of the evidence previously reported. It is known from the Drucker and Teitelbaum labs that acute treatment with EGF increases proliferation in the stem cell zone of the intestine. Furthermore, the

EGFR is an important component of the GLP-2 signaling pathway, without which, the growth effects of GLP-2 are modest.8,60,66 The Brubaker lab has also shown the importance of IGF-1 and

IGF-1R in the GLP-2 pathway. Both the lack of IGF-1 and the absence of IGF-1R produced modest GLP-2-mediated growth effect.2,3 However, it is still not clear how all three, EGF, IGF-1, and GLP-2 interact together to produce the profound proliferation which renders GLP-2 an ideal treatment for short-bowel syndrome. To shed light on the way GLP-2 acts to promote intestinal growth, this project was set to investigate the interactions between all three growth factors in the jejunum of control and knockout mice. The hypothesis of this study was that pre- and co- treatment with EGF enhances the IGF-1R dependent intestinal proliferative effects of GLP-2 in the murine model.

To investigate the hypothesis, the optimal timing for EGF-GLP-2 combination treatment was first determined. Mice were treated with different combinations of EGF and GLP-2 treatments, which gave insight into how EGF and GLP-2 work together in normal mice. A combination of EGF and GLP-2 treatment had a positive effect on gut growth as every EGF-

GLP-2 combination group had a significant increase in growth in crypt-villus height compared to vehicle, GLP-2 alone, and their respective EGF treatment alone. However, it was found that the best outcome was achieved with the pre-EGF+co-EGF+GLP-2 combination treatment. Kitchen et al.105 also reported a significant improvement to gut growth in response to EGF-GLP-2 combination treatment, albeit they reported a synergistic effect in parenterally fed rats. It is therefore, evident that EGF enhances the GLP-2-mediated growth seen in the intestine, in a manner not yet clear. In this study, chronic administration of 0.8µg/g of EGF subcutaneously to

C57Bl/6 mice increased crypt-villus height. Similarly, Feng et al. (2017)60 showed that 0.8µg/g

EGF orally increased crypt-villus in C57Bl/6J mice. However, Drucker et al. (1997)129 have treated CD1 mice for 14 days with 2 µg EGF subcutaneously and have shown no increase in villus height. Whether these differences are due to the dose of EGF and/or mouse strain utilized remains to be determined.

IE-IGF-1-R KO mice have previously been reported to have a reduced response to GLP-

2.3 However, the data reported here shows that, while their response to GLP-2 in the crypt-villus height of the jejunum was indeed reduced, these knockout mice have a response to an EGF-GLP-

2 combination treatment comparable to their control counterparts. These results suggest that the administration of exogenous EGF allows for the bypassing of the IE-IGF-1R signaling, thus promoting GLP-2-mediated proliferation despite the lack IGF-1R of the receptor in the intestinal epithelium. This result was also confirmed in vitro in the organoid model, as organoids derived from KO animals retained normal proliferation in vitro despite the lack of IGF-1R. While the

Brubaker lab has reported that IGF-1 and IGF-1R are essential for GLP-2 signaling,3,75,91 other labs have suggested that it is EGF, not IGF-1, that is essential for GLP-2-mediated intestinal growth. Yusta et al.8 have shown that IGF-1 treatment of normal mice does not change transcript levels of growth and transcription factors, while GLP-2 and EGF treatment does. Therefore, it is

54 possible that exogenous EGF treatment can override the absent IGF-1R signaling through the amplification of these growth transcription factors.

The proliferative effect of EGF and GLP-2 had surprising results. While GLP-2 increased proliferation in the TA zone in the normal mice, as expected2,3,58, EGF did not have an additive effect. This is contrary to what is known about the action of EGF in the intestine, as increased proliferation in the stem cell zone has been observed in mice receiving an acute treatment of

EGF.8 The difference in observation could be the result of the difference in treatment protocols, as chronic treatment with EGF may be activating a different pathway compared to acute treatment. In an attempt to investigate this, two apoptotic markers were measured, Bax and Bcl-

2. qRT-PCR data showed that there was a decrease in apoptosis in every treatment group receiving co-EGF treatment, suggesting that when administered chronically, EGF signaling favours reduction in apoptosis. EGF is known to be anti-apoptotic and proliferative in the intestine as shown in vivo by Feng et al.60,93 and in vitro by the Polk group130, as it counteracts the apoptotic effect of TNF. Interestingly, GLP-2 did not have a reduced effect on apoptosis.

This is in contrast to what has been observed in the Brubaker lab, where GLP-2 reduced the Bax-

Bcl-2 ratio.58 The Drucker group, however, observed the lack of Bax and Bcl-2 changes in response to GLP-2 treatment both in protein and mRNA quantification.8 Finally, and unexpectedly, in the knockout experiment, there was no significant increase in proliferation in the TA zone in the control group in response to GLP-2 and/or EGF. After further data analysis

(not shown), it was found that the females in this study did not respond appropriately to either

GLP-2 or EGF with respect to crypt-villus height and proliferation. The reason for this is currently unknown.

A paper by the Lund group has demonstrated that IGF-1 promotes proliferation differentially in vivo and in vitro. While IGF-1 promotes proliferation in active stem cells in vivo, organoid data shows that IGF-1 favours quiescent stem cell activation.27 The organoids data presented here supports the findings from the Lund group. IGF-1 did not have an additive effect to EGF, however, IGF-1 did restore proliferation to basal levels when EGF was absent from the media. This suggests that the stress undergone by the organoids due to lack of an important growth factor has activated the quiescent stem cell pool to start proliferating with the help of the addition of IGF-1. This finding contrasts previous findings in vitro that show that

EGF enhances IGF-1 proliferative action, albeit this was not tested in organoid models9,10,123–125

According to these studies, EGF and IGF-1 act to enhance each other’s receptors to amplify the signaling pathways that lead to proliferation. It is possible that the lack of this observation in our model is due to the type of cells involved, the complexity of the organoid architecture, and the various different cells that are present in these organoids, which in contrast to homogenous cell models previously used, such as the IEC-6 cell line. Of note, the organoids were able to proliferate further at a higher rate than 60% seen in the EGF-IGF-1 withdrawal experiments as can be seen in the EdU timepoint experiment, where proliferation reached 80%. Therefore, an additive effect of IGF-1 and EGF could have been potentially detected by proliferation.

Finally, qRT-PCR has shown that there is no significant difference in transcripts in mucosal scrapes from C57Bl/6 mice treated with vehicle, GLP-2, EGF or a combination of EGF and GLP-2. This was unexpected as it has been shown in vitro that who have shown that in vitro,

IGF-1R levels decrease in response to IGF-1, and are maintained at basal level with EGF treatment.9 Although in this project mice were not treated with IGF-1, it is known that GLP-2 decreases IGF-1R transcript levels in subepithelial myofibroblasts.76,78 Although there was a trend towards decrease in IGF-1R transcript levels in mice treated with GLP-2, and an increase

56 when EGF was given, there was no significance to the data. qRT-PCR data for organoids pooled from a control mouse verified that while GLP-2R is not present in the epithelium of the intestinal crypt, EGFR and IGF-1R are indeed present, as previously reported.17–19

4.2 Limitations

There are several limitations to this study. Chiefly, the investigation on how EGF and

GLP-2 affect intestinal stem cells is not direct as it is only looked through the effects the treatments had on growth without investigating the molecular mechanism governing the process.

Furthermore, the use of IE-IGF-1R-KO mice provide only one side of the story. It was determined that exogenous EGF administration is sufficient to maintain GLP-2-mediated proliferation in vivo in the absence of the IGF-1R, but it is unclear if this holds true with EGFR knockout mice treated with IGF-1 and GLP-2. The IE-IGF-1R KO mice are capable of some intestinal growth in response to GLP-2,3 however, it is unknown whether these mice can respond to other growth factors, such as other EGFR ligands and/or KGF. Therefore, it is unknown if the response to the EGF-GLP-2 treatment seen in the data presented here is appropriate and proportional compared to other growth factors.

Our organoid studies directly addressed the relationship between EGF and IGF-1. The data showed that there was no additive effect between IGF-1 and EGF on proliferation.

However, it is a possibility that an additive effect was not observed due to the organoids reaching their proliferative capacity. However, although we designed an experiment using FBS to induce further proliferation, the organoids did not survive (data not shown). The in vitro studies also used organoids that were only derived from Cre/Fl and Fl/Fl mice, but not Cre mice. A previous

57 report has shown that activation of the Cre enzyme by tamoxifen can be toxic for stem cells, and derivation of organoids can be challenging.131 Consequently, a strict protocol was followed where mice were given 7 days rest between the last tamoxifen injection and crypt extraction.

However, Cre organoids were not able to survive despite the protocol. Moreover, the organoids used in this study were derived strictly from stem cells, therefore lacking any subepithelial tissue and GLP-2R. Therefore, the effects of GLP-2 on proliferation in vitro in this model cannot be studied.

Finally, qRT-PCR studies were not conclusive as was expected. It is known from previous publications that there is fluctuation of some transcripts in response to GLP-2 and EGF treatments. It is possible that the lack of significance in the findings is due to the number of treatment groups that were used in this study, thus lowering the significance of the overall study.

Further, performing qRT-PCR in organoids is challenging due to the low transcript numbers. For the qRT-PCR experiment reported in this project, 6 wells were pooled to produce enough RNA to be read. Therefore, it was not feasible to perform qRT-PCR for the withdrawal and IGF-1 experiments.

4.3 Future Directions

To address the first limitation of the study, EGFR132 and IGF-1R reporter mice can be used to investigate receptor internalization in response to GLP-2, IGF-1, or EGF treatment.

Organoids could also be derived from these mice to trace internalization in vitro in real-time. Not only it would provide a clearer picture of upstream and downstream mediation of the process of

58 proliferation in response to GLP-2, it would allow for the investigation of the crosstalk between

EGF and IGF-1 in response to promote proliferation.

A second possible further direction in this study is to generate an IE-EGFR-KO mouse that can be treated with several combinations of IGF-1 and GLP-2 would be necessary to paint a more complete in the relationship between all three growth factors. In vitro studies with organoids can employ EGFR inhibitors to reach the same effect. The organoids used in this project still maintained proliferation despite lacking EGF in the withdrawal group. It is hypothesized that these organoids can produce and secrete EGF from the Paneth cells to compensate for the lack of EGF in the media28. Therefore, treating organoids with an EGFR inhibitor would be beneficial to achieve lower proliferation rate.

To investigate the potential growth of the intestine of IE-IGF-1R KO mice, other growth factors can be used alone or in combination with GLP-2. The expression of some growth factors such as amphiregulin and epiregulin, are modulated by GLP-2.8 Other growth factors, such as

KGF, are known to be downstream of GLP-2.7 Therefore, a study on these knockout animals employing these growth factors alone, or in combination with GLP-2, could provide a range of intestinal growth that these animals can reach that could perhaps extend beyond the growth effects seen in the data presented here.

To address the possibility of the organoids reaching maximal proliferation in the withdrawal study, several doses of EGF and IGF-1 can be given in combination. Titration of doses could determine the optimal dosage combination that might allow for additive or synergistic effects to be observed. Additionally, the organoids used in this study were only derived from Cre/Fl and Fl/Fl mice, but not Cre mice. A previous report has shown that activation of the Cre enzyme by tamoxifen can be toxic for stem cells, and derivation of

organoids can be challenging.131 Consequently, a strict protocol was followed where mice were given 7 days rest between the last tamoxifen injection and crypt extraction. However, Cre organoids were not able to survive despite the protocol. The lack of Cre organoids is challenging as it is unclear how tamoxifen treatment can affect proliferation in vitro in our study. As it is known that stem cells are generally affected by Cre activation, it could have ramifications to both our in vivo and in vitro studies. To improve our organoids experiment, and to test for any effects that Cre may have on proliferation in these organoids, a new protocol may have to be used, where a lower dosage of tamoxifen, or a longer wait period between the last tamoxifen administration and stem cell extraction may be required.

Another future direction would be to incorporate GLP-2 into the organoid studies. To do so, air-liquid interface organoids, which likely express the GLP-2 receptor due to the presence of supportive tissue, can be used.133 The use of these organoids would allow us to tease out the interactions of all three growth factors in vitro in different combinations to elucidate the mechanistic relationship. These organoids can be derived from both IGF-1R and EGFR knockout and reporter mice and used to measure proliferation in response to GLP-2 treatment.

Lastly, to address the issues arising with the qRT-PCR studies, the sample size for each mouse group can be increased to address the lack of significance in the qRT-PCR. Performing additional organoid experiments again to measure transcript levels, particularly the levels of

IGF-1R and EGFR, would be beneficial to have a more complete picture as to the mechanism by which proliferation is promoted, particularly whether EGFR and IGF-1R transcript fluctuate based on the treatment.

4.4 Conclusion

The limited knowledge on how GLP-2 acts to promote intestinal proliferation requires further study to improve the therapeutic outcome for patients with short-bowel syndrome.

Although it is known that GLP-2R is not expressed on intestinal stem cells of the intestine, the profound effect GLP-2 has on growth warrants investigation into the underlying mechanisms that govern this pathway, particularly as it was previously reported that rats and mice with induced colonic cancer can develop polyps that can lead to malignant tumours in mice and rats.134,135

EGF and IGF-1 have previously been shown to be effective for treating intestinal damage, so a possible combination of these treatments with submaximal dosage can be more effective and safer for patients.91,115 This study demonstrates that a combination treatment of EGF and GLP-2 can be more effective for the initiation of intestinal growth than GLP-2 by its own. This study also sheds a light on the possible mechanism that take place in response to GLP-2 administration.

Although further investigation into the specific mechanism that take place, this study confirms that exogenous EGF administration is sufficient to induce GLP-2-mediated intestinal growth in vivo, as IE-IGF-1R-KO mice responded similarly to control mice when treated with a combination of EGF and GLP-2. The in vitro model used confirms these in vivo data in KO organoids, where EGF was still able to promote proliferation in these organoids in a rate comparable to control organoids. This suggests that IGF-1 and EGF may act on different stem cells in the intestine. In conclusion, this study confirms that EGF is an important component of the GLP-2 pathway in vivo and can both improve the effects of GLP-2, as well as compensate for the lack of IGF-1R in vivo and in vitro. This is a step further in the understanding of GLP-2 action in the intestine.

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