Protein tyrosine phosphatase sigma (PTPσ) targets apical

junction complex proteins in the intestine and modulates

epithelial permeability.

by

Ryan Murchie

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Biochemistry

University of Toronto

© Copyright by Ryan Murchie 2013

Protein tyrosine phosphatase sigma (PTPσ) targets apical junction complex

proteins in the intestine and modulates epithelial permeability.

Ryan Murchie

Master of Science

Department of Biochemistry

University of Toronto

2012

Abstract

Protein tyrosine phosphatase sigma (PTPσ), encoded by PTPRS, was shown previously

by us to contain SNP polymorphisms that can confer susceptibility to inflammatory bowel

disease (IBD). PTPσ(-/-) mice exhibit an IBD-like phenotype and show increased susceptibility

to acute models of murine colitis. The function of PTPσ in the intestine is uncharacterized. Here,

I show an intestinal epithelial barrier defect in the PTPσ(-/-) mouse, demonstrated by a decrease

in trans-epithelial resistance and a leaky intestinal epithelium that was determined by in vivo

tracer analysis. We identified several putative PTPσ intestinal substrates; among these were

several proteins that form and regulate the apical junction complex, including ezrin. My results

show that ezrin binds to and undergoes tyrosine de-phosphorylation by PTPσ in vitro, suggesting

it is a direct substrate for this PTP. The results suggest a role for PTPσ as a positive regulator of

epithelial barrier integrity in the intestine. The proteins identified in the screen, including ezrin,

suggest that PTPσ may modulate epithelial cell adhesion through the targeting of AJC-associated

proteins, a process impaired in IBD.

ii

Acknowledgements

First and foremost, I would like to thank my supervisors Dr. Daniela Rotin and Dr. Aleixo Muise

for all of your guidance over the course of my project. Your encouragement and support were

essential to the success my endeavours. I would also like to thank my supervisory committee

members Dr. John Brumell and Dr. Christine Bear for their helpful guidance and suggestions

during my committee meetings.

To all the members of the Rotin and Muise labs, thank you for making my time in the lab a

memorable one. Your constant support always made the lab a welcoming place to work. Thank

you to Chong, Chen, Angie and Hui for all your invaluable technical support throughout my time

in the lab without which I would not have been successful. Special thanks to Melanie Gareau

from Dr. Phil Sherman’s lab for training on the Ussing Chamber experiments and to Ramzi

Fattouh from Dr. John Brumell’s lab for additional training. Lastly, thank you to my friends and

family for all of your support over the years!

iii

Table of Contents Acknowledgements ........................................................................................................................ iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii Abbreviations ............................................................................................................................... viii

Chapter 1: Introduction ................................................................................................................... 1

1) Tyrosine Phosphorylation ....................................................................................... 2 2) Protein Tyrosine Phosphatases ............................................................................... 2 3) Protein Tyrosine Phosphatase Sigma (PTPσ) ......................................................... 8 4) PTPσ(-/-) Mouse Model ........................................................................................ 11 5) Inflammatory Bowel Disease ................................................................................ 12 6) Protein Tyrosine Phosphatases in IBD ................................................................. 13 7) PTPσ in IBD ......................................................................................................... 14 8) Role of Intestinal Barrier Defence in IBD ............................................................ 19 9) Apical Junction Complex and Epithelial Barrier Function ................................... 20 10) PTPσ and the Apical Junction Complex ............................................................... 20 11) Rationale and Goals .............................................................................................. 26

Chapter 2: Materials & Methods................................................................................................... 27

1) Animal Experimentation ....................................................................................... 28 2) Constructs ............................................................................................................. 28 3) Tissue Preparation ................................................................................................. 28 4) Tandem immunoprecipitation of phosphotyrosine-mass spectrometry (TIPY-

MS) ....................................................................................................................... 29 5) MS/MS Data Analysis .......................................................................................... 29 6) In vitro substrate trapping assay ........................................................................... 30 7) Tissue culture ........................................................................................................ 31 8) para-Nitrophenyl phosphate (pNPP) phosphatase activity assay .......................... 31 9) In vitro dephosphorylation assay .......................................................................... 31 10) Ussing Chamber Studies ....................................................................................... 32 11) Macromolecular Permeability ............................................................................... 33 12) FITC-dextran assay ............................................................................................... 33 13) Dextran Sodium Sulfate (DSS) Model for IBD .................................................... 33 14) Immunohistochemistry ......................................................................................... 34 15) Transmission Electron Microscopy ...................................................................... 34

Chapter 3: Results ......................................................................................................................... 36

1) PTPσ(-/-) mice exhibit defects in intestinal barrier integrity ................................ 37 2) PTPRS is expressed in the crypts regions of the mouse intestine ......................... 40 3) Tyrosine phosphorylation is enriched in the crypts of PTPσ(-/-) mouse colon

and small bowel .................................................................................................... 40

iv

4) Identification of Villin and Ezrin as PTPσ binding partners by mass spectrometry .......................................................................................................... 45

5) Ezrin is a substrate of PTPσ .................................................................................. 49 6) E-cadherin and β-catenin colocalization and β-catenin localization to the

nucleus is unaffected in the PTPσ(-/-) mouse small bowel and colon .................. 54 7) Ezrin localization is altered in PTPσ(-/-) mouse small bowel after DSS

treatment ............................................................................................................... 58 8) Intestinal morphology is not disrupted in the small bowel and colon of

neonatal PTPσ (-/-) mice ....................................................................................... 61 9) Lysozyme immunostaining is reduced in the Paneth cells of the PTPσ(-/-)

mouse small bowel ................................................................................................ 64 10) Ki-67 immunostaining, a marker for cell proliferation, appears unchanged in

PTPσ(-/-) mouse small bowel and colon .............................................................. 64

Chapter 4: Discussion ................................................................................................................... 70

Role of PTPσ in intestinal epithelia .............................................................................................. 71

1) Evidence of defects in intestinal barrier integrity ................................................. 71 2) Role for E-cadherin and β-catenin ........................................................................ 72 3) Ezrin as a colonic PTPσ substrate ......................................................................... 73

Proposed Mechanism: PTPσ regulation of adherens junction proteins ........................................ 74 Implications for PTPσ in Paneth cell function .............................................................................. 78 PTPσ in IBD pathogenesis ............................................................................................................ 78 Future Directions .......................................................................................................................... 79

1) Further characterize the intestinal barrier defect in the PTPσ(-/-) mouse............. 79 2) Investigate the intestinal ligands of PTPσ ............................................................ 79 3) Evaluate the effect of PTPσ regulation on ezrin function ..................................... 80 4) Validate and characterize other putative substrates identified in MS screen ........ 80 5) Explore the role of PTPσ in other immune cells ................................................... 81 6) Explore the role of PTPσ in Paneth cell function ................................................. 81 7) Generate tissue specific, catalytically-inactive and ΔIg3 PTPσ mouse mutants .. 82

Summary ....................................................................................................................................... 83 Conclusion .................................................................................................................................... 83 References ..................................................................................................................................... 84

v

List of Tables Table 1: Proteins with increased tyrosine phosphorylation in the intestine of the PTPσ(-/-)

mice ............................................................................................................................. 48

vi

List of Figures Figure 1: Classifications and Substrate Specificities of PTPs ....................................................... 4

Figure 2: Domain Architecture and Ectodomain Shedding of LAR-PTP Family Members ......... 7

Figure 3: Summary of IBD Susceptibility Genes and Loci identified by Genome Wide Association Studies (GWAS) ...................................................................................... 16

Figure 4: Alternative splicing of PTPσ in UC ............................................................................. 18

Figure 5: SNP polymorphisms in CDH1 associate with CD and lead to the mislocalization of a truncated E-cadherin protein. ............................................................................... 22

Figure 6: Overview of the Apical Junction Complex (AJC) ....................................................... 24

Figure 7: PTPσ(-/-) mice have defects in epithelial barrier permeability .................................... 39

Figure 8: PTPσ is expressed in epithelial cells lining the crypt regions of the mouse small bowel and colon ........................................................................................................... 42

Figure 9: Tyrosine phosphorylation is enriched in the crypt regions of PTPσ(-/-) mouse colon and small bowel ................................................................................................. 44

Figure 10: Ezrin and villin precipitate with the D1 domain of PTPσ .......................................... 51

Figure 11: Ezrin undergoes dephosphorylation in the presence of D1 domain-containing PTPσ contructs ............................................................................................................ 53

Figure 12: E-cadherin and β-catenin colocalize to the plasma membrane along the basolateral border between enterocytes ....................................................................... 56

Figure 13: Ezrin localization is altered in the small bowel of PTPσ(-/-) mice after DSS treatment ...................................................................................................................... 60

Figure 14: Normal epithelial architecture but reduced Paneth cell number in PTPσ(-/-) neonatal mouse small bowel ........................................................................................ 63

Figure 15: Lysozyme expression is reduced in the Paneth cells of the PTPσ(-/-) small bowel .. 66

Figure 16: Ki-67 immunostaining, a marker for cell proliferation, appears unchanged in PTPσ(-/-) mouse small bowel and colon ..................................................................... 68

Figure 17: Proposed mechanism for PTPσ regulation of adherens junction stability ................. 77

vii

Abbreviations AJC – apical junction complex

ATP – adenosine tri-phosphate

CAM – cell adhesion molecule

CBC – crypt base columnar

CD – Crohn’s disease

CNS – central nervous system

CSPG – chondroitin sulfate proteoglycan

D1 – first catalytic PTPase domain of PTPσ

DSP – dual specificity phosphatase

DSS – dextran sodium sulfate

FNIII – fibronectin III-like

GH – growth hormone

GST – glutathione S-transferase

GWAS – genome wide association study

HNSCC – head and neck small cell carcinoma

HSPG – heparin sulfate proteoglycan

IBD – inflammatory bowel disease

Ig – immunoglobin-like

LAR – leukocyte antigen-related

MS – mass spectrometry

NGF – nerve growth factor

PNS – peripheral nervous system

PRL – prolactin

viii

PTB – phosphotyrosine binding domain

PtdIns3P – phosphatidylinositol 3-phosphate

PTP – protein tyrosine phosphatase

PTPδ – protein tyrosine phosphatase delta

PTPσ – protein tyrosine phosphatase sigma

RPTP – receptor protein tyrosine phosphatase

SH2 – Src homology 2 domain

SNP – single nucleotide polymorphism

SVZ – subventricular zone

TEER – transepithelial electrical resistance

UC – ulcerative colitis

ix

Chapter 1: Introduction

1

1) Tyrosine Phosphorylation

Tyrosine phosphorylation is a reversible post-translational protein modification involving

the covalent attachment of inorganic phosphate derived from ATP to the terminal hydroxyl

group on the side chain of the amino acid tyrosine. This modification can modulate enzymatic

activity[1, 2], dictate subcellular localization[3], or mediate protein-protein interactions through

specialized domains such as the SH2[4] or PTB domains[5]. Since its discovery in 1979[6], a

wide range of cellular processes have been characterized which feature regulation by tyrosine

phosphorylation. These processes include: signal transduction (especially growth factor kinase

signaling); cell proliferation; cell migration and adhesion; and cell differentiation (reviewed in

[7, 8]). The tyrosine phosphorylation of proteins is regulated by the activity of two classes of

enzymes: protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Enzymes in

these families work in opposition to modulate tyrosine phosphorylation levels in the cell. Given

the range of cellular functions mediated by tyrosine phosphorylation, it is not surprising that

dysfunction of PTKs and PTPs has been implicated in a wide range of human diseases including

cancer[9, 10], cardiovascular disease[11], obesity and diabetes[12], and inflammatory

disorders[13, 14].

2) Protein Tyrosine Phosphatases

I) Characteristics & Classifications There are 107 PTPs in the human genome[15]. These proteins can be classified

according to their specificity, molecular weight, and subcellular localization (Figure 1). The

catalytically active PTPase domains of PTPs are highly conserved and contain a signature motif

[(I/V)HCXAGXXR(S/T)G], which includes an essential cysteine residue necessary for the

2

Figure 1: Classifications and Substrate Specificities of PTPs. The four major classes of PTPs are color-coded. Class I PTPs (green) can be sub-classified into classical and VH-1 like dual specificity phosphatases (DSPs) according to substrate specificity. Classical phosphatases are tyrosine-specific and can be further classified into receptor (RPTP) and non-receptor (NRPTP) categories. The DSPs can target diverse non-tyrosine phosphorylated residues including phosphoinositols (PTENs). Class II PTPs (orange) are a family of low-molecular weight phosphatases. Class III PTPs (blue) are a group of three-related cell cycle regulators which target cyclin-dependent kinases (Cdks). Class IV PTPs (red) are unique in that they utilize an aspartic acid based catalytic mechanism and have been shown to target both phospho-tyrosine and phospho-threonine residues. (Adapted from Alonso et al. Cell. 2004)

3

Figure 1: Classifications and Substrate Specificities of PTPs

4

nucleophilic catalysis of the phosphotyrosine moiety through a cysteinyl-phosphate

intermediate[16-18]. Classical PTPs are Class I tyrosine-specific phosphatases which can be

divided into receptor or non-receptor categories according to domain architecture and subcellular

localization[15]. Receptor tyrosine phosphatases (RPTPs) are single-pass trans-membrane

proteins whose ectodomain domain can bind to ligands, transmitting extracellular signals to the

intracellular phosphatase machinery[19]. Receptor dimerization has been implicated in the

(in)activation and regulation of RPTPs[20, 21]. In addition, gatekeeping effects can be provided

by catalytically-inactive tandem PTPase domains which can bind and inhibit the active PTPase

domain[22, 23]; these domains may also play a role in dictating substrate specificity[24].

Reversible oxidation of the catalytically essential cysteine residue has also been implicated in the

regulation of PTPs[25] and specifically RPTPs[26].

II) LAR subfamily The leukocyte antigen-related (LAR) subfamily of RPTPs, comprised of LAR, PTPδ, and

PTPσ in humans, share a high degree of sequence identity (64%)[27]. Invertebrate orthologues

of this family include: hmLar1 and hmLar2[28] (H. medicinalis); DLAR[29, 30] and

DPTP69D[29, 31] (D. melanogaster); and PTP-3[32] (C. elegans). Generally, LAR family

receptors consist of a cell adhesion molecule (CAM)-like ectodomain with three immunoglobin-

like (Ig) domains and a variable number of fibronectin-III-like (FNIII) repeats; a short single

pass trans-membrane domain; and an intracellular region with two PTPase domains of which

only the first (D1) domain is catalytically active[27] (Figure 2A). Alternative splicing is known

to dictate the number of FNIII repeats[33]. These phosphatases are translated as proproteins

which undergo proteolytic processing at the membrane to form a non-covalently attached

5

Figure 2: Domain Architecture and Ectodomain Shedding of LAR-PTP Family Members. A) The domain architecture of the LAR subfamily of RPTPs. Proteins depicted include vertebrate and invertebrate orthologs. The alternatively spliced ‘short’ PTPσ isoform is depicted with FNIII repeats four through seven spliced out. B) Proteolytic processing of LAR family PTPs. After trafficking to the plasma membrane, LAR-PTPs undergo proteolytic processing to form a non-covalently attached complex consisting of a P-subunit and E-subunit. This complex can undergo further processing to disassociate the complex, thus solubilizing the E-subunit extracellularly and allowing the P-subunit to be internalized. (Adapted from Chagnon et al. Biochemistry and Cell Biology. 2004)

6

Figure 2: Domain Architecture and Ectodomain Shedding of LAR-PTP Family Members

7

complex consisting of two subunits, E-subunit and P-subunit[34, 35]. These subunits remain

bound a short distance upstream of the trans-membrane domain. This complex can undergo

further proteolytic processing to abrogate this interaction, solubilizing the E-subunit

extracellularly, thus allowing the P-subunit to be internalized: this process is known as

ectodomain shedding[34, 35] (Figure 2B). These processes may confer a regulatory effect to

these PTPs as these liberated subunits can either be redistributed within the cell, in the case of

the P-subunit, or go on to interact with other adhesion-like domains extracellularly, in the case of

the E-subunit[34].

3) Protein Tyrosine Phosphatase Sigma (PTPσ)

Protein-tyrosine phosphatase sigma (PTPσ) is encoded by the PTPRS gene in humans,

which is located at chromosome 19p13.3[15]. This phosphatase is evolutionarily conserved with

notable homologs in mice (M. musculus)[36], rats (R.norvegicus)[37], and zebrafish (D.

rerio)[38]. PTPσ has two major splice isoforms: the ‘long’ isoform, which contains 8 FNIII

repeats, is expressed in several tissue types including the thymus, colon, and small intestine,

whereas the ‘short’ isoform, which contains 4 FNIII repeats (FNIII repeats 4 through 7 are

spliced out), is expressed primarily in the nervous system[27]. Receptor homo-dimerization is

necessary for ligand binding to the PTPσ ectodomain in vitro with differential ligand binding

specificities observed for the two major splice variants[39]. Expression of PTPσ is

developmentally regulated with tightly controlled temporal and spatial distributions[37, 40]. Its

expression is strongest during embryonic development, where it is present in various epithelial

tissues and regions of the central (CNS) and peripheral (PNS) nervous systems, including the

cerebral cortex, cerebellum, hippocampus, brainstem, olfactory bulb, pituitary, spinal cord,

dorsal root ganglia, and sciatic nerve[41-45]. Expression levels of PTPσ decline after birth,

8

where its localization becomes restricted to the olfactory bulb, cerebellum, and hippocampus in

the nervous system and in epithelial cells in several tissue types [41-45].

I) Functions of PTPσ The most extensively described function for PTPσ involves its regulation of neuronal and

neuroendocrine processes. PTPσ has been implicated in the maturation of the CNS.

Architectural distortions including hippocampal dysgenesis and reductions in corpus callosum

and cerebral cortex thickness are present in the CNS of the PTPσ(-/-) mouse[46]. In the PNS,

electrophysiological analysis of peripheral motor nerves in the PTPσ(-/-) mouse revealed reduced

conduction velocities compared to wild-type controls, an effect linked to increased proportions

of slow conducting, small-diameter myelinated fibres and relative hypomyelination[47]. During

axon development and neurite outgrowth, PTPσ acts as an inhibitor of axonal regeneration and

as a mediator of axon guidance. PTPσ(-/-) mice subjected to sciatic nerve crush injury

(axonotmesis) or sciatic nerve transection show increased peripheral nerve regeneration

compared to wild-type control[48]. PTPσ(-/-) mice also show increased rates of functional

recovery following facial nerve crush injury indicative of increased neuronal regeneration[49].

Damaged cortico-spinal tract (CST) axons in PTPσ(-/-) mice are able to regenerate and show

increased extension (compared to wild-type controls) after thoracic spinal injury[50].

Furthermore, neural stem cells cultured from the subventricular zone (SVZ) of the PTPσ(-/-)

mouse show altered migration patterns and enhanced neurite outgrowth[51]. Directional defects

in axonal pathfinding are present in PTPσ(-/-) mice during axon regeneration following nerve

transection[48]. This parallels observations made with the LAR family ortholog DLAR, which

functions to regulate motor axon guidance in Drosophila[52].

9

PTPσ regulates elements of the neuroendocrine system, especially along the

hypothalamic-pituitary axis[53, 54]. Development of the somatotroph-lactotroph lineage of the

anterior pituitary requires PTPσ expression[54]. Adenohypophyses derived from the pituitary

gland of PTPσ(-/-) mice show reduced size and decreased growth hormone (GH) and prolactin

(PRL) immunoreactivity[54]. In addition, pancreatic islets in PTPσ(-/-) mice show hypoplasia

and reduced insulin immunoreactivity while gut enteroendocrine hormones are abnormally

expressed compared to wild-type controls[54]. Recently, PTPσ was implicated in the regulation

of autophagy, as RNA interference of PTPσ expression led to increased cellular levels of

PtdIns3P-positive vesicles and hyperactivation of constitutive and induced autophagy[55].

II) Ligands and Substrates for PTPσ To date, the identified ligands and substrates for PTPσ are predominantly associated with

its role in the nervous system. At sites of axon growth, PTPσ functions bimodally through

interactions with its ligands heparin sulfate proteoglycans (HSPGs) and chondroitin sulfate

proteoglycans (CSPGs) to promote or inhibit, respectively, the extension of sensory neurons[56].

In avian retinal basal lamina, the heparin sulfate proteoglycans, agrin and collagen XVIII, were

shown to bind to the first Ig (Ig1) domain on the extracellular region of PTPσ[57, 58]. Netrin-G

ligand-3 (NGL-3) interacts with the first two FNIII repeats on PTPσ to regulate excitatory

synapse formation in a bidirectional manner[59]. In the developing muscle, the ‘short’ PTPσ

isoform interacts with nucleolin on the surface of developing myotubes[39, 57, 60]. PTPσ was

also shown to form complexes with the neurotrophin receptors, TrkA and TrkC, facilitating

receptor dephosphorylation, thus attenuating downstream signaling in the presence of nerve

growth factor (NGF)[61].

10

PTPσ also interacts and targets proteins associated with cellular adhesion. The

cytoskeletal modulators Liprinα-4, Trio, p130CAS were identified as PTPσ interactors,

independent of tyrosine phosphorylation, through a modified yeast two-hybrid ‘substrate

trapping’ screen conducted on a mouse cDNA library[62]. p250GAP was identified as a PTPσ

substrate with its activation increasing in the presence of PTPσ, leading to down-regulated Rac

signaling[62]. Our group identified the adherens junction proteins N-cadherin and β-catenin as

PTPσ substrates in the brain[63].

Outside of the nervous system, PTPσ was shown to attenuate epidermal growth factor

receptor (EGFR) signaling in the A431 epithelial cell line after treatment with the ganglioside

GM3[64] or staphylococcal α-hemolysin[65]. Genomic copy number assessment in patients

with head and neck squamous cell carcinoma (HNSCC) revealed frequent intragenic

microdeletions in PTPRS which lead to increased EGFR/PI3K pathway activation[66]. These

deletions also correlated with poor prognostic outcomes and reduced survival in patients with

EGFR-activating oncogenic mutations[66]. In the context of autophagy, the substrates of PTPσ

are not clear, however, several targets have been suggested including: direct regulation through

dephosphorylation of PtdIns3P, regulation of the PI3 kinase, Vps34, or through the regulation of

autophagy initiation through Atg1/Ulk1[55].

4) PTPσ(-/-) Mouse Model

To investigate the function of PTPσ in vivo, our group[47] and others[67] had generated

PTPσ knockout (PTPσ(-/-)) mice. These mice are developmentally delayed with various

neurological and neuroendocrine defects (described above) and cachexia[46-48, 63]. After birth,

the PTPσ(-/-) mice can be separated into three cohorts: a high proportion (60%) die within hours

of birth due to hypoglycemia caused by insufficient production of GH; 37.5% demonstrate

11

growth defects and wasting syndrome, expiring 2 to 3 wks post-birth; while 2.5% reach

adulthood, although with a 20% - 50% size reduction compared to wild-type littermates[47].

Analysis of the intestinal tissue in surviving mice revealed the presence of mucosal

inflammation, intestinal crypt branching, and villus blunting: all features of a colitis phenotype

similar to the enteropathy associated with human inflammatory bowel disease (IBD)[13].

Notably, PTPσ(-/-) mice also showed increased susceptibility to chemical and infectious models

of murine colitis, specifically treatment with dextran sodium sulfate (DSS) and infection with

Citrobacter rodentium[13]. The intestinal phenotype in the mice strongly inferred a connection

between PTPσ and IBD.

5) Inflammatory Bowel Disease

I) Current Disease Model The inflammatory bowel diseases (IBDs) are chronic, idiopathic, relapsing disorders

affecting the gastrointestinal tract, where Crohn’s disease (CD) and ulcerative colitis (UC) are

the two major forms[68]. These two diseases share similar features but often different genetic

backgrounds and can be differentiated histopathologically by the location and depth of

inflammation and by the presence or absence of granulomas[68-70]. IBD patients typically

present with a combination of intestinal inflammation or ulceration, bowel obstruction, strictures

or fistulae and can experience diarrhea, abdominal pain, gastrointestinal bleeding, and weight

loss, as well as other extra-intestinal manifestations including anemia[71, 72]. Current treatment

options include anti-inflammatory and immunosuppressive therapies (5-aminosalicylates[73],

corticosteroids[74], azathioprine[75]), biologics (namely anti-tumor necrosis factor alpha(TNF-

α) monoclonal antibodies such as infliximab[76] or adalimumab[77]), or, in severe cases,

surgical interventions including bowel resection[78] or bone marrow transplants[79]. The

12

current model for IBD pathogenesis is that in the presence of environmental factors,

polymorphisms in IBD susceptibility genes cause an abnormal innate and adaptive host immune

response to commensal gut bacteria, leading to sustained and deleterious inflammation[80, 81],

chronic infection[82, 83], dysbiosis[83], defective mucosal barrier defense[84], and insufficient

microbial clearance[83]. The disease is known to have a strong genetic component as evidenced

by specific populations exhibiting disproportionately high disease incidences[85] and the high

disease concordance between monozygotic twins[86-88].

II) Susceptibility Genes Given the importance of genetic predisposition to the development of IBD, recent work

has focussed on identifying susceptibility genes to better understand disease pathogenesis. Early

genome wide scanning for microsatellite markers in linkage disequilibrium in patients with UC

or CD initially identified 9 loci (termed IBD1 through IBD9) with significant linkage[89-96].

(Figure 3A). Over the past decade, successive genome wide association studies (GWAS) and

meta-analyses conducted on case-control patient cohorts have increased the number of identified

susceptibility loci to 163, with 110 loci shared between UC and CD[97-99]. From these studies,

a wide range of pathways have been implicated in UC and CD including: autophagy

(ATG16L1[13, 97], IRGM[13, 97]), IL-23 signalling (IL23R[13, 100], STAT3[13]), and IL-10

signalling (IL10[97, 101]) (Figure 3B)

6) Protein Tyrosine Phosphatases in IBD

Several protein tyrosine phosphatases have been identified as IBD susceptibility genes

through both GWAS and functional analyses. PTPN2, also known as T-cell protein tyrosine

phosphatase (TC-PTP), was first associated with Crohn’s disease in a GWAS conducted by the

Wellcome Trust Case Control Consortium[102] and subsequently replicated in several

13

independent studies[103-106]. PTPN2 is known to regulate pro-inflammatory cytokine signaling

and loss of PTPN2 expression in mice leads to systemic inflammation[107]. Furthermore,

PTPN2(-/-) mice have increased susceptibility to induced colitis following treatment with

DSS[108]. PTPN11, which encodes for SHP-2, contains polymorphisms associated with

ulcerative colitis[109] and functions to attenuate STAT3-mediated inflammation[110] and to

regulate barrier function through its role in modulating the expression of claudins in the intestinal

epithelia[111] . PTPN22 (Lymphoid protein tyrosine phosphatase), a negative regulator of T-cell

activation, has been implicated in a range of autoimmune disorders[112-115] including both

Crohn’s disease[105, 116] and ulcerative colitis[116]. The identification of these PTPs as IBD

susceptibility genes demonstrates the importance of PTPs in the pathogenesis of IBD, and

underscores how investigations into the function of PTPs in the intestine can contribute to our

understanding of the disease.

7) PTPσ in IBD

PTPRS resides within the IBD6 susceptibility locus[94]. Through single nucleotide

polymorphism (SNP) analysis of IBD patients, our group has shown that PTPRS is genetically

associated with ulcerative colitis[13]. The identified SNP polymorphism leads to alternative

splicing in the extracellular region of the epithelial ‘long’ isoform of PTPσ leading to a loss of

micro-exon domain B (meB) resulting in the absence of the third immunoglobin-like domain

(Ig3) in the final protein[13] (Figure 4). This splicing might potentially lead to altered ligand

recognition or may affect receptor dimerization[39]

14

Figure 3: Summary of IBD Susceptibility Genes and Loci identified by Genome Wide Association Studies (GWAS) A) Schematic representation of first identified IBD susceptibility loci. These regions, termed IBD1 through IBD9, represent genomic areas containing microsatellite markers in strong linkage disequilibrium in IBD patients compared to healthy controls. Regions in which candidate susceptibility genes were further identified and validated are colored magenta. B) Overview of GWAS-identified IBD susceptibility genes. Susceptibility genes are classified according to cellular function. Genes are color-coded according to disease classification in which association was observed (blue: ulcerative colitis, red: Crohn’s disease, black: unspecified IBD). Underlined genes indicate genes with characterized cis-acting expression quantitative trait loci (cis-eQTL) indicating allele specific gene expression changes. Asterisks indicate genes with known coding region SNPs. The labels ‘P’ and ‘G’ denote Paneth and goblet cells respectively. (Adapted from Gaya et al. The Lancet. 2005 & Khor et al. Nature. 2011)

15

Figure 3: Summary of IBD Susceptibility Genes and Loci identified by Genome Wide Association Studies (GWAS)

A

B

16

Figure 4: Alternative splicing of PTPσ in UC. A) PCR analysis demonstrating alternative splicing of isoform 1 of PTPσ in individuals with the UC-associated risk haplotype. mRNA was isolated from lymphoblast cell lines derived from three healthy controls (labeled 1 through 3) and three IBD patients possessing the risk alleles (labeled 4 through 6). A DNA fragment comprising amino acids 191 through 371 of PTPσ was amplified by PCR and separated by gel electrophoresis. The top bands represent the full 168 amino acid segment found in isoforms 2 and 3 of PTPσ. The lower bands represent the alternatively spliced isoform 1 lacking exons 8 and 9. B) Schematic representation of alternative splicing of PTPσ in UC. The meB domain (exon 8) and the third Ig domain (exon 9) are spliced out in the individuals possessing the disease haplotype. This splicing is only in frame in the ‘long’ PTPσ isoform, which is expressed in the intestine. (Adapted from Muise et al. Current Biology. 2007)

17

Figure 4: Alternative splicing of PTPσ in UC

18

8) Role of Intestinal Barrier Defence in IBD

Intestinal barrier defence refers to the coordinated action of several innate immune

pathways to protect the interior of the body from both commensal and pathogenic organisms

present in the intestinal lumen (reviewed in [117]). These pathways include: the production of

antimicrobial peptides and immunoglobins by specialized Paneth cells at the base of the

intestinal crypts; the secretion of mucins by goblet cells, and the restriction of access to the

lamina propria provided by a polarized epithelial cell monolayer. Mucosal barrier dysfunction

has been previously associated with the development of IBD[84]. In IBD patients, evidence of

impaired barrier function, in the form of increased intestinal permeability, further underscores

the importance of innate immunity to IBD pathogenesis[118-123]. Genetic studies have revealed

several IBD susceptibility genes that play important roles in mediating this defence. One of the

earliest identified IBD susceptibility factors, NOD2, functions in Paneth cells through the

activation of the NF-κB pathway to regulator defensin production in the presence of the bacterial

proteoglycan component muramyl dipeptide (MDP)[124, 125]. MYO9B[126], PARD3[121],

MAGI2[121], three proteins known to mediate tight junction dynamics, were identified in

European GWAS and replicated in North American cohorts. Our group has identified a SNP

haplotype polymorphism in CDH1 that associates with Crohn’s disease and leads to a truncated

E-cadherin protein that fails to localize to the plasma membrane[127]. The mislocalization of E-

19

cadherin and its binding partner β-catenin was observed in IBD patient biopsy samples[127]

(Figure 5). CDH1 was also identified in a recent GWAS as an UC susceptibility gene [81].

Several of these identified proteins implicate the apical junction complex as a system whose

dysfunction can contribute to IBD pathogenesis.

9) Apical Junction Complex and Epithelial Barrier Function

The apical junction complex (AJC) is essential for the formation of the epithelial cell

monolayer in the intestine as it mediates epithelial cell adhesion and polarity through two distinct

junction complexes: the tight junctions or zonula occludins and the adherens junctions or zonula

adherens (Figure 6A). These complexes form intercellular junctions through claudin-occludin

and cadherin-catenin homophilic interactions, respectively, and anchor to the actin cytoskeleton

to provide mechanical support for the epithelial cell and to maintain proper polarized

morphology (Figure 6B). In addition, these complexes restrict fluid access from the intestinal

lumen to the lamina propria except through regulated paracellular transport[128]. The defective

regulation of AJC-associated proteins creates disrupted epithelial barriers leading to permeability

defects and aberrant intestinal morphology[128, 129]. Expression of a dominant negative N-

cadherin chimera in mice was shown to induce a CD-like phenotype in the intestine[130]. The

similarity between the intestinal pathologies associated with IBD and apical junction complex

disruption suggests that AJC proteins may be potential IBD susceptibility genes.

10) PTPσ and the Apical Junction Complex

PTPσ colocalizes with plakoglobin (γ-catenin) which is known to localize to adherens

and desmosome junctions along the basolateral plasma membrane in epithelia[131]. The

localization of PTPσ to sites of intercellular junctioning, coupled with the previous identification

of N-cadherin as a neural PTPσ substrate by our group, suggest that PTPσ may regulate cell

20

Figure 5: SNP polymorphisms in CDH1 associate with CD and lead to the mislocalization of a truncated E-cadherin protein. A) Schematic representation of the truncated E-cadherin mutant found in CD patients with the disease associated haplotype. A 72 amino acid truncation at the N-terminus (Δ72) leads to a loss of the signal peptide region (S) and a portion of the precursor domain (Pre). Cad, cadherin domain; TM. transmembrane domain; ICD, intracellular domain. B) Immunofluorescence microscopy demonstrating that the Δ72 E-cadherin protein accumulates in the cytoplasm. mCherry-tagged wild-type (A) and Δ72 E-cadherin (B) plasmids were transfected into MDCK cells. Zo-1 and calnexin (green) were co-stained to act as markers for plasma membrane and cytoplasmic localization respectively. β-catenin was also co-stained and was shown to accumulate in the cytoplasm along with Δ72 E-cadherin. Scale bars = 20 μm. C) Immunohistochemical staining for E-cadherin in intestinal biopsies from patients with (E-H) or without (A-D) the CD-associated risk haplotype. DAB staining (brown) showed strong accumulation in the cytoplasm for patients with the risk allele compared to controls. Nuclei are counterstained in blue. (Adapted from Muise et al. Gut. 2009)

21

Figure 5: SNP polymorphisms in CDH1 associate with CD and lead to the mislocalization of a truncated E-cadherin protein.

A

B

C

22

Figure 6: Overview of the Apical Junction Complex (AJC). A) Schematic representation of junctioning complexes throughout the intestinal epithelial cells. Enterocytes adopt a polarized morphology as mediated by the apical junction complex partitioning the plasma membrane into distinct apical and basolateral compartments. The tight junctions restrict access to the lamina propria through claudin-occludin interactions. The adherens junctions mediate intercellular adhesion and provide structural support through interactions with the actin cytoskeleton. Microvilli protrude from the apical plasma membrane into the lumen of the small intestine. B) Schematic of principal mediators of adherens and tight junctioning. At the adherens junction, cadherins forms calcium dependent homophilic interactions with cadherin complexes on opposing cells. The cytoplasmic tail of the cadherin interacts with adapter catenin proteins, which mediates interactions with the actin cytoskeleton and other regulatory molecules. At the tight junction, claudins in conjunction with occludins and junction associated molecules (JAMs) form tight intercellular interactions and interact with the actin cytoskeleton through the tight junction proteins ZO-1 and ZO-2.

(Adapted from Guttman et al. BBA-Biomembranes. 2009 & Nyqvist et al. EMBO Reports 2008)

23

Figure 6: Overview of the Apical Junction Complex (AJC)

A

B

24

adhesion through targeting elements of the apical junction complex, specifically the adherens

junction. Through an interaction-trap assay, E-cadherin (CDH1) and β-catenin (CTNNB1) were

identified as intestinal substrates of PTPσ by our group[13]. These identified substrates further

support a role of PTPσ in cellular adhesion. Tyrosine phosphorylation is known to play an

important role in mediating the stability of cadherin-catenin interactions. Tyrosine

phosphorylation of VE-cadherin at sites Y658 and Y731 has been shown to abrogate binding to

p120 and β-catenin[132]. p120-catenin, which is known to regulate E-cadherin

internalization[133], contains eight tyrosine residues that can be phosphorylated by Src[134],

leading to the recruitment of various downstream effectors as well as modulating the stability of

its interaction with E-cadherin. Increased tyrosine phosphorylation of AJC proteins has also

been linked to decreased transepithelial resistance (TER) and increased paracellular

permeability[135-137]. Other PTPs, including PTPμ[61] and LAR[138], have been implicated

in modulating the tyrosine phosphorylation of cadherins and catenins in vivo. Tyrosine

phosphorylation is also implicated in the potentiation of signals that regulate adherens junction

formation. The receptor tyrosine kinase EGFR, which is a known substrate for PTPσ, modulates

adherens junction remodeling[139, 140]. Rac1, a Rho GTPase implicated in adherens junction

regulation, can become activated through phosphotyrosine mediated interactions with scaffold

proteins such as p130CAS[141] (another known PTPσ interactor) or ezrin[142, 143]. The link

between the effects of tyrosine phosphorylation on AJC proteins and the modulation of epithelial

barrier integrity, as well as our lab’s identification of E-cadherin as an intestinal substrate for

PTPσ, suggest that PTPσ may serve as a regulator of intestinal permeability.

25

11) Rationale and Goals

The phenotype observed in the PTPσ(-/-) mouse coupled with the genetic association

with UC implicates PTPRS as a IBD susceptibility gene. However, the role and regulatory

targets of PTPσ in the intestine have been largely uncharacterized. The identification of E-

cadherin and β-catenin as colonic PTPσ substrates by our group suggest a role for PTPσ in the

regulation epithelial cell adhesion. Given that defects in epithelial barrier function have been

observed both experimentally and clinically in IBD patients, we postulate that PTPσ regulates

epithelial barrier integrity through the regulation of apical junction complex proteins and that

defective PTPσ function can contribute to the pathogenesis of IBD. To test this hypothesis, the

intestinal epithelial barrier integrity will be characterized in the PTPσ(-/-) mouse to determine if

permeability is comprised. To understand the downstream effects of PTPσ regulation, putative

intestinal substrates will be identified through mass spectrometry and substrate trapping then

validated through in vitro assays. Finally, the intestinal phenotype of the PTPσ(-/-) mouse will

be further characterized through histopathology to determine if other factors are contributing to

the observed phenotype.

Thus, this thesis has three principal goals:

1. Characterize intestinal epithelial barrier function in the PTPσ(-/-) mouse.

2. Identify and validate intestinal PTPσ substrates.

3. Investigate the histopathology of PTPσ(-/-) mouse intestine.

These goals will further characterize the role of PTPσ in the intestine with the wider

purpose of elucidating the cellular events underlying IBD pathogenesis.

26

Chapter 2: Materials & Methods

27

1) Animal Experimentation

PTPσ(-/-) mice were generated and backcrossed onto a pure C57Bl/6 background as

described previously[47]. Mice used for experiments were between 8 to 12 weeks old and all

control mice were littermates to the PTPσ(-/-) mice. Mice were housed in a specific-pathogen

free environment with a standard diet and free access to water. Animal care and experimental

protocols were approved by the Animal Care Committee panel at the Hospital for Sick Children

(Toronto, ON).

2) Constructs

Full-length human ezrin (hEZR) cDNA was obtained from SIDNET (Hospital for Sick

Children, Toronto, ON) from the Mammalian Gene Collection (MGC) on a pOTB7 plasmid.

This cDNA was cloned into the N-terminal FLAG epitope tagged mammalian expression

plasmid, pcDNA 3.1/nFLAG, using the Gateway system (Invitrogen, Carlsbad, CA).

Glutathione S-transferase (GST) fusion constructs for the intracellular PTPase domains of

rat PTPσ (GST-PTPσ-D1, GST-PTPσ-D1D2, GST-PTPσ-D2, GST-PTPσ-D1(D1478A)) were

previously generated on a bacterial expression plasmid, pGEX-2T as described[63].

3) Tissue Preparation

Large bowel tissue was collected from PTPσ(-/-), PTPσ(+/-), and PTPσ(+/+) littermates,

washed with phosphate buffered saline (PBS), and placed in 5mL ice cold lysis buffer (150mM

NaCl, 50mM HEPES, 1% Triton X-100, 10% glycerol, 1.5mM MgCl2, 1.0mM EGTA)

supplemented with protease and phosphatase inhibitors (aprotinin (1µM), leupeptin (100µM),

pepstatin A (1µM), PMSF (1µM), sodium orthovanadate (Na3VO4; 1mM)). The tissue was

homogenized using a handheld rotor-stator homogenizer set to medium intensity and left on ice

28

for 30 min. The resulting slurry was centrifuged at 10,000 rpm at 4°C for 30 min and the

supernatant was clarified through a 0.45µM filter.

4) Tandem immunoprecipitation of phosphotyrosine-mass spectrometry (TIPY-MS)

Mouse colon tissue homogenate was incubated overnight at 4°C with a commercial

phosphotyrosine (pTyr) immunoprecipitation (IP) kit (Sigma, St. Louis, MO) with anti pTyr-

agarose. The beads were washed twice with IP wash buffer (20mM HEPES, pH 7.5, 10%

glycerol, 0.1% Triton X-100, and 150mM NaCl) and twice with HPLC-grade water. The bound

proteins were eluted using 0.1% trifluoroacetic acid (TFA). Eluted proteins were digested with

trypsin, desalted, and the resultant peptide mixtures were separated on an automated nanoliter-

scale liquid chromatography (LC) system (Easy-nLC, ProxeonBiosys tems A/S, Odense,

Denmark) then detected using a Thermo-Fisher linear ion trap mass spectrometer system (LTQ,

Thermo, San Jose, CA), as described[144].

5) MS/MS Data Analysis

Tandem mass spectra data obtained were extracted using BioWorks (Thermo Scientific,

Waltham, MA). To determine the identity of the peptide fragments, MS/MS samples were

analyzed using SEQUEST (Thermo Finnigan, version 27) set to search the Swiss-Prot database

assuming trypsin digestion and with the dynamic modification: phosphorylation (Ser, Thr, Tyr).

The precursor mass tolerance was set to 1.08 Da and two missed tryptic cleavages were allowed.

Scaffold (version Scaffold-01.06.05, Proteome Software Inc., Portland, OR) was used to confirm

and validate MS/MS-based peptide and protein identifications. Probability thresholds for peptide

and proteins identification were set at ≥95% and ≥90%, respectively, as determined through the

ProteinProphet™ algorithm [Nesvizhskii et al. Analytical Chemistry. 2003]. Post-translational

29

modifications were assigned according to results obtained from the search engines described

above.

6) In vitro substrate trapping assay

Tissue homogenate from PTPσ(-/-) mouse colon and small bowel was obtained as above.

DH5α Escherichia coli transformed with the GST-fusion constructs was grown in 1L Lennox

broth (LB) at 37°C to an OD590 of 0.7, followed by addition of 1mM IPTG to induce expression.

After 2 hrs of induction, the cells were collected by centrifugation, re-suspended in PBS

supplemented with protease inhibitors (as above) and 1mM lysozyme, and lysed by sonication.

After clarification by centrifugation at 12,500 x g for 30 min at 4°C, the GST-fusion proteins

were purified through incubation with 200µL GST-agarose (Sigma) for 1 hour.

To perform the substrate trapping, 5mg of PTPσ(-/-) mice colonic tissue homogenate in

1mL of lysis buffer was incubated with 20µL of the GST-agarose bound to the GST-fusion

proteins. Bound proteins were washed twice with lysis buffer, then twice with low-salt HNTG

buffer (20mM HEPES, 10% glycerol, 0.1% Triton X-100, 150mM NaCl). The beads were

compacted by centrifugation, the supernatant was aspirated, and 30µL of 1x SDS sample buffer

was added. Following a brief incubation at 95°C, the proteins were separated using SDS-PAGE,

and transferred to a nitrocellulose membrane (GE Healthcare, Waukesha, WI) for

immunoblotting.

The antibodies used for immunoblotting were: mouse anti-ezrin (1:1000; 3C12

Invitrogen), mouse anti-villin (1:1000; BD Biosciences, Franklin Lakes, NJ), mouse anti-

phosphotyrosine (1:1000; 3G10 Invitrogen), mouse anti-E-cadherin (1:1000; BD Biosciences),

and mouse anti-p130cas (1:1000, BD Biosciences). Following a 1 hour incubation with primary

antibody, the blots were incubated with horseradish-peroxidase (HRP) conjugated goat anti-

30

mouse secondary antibody (1:10,000, Covance) in PBS-T and developed with Western

Lightning® Plus ECL (Perkin-Elmer, Waltham, MA)

7) Tissue culture

Human embryonic kidney 293T (HEK-293T) were maintained in Dulbecco’s minimum

essential minimum (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%

penicillin/streptomycin at 37°C and 5% CO2. Twenty µg of DNA for each of the FLAG-tagged

constructs was transiently expressed using the calcium phosphate transfection method onto

100mm dishes. After two days, cells were treated for 10 min with 40µL sodium pervanadate

prepared by adding equal volumes 1mM sodium orthovanadate and 1% hydrogen peroxide, to

enrich for tyrosine phosphorylated proteins. Cells were lysed using 1mL lysis buffer

supplemented with protease inhibitors (as above).

8) para-Nitrophenyl phosphate (pNPP) phosphatase activity assay

GST-fusion proteins were purified as above. Thirty µL of the GST-agarose bound to the

GST-fusion proteins were added to 300µL of PBS with 1mg/mL pNPP (Sigma) in a 96-well

plate. The plate was incubated at 37°C for 30 min then the absorbance was read at 405nm.

Absorbance readings were normalized to zero values taken at start of incubation. Readings

displayed on Figure 2A are at the 30 min time point.

9) In vitro dephosphorylation assay

Activity of GST-fusion proteins was confirmed using pNPP assay above. FLAG-tagged

proteins were precipitated from cell lysate using 20µL Anti-M2 FLAG-agarose (Sigma) for 1

hour at 4°C. Bound proteins were washed twice with lysis buffer and twice with low-salt

HNTG. The GST and FLAG fusion proteins were eluted from the beads using 50mM Tris-HCl

31

pH 8.0 with 10mM reduced glutathione and 0.1M glycine pH 3.5 quenched with 10% v/v

100mM Tris-HCl pH 8.0, respectively. 50µL of each of the eluted GST-fusion proteins and the

eluted FLAG-fusion proteins were combined in 50µL PBS and incubated for 30 min at 37°C.

The reaction was stopped by the addition of 30µL 5x SDS sample buffer. Proteins were

separated on SDS-PAGE gel and immunoblotted as described above.

10) Ussing Chamber Studies

Mice were sacrificed by cervical dislocation and the colon and small bowel were excised

and placed into oxygenated Kreb’s buffer (115mM NaCl, 1.25mM CaCl2, 1.2mM MgCl2,

2.0mM KH2PO4 and 25mM NaHCO3 at pH 7.35) at 37°C. Small bowel sections were obtains

proximal to the cecum while large bowel sections were obtained proximal to the anus. Excised

tissue were opened by dissection along the mesentery axis and mounted on 2.8m x 11.2 mm

oblong sliders (P2304; Physiological Instruments, San Diego, CA) with the luminal side down.

The sliders were loaded into a four-chamber Ussing chamber system (EM-CSYS-4;

Physiological Instruments) that was pre-calibrated. Kreb’s buffer was added to both chambers

while 10mM glucose was added to the serosal side as an energy source and 10mM mannitol was

added to the luminal side to maintain osmotic balance. Agar-salt bridges were used to both

monitor potential difference across the membrane and to apply the appropriate short-circuit

current (Isc) to maintain the potential difference at zero as controlled through an automated

voltage clamp. The system is controlled through the Acquire & Analyze software (Physiological

Systems), which modulates voltage and current controls remotely. The tissue was allowed to

equilibrate for 15 min before Isc readings were taken.

32

11) Macromolecular Permeability

After the tissues have equilibrated in the Ussing chamber and baseline Isc readings were

acquired, 10-5 M horseradish peroxidase (HRP) (Type VI, Sigma) was added to the luminal

chambers to act as a probe for macromolecular permeability. 500µL samples were taken from

the serosal chambers at 30 min intervals for 2 hrs and replaced by 500µL of fresh Kreb’s buffer

to maintain constant volume. A modified Worthington method was used to evaluate the

enzymatic activity of the HRP in the serosal samples as described [145].

12) FITC-dextran assay

PTPσ(-/-), PTPσ(+/-), and PTPσ(+/+) littermates were fasted for 4 hrs and then

administered 15 mg of fluorescein isothyanate (FITC)-dextran (MW 3000-5000Da and

40,000Da; Sigma) by orogastric gavage. Needle size and gavage volume used were determined

according the weight of the mouse based on standard practices. After 4hrs, the mice were

sacrificed by cervical dislocation and bled by cardiac puncture. Serum was isolated using

centrifugation and serum FITC levels were evaluated using fluorometry.

13) Dextran Sodium Sulfate (DSS) Model for IBD

PTPσ(-/-), PTPσ(+/-), and PTPσ(+/+) littermates were treated with 3% dextran sodium

sulfate (DSS) (Sigma) in their drinking water ad libitium for 4 days. The mice were monitored

daily for weight loss, stool consistency, rectal bleeding, and general health. On the fourth day,

the mice were sacrificed and the small bowel and colon were excised and fixed using 4% w/v

paraformaldehyde (PFA) in phosphate buffered saline (PBS).

33

14) Immunohistochemistry

Fixed tissue sections were dissected to isolate regions of interest then embedded in

paraffin. Five µm sections were obtained using a microtome and placed on Superfrost Plus

(Fisher Scientific, Waltham, MA) microscope slides. Following incubation at 60°C for 30 min,

the tissues sections were deparaffinized and rehydrated using sequential washes (100% xylenes,

5min x 2; 50% xylenes:50% ethanol, 5 min; 100% ethanol, 5 min x 2; 95% ethanol, 3 min; 75%

ethanol, 3min; 50% ethanol, 3 min; cold H2O, 5 min). Antigen retrieval was performed using

sodium citrate buffer (pH 6.0) heated to 95°C in a pressure cooker for 35 min. Membrane

solubilization and blocking was performed using SS-PBS (PBS + 0.1% saponin, 10% goat

serum). Primary antibodies were incubated overnight at 4°C in a humidified chamber. Primary

antibodies used were: mouse anti-ezrin (1:300; 3C12 Invitrogen), mouse anti-villin (1:350; BD

Biosciences), mouse anti-E-cadherin (1:400; BD Biosciences), and rabbit anti-β-catenin (1:1000;

Cell Signaling Technologies, Beverly, MA). Following washes in PBS, the slides were

incubated with secondary antibodies in SS-PBS for 1 hour at room temperature. Secondary

antibodies used were: goat anti-rabbit Alexa488 (1:1000, Invitrogen) and goat anti-mouse

Alexa555 (1:1000, Invitrogen). Slides were further stained with DAPI (1mg/mL) and coverslips

were mounted using Dako Fluorescent Mounting reagent (Dako, Glostrup, Denmar

15) Transmission Electron Microscopy

PTPσ(-/-) one day old neonates and littermates were sacrificed and the small bowel was

removed by dissection. Tissues were immediately place in aldehyde fixation buffer (2%

paraformaldehyde in 100 mM cacodylate buffer (pH 7.0) with 2 mM CaCl2) overnight at 4°C.

Samples were washed with 100 mM cacodylate buffer (pH 7.0), then post-fixed in 1% osmium

tetroxide in 100 mM cacodylate buffer (pH 7.0) for 2 h at 4°C. After a ddH2O wash, samples

34

were stained in 2.0% aqueous uranyl acetate for 2h at 4°C. Stained samples were dehydrated

through increasing percentage acetone washes followed by propylene oxide mixed in increasing

ratios of embedding resin. Embedded tissues were sectioned on an ultra-microtome and

visualized using a transmission electron microscope.

35

Chapter 3: Results

All work presented in this chapter was completed by myself with the exception of the initial

tandem MS screen which was conducted by Cong-hui Guo

36

1) PTPσ(-/-) mice exhibit defects in intestinal barrier integrity

Given the prior identification of the adherens junction components, E-cadherin and β-

catenin as colonic PTPσ substrates, and in light of their importance in mediating epithelial cell

adhesion, the integrity of the intestinal epithelial layer in our PTPσ(-/-) mice was investigated by

evaluating the permeability and electrophysiology of the small bowel and colon ex vivo and

performing in vivo tracer studies. Small bowel and colon sections from PTPσ(+/+) and

PTPσ(-/-) mice were analyzed in an Ussing Chamber. PTPσ(-/-) mouse colon and small bowel

both showed decreased trans-epithelial electrical resistance (TEER) (Figure 7A) and increased

macromolecular flux (Figure 7B) as compared to PTPσ(+/+) littermates. PTPσ(+/-) mice were

similar to wild-type controls. These data suggest that loss of PTPσ function leads to loss of

epithelial resistance that could indicate a defect in intestinal permeability. To confirm these

observations in vivo, mice were gavaged with fluorescein-isothiocyanate (FITC)-conjugated

dextran that acts as a molecular probe to evaluate epithelial barrier permeability. Two different

size dextran conjugates were used, 4.4 kDa and 40 kDa, to assess both paracellular and

macromolecular permeability, respectively. PTPσ(-/-) mouse small bowel and colon tissue

showed increased FITC serum levels for both dextran conjugates as compared to PTPσ(+/+)

littermates (Figure 7C,D). PTPσ(+/-) mouse tissue showed slightly elevated levels but were not

significantly different from their PTPσ(+/+) littermates. These data support the Ussing chamber

results and further suggests a role for PTPσ in the maintenance of epithelial barrier integrity and

defense.

37

Figure 7: PTPσ(-/-) mice have defects in epithelial barrier permeability. A) Trans-epithelial electrical resistance (TEER) was measured ex vivo in whole intestinal tissue sections mounted in an Ussing chamber. PTPσ(-/-) mice showed decreased resistance in both the small bowel and colon compared to wild-type littermates. B) Macromolecular flux across the epithelia was evaluated by adding horseradish peroxidase (HRP) as a tracer to the mucosal side of the Ussing chamber with tissues mounted. Measurement of the concentration of HRP in the serosal chambers at thirty minute intervals revealed an increased flux in the PTPσ(-/-) mice compared to wild-type littermates. To evaluate permeability in vivo, PTPσ(-/-) mice and controls were orogastrically gavaged with the molecular tracer FITC-dextran. C) PTPσ(-/-) mice showed increased serum levels of FITC-dextran 4kDa compared to wild-type littermates. D) PTPσ(-/-) mice showed increased serum levels of FITC-dextran 40kDa compared to wild-type littermates. * denotes p<0.05 versus PTPσ(+/+) littermate controls (n=8 to 11 mice, Student’s t-test). Error bars represent mean +/- SE.

38

Figure 7: PTPσ(-/-) mice have defects in epithelial barrier permeability

39

2) PTPRS is expressed in the crypts regions of the mouse intestine

The gene inactivating cassette used to generate our PTPσ(-/-) mice contains a β-

galactosidase reporter, which is thus expressed from the endogenous PTPRS promoter [47]. To

determine where PTPRS (PTPσ) is expressed in the intestine, we performed X-gal staining on

frozen tissue sections from PTPσ(+/-) mice and littermate controls. Dark blue/black staining

indicative of PTPσ expression was observed in the epithelial cells lining the crypt region of the

small bowel and colon in the heterozygous mice that was not present in the wild-type (Figure 8).

This suggests the PTPσ is expressed in epithelia lining the intestinal crypts, an area of rapid cell

proliferation. PTPσ expression was also observed (based on morphology) in the region at the

base of the small bowel crypts known to contain Paneth cells and crypt base columnar (CBC)

intestinal stem cells (Figure 8E). In addition, we also observed PTPσ expression in cells present

in the lamina propria regions of the small bowel and colon (Figure 8F).

3) Tyrosine phosphorylation is enriched in the crypts of PTPσ(-/-) mouse colon and small bowel

To determine how loss of PTPσ regulation affects tyrosine phosphorylation in the

intestine, we investigated the localization and extent of tyrosine phosphorylation in PTPσ(-/-)

mouse colon and small bowel using immunofluorescence microscopy. Colon and small bowel

sections from both untreated and DSS-treated PTPσ(+/+) and PTPσ(-/-) mice were

immunostained using anti-phosphotyrosine antibody (Figure 9). In the untreated mice, the villi

in the small bowel did not show significant staining for phoshotyrosine in either the PTPσ(+/+)

or PTPσ(-/-) sections. The crypt regions of the small bowel of the PTPσ(-/-) mice, however,

showed strong phosphotyrosine staining compared to PTPσ(+/+) controls (Figure 9A). A similar

crypt staining pattern was also observed in the colon of PTPσ(-/-) mice (Figure 9B). These

results support the X-gal staining data shown in Figure 6, as the increase in tyrosine

40

Figure 8: PTPσ is expressed in epithelial cells lining the crypt regions of the mouse small bowel and colon. X-gal staining was performed on frozen tissue sections from PTPσ(+/+) and PTPσ(+/-) mice. A,B) X-gal crystal formation was observed in cells in the crypt regions of the small bowel of the PTPσ(+/-) mice. Strong staining appears at the base of the intestinal crypts where the Paneth and intestinal stem cells reside. Background signal in the PTPσ(+/+) section represents non-specific staining. Scale bar = 40μm C,D) X-gal staining was present in the intestinal cells lining the crypts in the colon tissue of the PTPσ(+/-) mice but not the PTPσ(+/+) mouse. Scale bar = 40μm. E) X-gal staining is present in cells at the base of the small bowel crypts. Scale bar = 40μm. F) Cross-sections of the intestinal crypts reveal X-gal positive staining on cells inside the lamina propria region of the PTPσ(+/-) mouse.

41

Figure 8: PTPσ is expressed in epithelial cells lining the crypt regions of the mouse small bowel and colon

42

Figure 9: Tyrosine phosphorylation is enriched in the crypt regions of PTPσ(-/-) mouse colon and small bowel. Tissue sections from both naïve and DSS-treated mice were immunostained with a phosphotyrosine antibody and visualized using confocal microscopy. A,B) Tyrosine phosphorylation is enriched in the crypt regions of PTPσ(-/-) mouse small bowel and colon as compared to littermate controls. Increased tyrosine phosphorylation is also observed in cells contained within the lamina propria regions of PTPσ(-/-) mice. Scale bar = 50 μm. C,D) DSS-treatment of the PTPσ(-/-) mice and littermates leads to increased tyrosine phosphorylation in both knock-out and wild-type small bowel and colonic tissue sections. Overall tyrosine phosphorylation levels appear elevated in the PTPσ(-/-) mouse compared to the wild-type controls. Scale bar = 50 μm.

43

Figure 9: Tyrosine phosphorylation is enriched in the crypt regions of PTPσ(-/-) mouse colon and small bowel

44

phosphorylation is located in regions positive for PTPσ expression. In addition, cells in the

lamina propria along the crypt-villus axis showed increased tyrosine phosphorylation in the

PTPσ(-/-) mice (Figure 9A). These cells include lymphocytes, which mediate immune function

in the intestine and are known to be present in large numbers in the lamina propria region.

In the DSS-treated mice, phosphotyrosine staining was found to be enriched in all

genotypes in both the small bowel (Figure 9C) and colon (Figure 9D). Notably, the staining was

increased in the PTPσ(-/-) mice relative to DSS-treated control littermates.

4) Identification of Villin and Ezrin as PTPσ binding partners by mass spectrometry

The altered permeability and increased tyrosine phosphorylation present in the PTPσ(-/-)

mouse intestine suggest that PTPσ is regulating proteins associated with the function and

maintenance of the epithelial barrier through its phosphatase activity. Given that E-cadherin and

β-catenin are the only intestinal substrates of PTPσ identified to date[13], we sought to

characterize the intestinal substrates of PTPσ to determine the mediators through which PTPσ

may be regulating intestinal barrier integrity. In order to identify such putative substrates, tandem

mass spectrometry was performed on phospho-tyrosine containing proteins immunoprecipitated

from colon tissue homogenate from PTPσ(-/-) and PTPσ(+/+) littermates, with the aim of

identifying hyper-tyrosine phosphorylated proteins in the PTPσ(-/-) intestine. Isolated proteins

were digested with trypsin to generate peptide fragments and analyzed by MS/MS to identify

pTyr containing fragments. Over 3600 unique spectral hits were identified in each of the tissues.

To refine this list to isolate potential PTPσ substrates, spectral hit counts were compared between

PTPσ(-/-) and PTPσ(+/+) samples, as we have demonstrated by immunofluorescence that loss of

PTPσ function leads to an increase in tyrosine phosphorylation in the intestine. Proteins were

ranked according to the difference in unique spectral counts between samples and a minimum

45

threshold of four was assigned to select specifically for phospho-proteins enriched in the PTPσ(-

/-) mouse tissue. Thirty-seven proteins which were enriched in tyrosine phosphorylation in the

PTPσ(-/-) colon and met the threshold requirement are listed in Table 1.

E-cadherin (Cdh1) and β-catenin (Cttnb1) were previously identified as colonic PTPσ

substrates by our group[13]. p130CAS (Bcar1) was recently identified as a PTPσ interacting

partner in a yeast-two hybrid screen[62]. Furthermore, EGFR was also previously shown to be

regulated by PTPσ[65] and that loss of PTPσ function leads to sustained EGFR activation[66].

The presence of these previously identified substrates in the hit list (Table 1) provided

confidence in our the MS analysis. A large proportion of the putative substrates identified in this

analysis are known to play a role in cell adhesion and cell junction pathways, specifically the

apical junction complex (AJC). In addition, many of the proteins are regulators of cytoskeletal

dynamics either by binding or processing actin directly or through downstream signaling

pathways. Given the number of cell adhesion and cell junction proteins identified in the screen,

we chose to focus on those putative substrates that are associated with epithelial barrier structure

and maintenance. Two proteins: ezrin and villin, were selected for further validation. Ezrin is a

scaffold protein implicated in the organization of the apical web region and participates in a

range of signaling pathways including adherens junction remodeling[143, 146]. Interestingly,

ezrin is known to modulate E-cadherin trafficking through activation of the Rho-GTPase Rac1 in

a tyrosine phosphorylation-mediated interaction. Furthermore, ezrin regulates villus

morphogenesis and Ezr(-/-) mice have defects in intestinal epithelial architecture including

atrophic villi[129]. Villin binds caps and severs actin filaments and regulates cell morphology

46

Table 1: Proteins with increased tyrosine phosphorylation in the intestine of the PTPσ(-/-) mice. This list was generated by comparing the unique spectral hits counts observed for each protein between the PTPσ(-/-) and PTPσ(-/-) mouse tissue samples. Hits that contained three or greater spectral counts in the PTPσ(-/-) sample compared to littermate controls were included. These represent the subset of proteins with increased tyrosine phosphorylation in the PTPσ(-/-) intestine. Proteins highlighted in red are known to be involved in the formation or regulation of the apical junction complex.

47

Table 1: Proteins with increased tyrosine phosphorylation in the intestine of the PTPσ(-/-) mice

Gene Protein Difference in Unique Spectral

Counts PTPσ(-/-) vs. PTPσ(+/+)

Myh11 myosin, heavy chain 11, smooth muscle 244 Myh9 myosin, heavy polypeptide 9, non-muscle 67 Vil1 villin 1 36 Gsn gelsolin 24 Myh14 myosin, heavy chain 14, non-muscle 24 Acta1 actin, alpha 1, skeletal muscle 23 Actb actin, beta 16 Bcar1 (p130CAS)

breast cancer anti-estrogen resistance 1, CRK-associated substrate

16

Myl6 myosin, light polypeptide 6, alkali, smooth muscle and non-muscle

15

Myh10 myosin, heavy chain 10, non-muscle 13 Hist1h1e histone cluster 1, H1e 10 Tubb5 tubulin, beta 5 class I 10 Cttn cortactin 7 Mylc2b myosin light chain, regulatory B 7 Dbn1 drebrin 1 6 Lpp LIM domain containing preferred translocation partner

in lipoma 5

Tuba1b tubulin, alpha 1B 5 Lasp1 LIM and SH3 protein 1 4 Ezr ezrin 4 Cdh1 E-cadherin 4 Ctnnb1 β-catenin 4 Inpp5d inositol polyphosphate-5-phosphatase 4 Ptpn18 protein tyrosine phosphatase, non-receptor type 18 4 Egfr epidermal growth factor receptor 4 Flt3 FMS-like tyrosine kinase 3 4 Tpm1 tropomyosin 1, alpha 4 Hcls1 hematopoietic cell specific Lyn substrate 1 4 Nedd9 neural precursor cell expressed, developmentally

down-regulated gene 9 4

Actr3 ARP3 actin-related protein 3 homolog 3 Pecam1 platelet/endothelial cell adhesion molecule 1 3 Krt8 keratin 8 3 Hnrpab heterogeneous nuclear ribonucleoprotein A/B 3 Fau Finkel-Biskis-Reilly murine sarcoma virus (FBR-

MuSV) ubiquitously expressed 3

Tjp2 tight junction protein 2 3 Tpm3 tropomyosin 3, gamma 3 Eps8 epidermal growth factor receptor pathway substrate 8 3

48

and microvilli formation[147]. Loss of villin function has been shown to increase colonic

susceptibility to the DSS model of murine colitis[148].

To confirm these putative substrates can bind to PTPσ, an in vitro substrate trapping

assay was performed using glutathione S-transferase (GST) fusion proteins that were previously

generated by our group[63], which contain the catalytically active D1 domain of PTPσ. A site-

directed aspartic acid to alanine mutation at amino acid 1472 (GST-PTPσD1(D1472A)) was

introduced to create as a substrate trapping mutant, as described[63, 149]. Figure 10 shows that

ezrin and villin can bind to the D1 domain of PTPσ. To ensure that the binding was not GST-

mediated, a pull-down control of GST-agarose alone was used and the substrates showed no

interaction.

5) Ezrin is a substrate of PTPσ

To test whether ezrin and villin are substrates of PTPσ, an in vitro dephosphorylation

assay was performed. GST-fusion proteins containing the intracellular PTPase domains of PTPσ

were purified and their phosphatase activity was confirmed through incubation with pNPP, a

generic phosphotyrosine substrate (Figure 11A). Only the GST-fusion proteins containing the

D1 domain, namely GST-PTPσD1 and GST-PTPσD1D2, showed an increase in absorbance at

405nm, indicative of PTPase activity. This is expected considering that the D1 domain is the

catalytically active domain[150]. The GST-PTPσD1(D1472A) mutant showed no activity,

which was consistent with the substrate trapping mutation abrogating phosphatase activity.

The purified domains were incubated for 30min with an immunoprecipitated FLAG-

ezrin protein. Immunoblotting for phosphotyrosine revealed significantly decreased tyrosine

phosphorylation levels of ezrin in those incubations containing the catalytically active domain of

PTPσ (Figure 11B). No change in tyrosine phosphorylation levels was observed for the GST-

49

Figure 10: Ezrin and villin precipitate with the D1 domain of PTPσ. Lysates obtained from the intestine of PTPσ(-/-) mice were incubated with wild-type (GST-PTPσD1-WT) or substrate trapping (GST-PTPσD1-(D1472A) constructs bound to GST-agarose containing the catalytic D1 domain of PTPσ. Binding partners were separated on SDS-PAGE and immunoblotted. GST-agarose alone was included as a negative control to ensure binding is not GST-dependent. A) Substrate trapping assay revealing ezrin binding to the D1 domain of PTPσ. E-cadherin was included as a positive control. B) Substrate trapping assay revealing villin binding to the D1 domain of PTPσ. For labeling, ‘σD1-WT’ and ‘σD1-DA’ refer to GST-PTPσD1-WT and GST-PTPσD2(D1472A), respectively.

50

Figure 10: Ezrin and villin precipitate with the D1 domain of PTPσ

51

Figure 11: Ezrin undergoes dephosphorylation in the presence of D1 domain-of PTPσ. GST-fusion constructs for the intracellular domains of PTPσ were incubated with FLAG-tagged ezrin. A) para-nitrophenyl phosphate (pNPP) phosphatase assay demonstrating activity of GST-fusions constructs. B) Incubation with GST-PTPσD1 or GST-PTPσD1D2 leads to decreased tyrosine phosphorylation of FLAG-ezrin. No change in tyrosine phosphorylation was observed for incubations with GST-PTPσD2 or GST-PTPσD1(D1472A). C) Incubation with GST-PTPσD1 or GST-PTPσD1D2 does not change tyrosine phosphorylation levels of FLAG-villin. No change in tyrosine phosphorylation was observed for incubations with GST-PTPσD2 or GST-PTPσD1(D1472A). D) Histogram depicting the change of in tyrosine phosphorylation of ezrin during incubation with the various GST-fusion proteins as compared to the catalytically inactive D1 mutant. All values are normalized to total protein levels by densitometry. (n=4, Student’s t-test, p<0.05). Error bars represent mean +/- SE.

52

Figure 11: Ezrin undergoes dephosphorylation in the presence of D1 domain-containing PTPσ

contructs

53

PTPσD2 or the GST-PTPσD1(D1472A) proteins. This assay was repeated and the cumulative

histogram is depicted in Figure 11D, demonstrating a statistically significant change in tyrosine

phosphorylation levels between the D1-domain containing samples and the catalytically in-active

mutant. These data suggest that ezrin can interact with and undergo tyrosine dephosphorylation

by PTPσ in vitro, supporting its role as a PTPσ substrate. No change in tyrosine phosphorylation

of villin was observed in this assay (Figure 11C), suggesting that villin is not an in vitro (direct)

substrate of PTPσ but may interact with this PTP as part of a complex.

6) E-cadherin and β-catenin colocalization and β-catenin localization to the nucleus is unaffected in the PTPσ(-/-) mouse small bowel and colon

As described earlier, tyrosine phosphorylation plays a key role in the regulation of the E-

cadherin-β-catenin interaction and increased tyrosine phosphorylation is associated with complex

disassembly[151]. To determine if the adherens junctions are disrupted by loss of PTPσ

function, the localization of E-cadherin and β-catenin was assessed in our PTPσ(-/-) mice both

naïve and after DSS treatment using immunofluorescence microscopy. Under normal

conditions, E-cadherin localized to the basolateral region of the enterocytes in PTPσ(+/+) and

PTPσ(-/-) mouse small bowel and colonic sections (Figure 12A,B). β-catenin colocalized with

the E-cadherin and was also present in the nucleus. No significant difference was found between

the PTPσ(+/+) and PTPσ(-/-) tissue sections suggesting that E-cadherin localization is not

affected by loss of PTPσ (Figure 12E). This result contradicts the permeability defect observed

earlier as we would expect a disruption of the adherens junction if E-cadherin and β-catenin are

dysregulated. This would suggest that the increased permeability observed in the PTPσ(-/-)

mouse is either independent of PTPσ mediated E-cadherin- β-catenin regulation or that PTPσ

does not regulate E-cadherin- β-catenin complex internalization. E-cadherin internalization via

endocytosis has been implicated in the regulation of cellular adhesion[152].

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Figure 10: E-cadherin and β-catenin colocalize to the plasma membrane along the basolateral border between enterocytes. E-cadherin and β-catenin localization was evaluated in the small bowel and colon of both naïve and DSS-treated PTPσ(-/-) mice and littermates using immunofluorescence microscopy. E-cadherin and β -catenin colocalized to the basolateral plasma membrane region between the enterocytes lining the villi (A) and the crypts (B) in the small bowel. β-catenin staining was also present in the nuclei of the enterocytes. DSS-treatment of the PTPσ(-/-) mice and littermates did not disrupt the colocalization of E-cadherin and β -catenin in the small bowel (C) or colon (D). Scale bars = 45μm. E) Evaluation of the colocalization of E-cadherin and β-catenin using Pearson’s coefficient revealed no significant difference between PTPσ(-/-) mice and littermate controls. (n=75, Student’s t-test, p>0.05). Error bars represent mean +/- SE. F) Evaluation of the colocalization of the nuclear marker DAPI (blue) and β-catenin revealed no significant difference between PTPσ(-/-) mice and littermate controls (n=67, Student’s t-test, p>0.05). Error bars represent mean +/- SE.

55

Figure 12: E-cadherin and β-catenin colocalize to the plasma membrane along the basolateral border between enterocytes

56

E

F

57

Following treatment with DSS, E-cadherin again localized predominantly to the plasma

membrane along basolateral region between enterocytes in both PTPσ(-/-) and PTPσ(+/+) mouse

small bowel (Figure 12C) and colon tissue (Figure 12E). β-catenin immunostaining mirrored a

similar pattern to the E-cadherin in addition to being present in the nucleus. Quantification of

the colocalization between E-cadherin and β-catenin showed no significant difference between

the PTPσ(-/-) and PTPσ(+/+) samples (Figure 12E). β-catenin localization to the nucleus is

indicative of Wnt signaling pathway activation, which is known to play an important role in

enterocyte differentiation along the crypt-villus axis[153, 154]. We evaluated the colocalization

of β-catenin with the nuclear marker DAPI in immunostained tissue sections from our PTPσ(-/-)

mice and littermates (Figure 12F). There was no significant difference in nuclear localization of

β-catenin between the PTPσ(-/-) and PTPσ(+/+) tissue sections, suggesting the β-catenin

mediated Wnt signaling is not affected by loss of PTPσ.

7) Ezrin localization is altered in PTPσ(-/-) mouse small bowel after DSS treatment

Tyrosine phosphorylation has been implicated in the trafficking of ezrin to the plasma

membrane[155, 156]. To determine if loss of PTPσ activity disrupts ezrin localization, we

performed immunofluorescence on mouse tissue sections. Small bowel and colonic tissue was

acquired from both naïve and DSS-treated PTPσ(-/-), and PTPσ(+/+) mice. Ezrin localization in

naïve mouse colon and small bowel revealed no difference between PTPσ(-/-) and PTPσ(+/+)

sections (Figure 13A,B). However, differences in ezrin localization were observed between

PTPσ(-/-) and PTPσ(+/+) small bowel sections after DSS treatment (Figure 13C,D). The

PTPσ(+/+) small bowel tissue showed ezrin localization in both the cytoplasm and at the plasma

membrane, in contrast to the PTPσ(-/-) tissue, which showed plasma membrane staining almost

exclusively. This provides evidence that PTPσ may regulate ezrin trafficking, and that loss of

58

Figure 13: Ezrin localization is altered in the small bowel of PTPσ(-/-) mice after DSS treatment. Ezrin localization was evaluated in small bowel and colon tissue sections from both naïve and DSS-treated PTPσ(-/-) mice and littermates using immunofluorescence microscopy. A) Ezrin localization was present at the apical plasma membrane of enterocytes in the small bowel in PTPσ(-/-) mice and controls. Scale bar = 30μm. B) Ezrin localized to the apical surface of the enterocytes in the colon and there was no observed difference between PTPσ(-/-) mice and littermate controls. Scale bar = 30μm. C) Ezrin localization is disrupted in the small bowel of PTPσ(-/-) mice treated with DSS. D) DSS-treatment has no effect on the localization of ezrin in the colon. Scale bar = 30μm.

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Figure 13: Ezrin localization is altered in the small bowel of PTPσ(-/-) mice after DSS treatment

60

PTPσ may lead to accumulation of ezrin at the plasma membrane under acute inflammatory

conditions. However, given the severity of the intestinal tissue disruption in the PTPσ(-/-) mouse

after DSS treatment, it is possible that the observed changed in ezrin localization is the result of

inflammation as opposed to a contributing factor causing inflammation. Consequently,

determining if PTPσ mediates ezrin localization to the plasma membrane will require more

robust experimental techniques.

8) Intestinal morphology is not disrupted in the small bowel and colon of neonatal PTPσ (-/-) mice

Ezrin is known to play an important role in the architectural development of the intestine,

mediating villus morphogenesis and microvilli formation[129, 157]. Loss of ezrin activity has

been linked to villus blunting, a phenotype observed in the small bowel of the PTPσ knockout

mouse [129]. Given that PTPσ expression is known to be developmentally regulated and that

loss of PTPσ function has been linked to developmental delays in other tissue types, it is possible

that loss of PTPσ regulation of ezrin may affect small bowel morphology during intestinal

development in the PTPσ(-/-) mouse. To determine if intestinal morphology is altered after

birth, small bowel tissue sections from one day old PTPσ(-/-) neonates were analysed by electron

microscopy. The structure and morphology of the enterocytes lining the PTPσ(-/-) small bowel

appear normal as compared to wild-type littermates (Figure 14A,B). Microvilli and the apical

junction complex region also appear similar to wild-type controls (see indicated). However, the

number of Paneth cells present at the base of the small bowel crypts in the PTPσ(-/-) mouse

appeared reduced compared to wild-type littermates. To determine if Paneth cell function was

affected in the PTPσ(-/-) neonates, frozen small bowel neonatal tissue sections were tested for

immunoreactivity to the anti-microbial factor lysozyme, which serves as a marker for Paneth

cells. DAB staining at the base of the intestinal crypts appears reduced in the PTPσ(-/-) small

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Figure 14: Normal epithelial architecture but reduced Paneth cell lysozyme immunoreactivity in PTP(-/-) neonatal mouse small bowel. A) The morphology of the intestinal epithelial cells in the small bowel of the PTPσ(-/-) neonates and littermate controls characterized by electron microscopy. Tissue sections were fixed by aldehyde fixation, embedded, and sectioned on an ultramicrotome before visualization by TEM. Cell morphology in the PTPσ(-/-) mouse appears like wild-type. Notably, the microvilli length and distribution appears normal and junction complexes have proper size and alignment. Scale bars = 2μm B) Evaluation of lysozyme expression in the PTPσ(-/-) mouse small bowel. Frozen tissue sections from PTPσ(+/+) and PTPσ(-/-) neonates were stained for lysozyme and visualized by DAB reaction. Comparison of staining intensity in the crypt regions of the small bowel reveals reduced DAB color intensity in the PTPσ(-/-) neonates. Negative control represents DAB reaction in absence of antibody. Scale bar = 40μm.

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Figure 14: Normal epithelial architecture but reduced Paneth cell number in PTPσ(-/-) neonatal mouse small bowel

]

63

bowel compared to wild-type littermates (Figure 14C,D). This suggests that PTPσ may play a

role in the regulation of Paneth cells. .

9) Lysozyme immunostaining is reduced in the Paneth cells of the PTPσ(-/-) mouse small bowel

Paneth cells are specialized secretory epithelial cells responsible for the production of

various antimicrobials factors including α-defensins (cryptidins)[158, 159], lysozyme[160], and

phospholipase A2[161]. The observed LacZ staining pattern in the small bowel of the PTPσ(-/-)

mouse suggests that PTPσ is expressed in the Paneth cells, as evidenced by strong staining at the

base of the crypts. The reduced immunoreactivity to lysozyme observed in the PTPσ(-/-)

neonates further supports a role for PTPσ in Paneth cell function. Given that Paneth cell

dysfunction has previously been implicated in IBD, evaluating indicators of Paneth cell function

in the adult PTPσ(-/-) mice will help to determine if loss of PTPσ is impairing the innate immune

function of the Paneth cell. Using confocal microscopy, small bowel tissue sections from 2 to 3

wk old PTPσ(-/-) mice and littermates were immunostained for lysozyme. Lysozyme

immunostaining, as measured by fluorescence intensity per unit area, was reduced in the PTPσ(-

/-) mouse small bowel compared to wildtype controls (Figure 15A). To quantitate these results,

fluorescence intensity measurements were taken for approximately 95 crypts from both

PTPσ(+/+) and PTPσ(-/-) small bowel sections. These results were tabulated and the difference

in fluorescence intensity between the two genotypes was shown to be statistically significant

(Figure 15B).

10) Ki-67 immunostaining, a marker for cell proliferation, appears unchanged in PTPσ(-/-) mouse small bowel and colon

The crypts of the small bowel and colon are regions of high cell proliferation as intestinal

progenitor cells migrate toward the lumen along the crypt-villus axis. Several pathways are

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Figure 15: Lysozyme expression is reduced in the Paneth cells of the PTPσ(-/-) small bowel. A) Lysozyme immunostaining in the crypt regions of the small bowel of PTPσ(+/+) and PTPσ(-/-) mice. Paraffin-embedded small bowel tissues sections were obtained for PTPσ(-/-) mice and littermates controls and immunostained using lysozyme primary antibody. Staining was visualized using Alexa565 secondary antibody and imaged by confocal immunofluorescence microscopy. Staining intensity in the PTPσ(-/-) mouse small bowel appears reduced compared to wild-type littermates. Scale bar = 45μm. B) Quantification of lysozyme staining intensity in the PTPσ(+/+) and PTPσ(-/-) mice. Using the image processing software Velocity, areas presenting the base of the intestinal crypts were defined and the mean fluorescence intensity per unit was calculated. Statistical comparison by Student’s t-test reveals a statistically significant decrease in staining intensity in the PTPσ(-/-) mouse (n=95 crypts, Student’s t-test, p<0.05). Error bars represent mean +/- SE.

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Figure 15: Lysozyme expression is reduced in the Paneth cells of the PTPσ(-/-) small bowel

66

Figure 14: Ki-67 immunostaining is similar between PTPσ(-/-) and PTPσ(+/+) mouse small bowel and colon. A) Ki-67 immunostaining in the crypt region of paraffin-embedded tissue sections from PTPσ(+/+) and PTPσ(-/-) mice small bowel reveals no difference in staining pattern or intensity. Scale bars = 90μm. B) Ki-67 immunostaining in the crypt regions of the colon of PTPσ(+/+) and PTPσ(-/-) shows no apparent difference. Scale bars = 90 μm.

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Figure 16: Ki-67 immunostaining, a marker for cell proliferation, appears unchanged in PTPσ(-/-) mouse small bowel and colon

68

implicated in modulating this proliferation including Wnt, Notch, and EGFR signaling. It is also

known that tyrosine phosphorylation is an important mediator in these pathways, and that

increased tyrosine phosphorylation is often associated with increased cell proliferation.

Furthermore, PTPσ is known to directly target key factors in these pathways, namely β -catenin

in Wnt signalling and EGFR. To determine if the observed phosphotyrosine staining pattern in

the PTPσ(-/-) mice is indicative of increased cell proliferation, immunostaining for the nuclear

proliferation marker Ki-67 was performed on small bowel and colon tissue sections from PTPσ(-

/-) mice and wild-type littermates (Figure 14). No difference in Ki-67 staining was observed

between PTPσ(+/+) and PTPσ(-/-) small bowel and colonic tissue sections. This would suggest

that PTPσ is not mediating cell proliferation in the intestinal crypts. Further analysis of cell

proliferative markers will be necessary to fully conclude that cell proliferation is unaffected.

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Chapter 4: Discussion

70

Role of PTPσ in intestinal epithelia

1) Evidence of defects in intestinal barrier integrity

The work presented here demonstrates a defect in the intestinal barrier integrity of the

PTPσ(-/-) mice. The reduced epithelial resistance (Figure 1A) and increased macromolecular

flux (Figure 1B) observed suggest a destabilization of the epithelial monolayer that forms the

barrier between the intestinal lumen and lamina propria. The presence of compromised barrier

integrity is further supported by the increased intestinal permeability revealed by in vivo tracer

studies (Figure 1C,D). Cell adhesion, mediated by the apical junction complex, is essential to

the barrier function of polarized epithelia[128]. Interestingly, the mass spectrometry analysis

(Table 1) identified numerous hyper-tyrosine phosphorylated proteins in the PTPσ(-/-) mouse

intestine with functions associated with the apical junction complex. These hits include proteins

that mediate intercellular junctioning (E-cadherin, β-catenin, Tjp-2), proteins which regulate

interactions with the actin cytoskeleton (villin, ezrin, gelsolin) and proteins which regulate

signaling pathways that modulate complex formation and remodeling (EGFR, ezrin). In the

context of the adherens junction specifically, 18 putative substrates were identified in our screen

that directly modulate junction formation and stability (Table 1; red highlight). Given that PTPσ

is known to localize to the adherens junctions[131], the data suggest that PTPσ may regulate

adherens junction proteins directly at sites of intercellular junctions. The observed defect in

permeability and the proteins identified in the MS screen support our hypothesis of PTPσ

functioning as a positive regulator of epithelial barrier integrity in the intestine through the

regulation of proteins associated with the apical junction complex.

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2) Role for E-cadherin and β-catenin

E-cadherin and β-catenin form the essential core of the adherens junction and were

previously validated as colonic PTPσ substrates by our group[13]. Dysregulation of E-cadherin

has been shown to decrease epithelial cell adhesion and increase paracellular permeability[162,

163]. Studies have also demonstrated that tyrosine phosphorylation mediates the cadherin-

catenin interactions and that enrichment of pTyr is associated with complex disassembly[136,

164]. Other PTPs, such as PTPµ[61], have been shown to modulate E-cadherin – β-catenin

binding at the adherens junction through regulation of these pTyr residues. The tandem MS

screen indicates increased tyrosine phosphorylation on E-cadherin and β-catenin in the PTPσ(-/-)

mouse intestine (Table 1). Interestingly, we observed increased tyrosine phosphorylation at the

plasma membrane in the PTPσ(-/-) mouse intestine compared to wild-type controls (Figure 3).

However, we did not observe a significant difference in E-cadherin- β-catenin colocalization at

the basolateral plasma membrane in the PTPσ(-/-) mouse intestine compared to wild-type

littermates (Figure 9). These results taken together could suggest several different scenarios.

PTPσ may regulate the stability of the adherens junction by targeting pTyr residues on E-

cadherin and β-catenin that modulate the cadherin-catenin interaction but not complex

internalization. This would alter cellular adhesion but would presumably not affect the

localization of E-cadherin and β-catenin to the plasma membrane. Further, immunofluorescence

microscopy may not be sensitive enough to observe complex destabilization which is evident

through functional analyses. In this scenario, loss of PTPσ regulation on E-cadherin- β-catenin

could explain the defective barrier phenotype observed in the PTPσ(-/-) mouse. However, in

contrast, it is possible that the permeability defect in the PTPσ(-/-) intestine is not related to the

adherens junction, as the colocalization data does indicate that E-cadherin and β-catenin are at

their appropriate subcellular location. The observed plasma membrane staining for pTyr in the

72

intestinal crypts may not be related to E-cadherin or β-catenin, but could represent other PTPσ

targets at the membrane. Given the multitude of AJC proteins identified in the MS screen, it is

also possible that PTPσ is influencing the function of the adherens junction through other

effectors besides E-cadherin and β-catenin.

It is important to note that these results assert that epithelial cell adhesion is not

completely abrogated by loss of PTPσ, which is clear given that total loss of AJC-mediated

adhesion would prevent epithelial layer formation, collapsing the overall morphology of the

colon and small bowel. Alternatively, it is possible that changes in epithelial permeability in the

PTPσ knockout intestines may not be the result of disrupted junctioning but are instead the result

of dysregulated paracellular trafficking, which is known to be mediated by the AJC.

The lacZ (Figure 8) and phosphotyrosine (Figure 9) immunostaining suggest that PTPσ is

expressed in cells within the lamina propria regions of the small bowel and colon. Lymphocytes

are known to be present in these regions, especially at sites of inflammation[165]. Importantly,

several T-cell secreted pro-inflammatory cytokines, including TNF-α[166, 167], IL-4[166, 168],

and IL-6[169], have been shown to modulate AJC stability which can influence the barrier

integrity of the intestine. It is possible that the disrupted barrier integrity observed in the PTPσ(-

/-) mouse may be the result of dysregulated pro-inflammatory cytokine secretion from these T-

cell populations. Further experimentation will be necessary to differentiate which effects are

immune-driven and which are adhesion-mediated.

3) Ezrin as a colonic PTPσ substrate

My results demonstrate that ezrin is a direct intestinal substrate of PTPσ (Figure 5), and

that ezrin localization is altered in the PTPσ(-/-) mouse following DSS-treatment (Figure 6). This

change in localization observed in the DSS-treated PTPσ(-/-) mouse small bowel suggests that

73

PTPσ regulates the pTyr phosphorylation status of ezrin, which is known to dictate plasma

membrane targeting. The presence of this mislocalization only during DSS-treatment does

suggest that other PTPs are likely also involved in the regulation of ezrin trafficking, given that

we still observe ezrin at the plasma membrane in the untreated mice. Tyrosine phosphorylation

is also essential to the function of ezrin as a scaffold protein. Several binding partners of ezrin,

including Rac1[143], Src[170], and p85[171] bind through SH2 domain-mediated interactions.

Once bound, these effectors can become activated through interactions with other proteins that

have also been recruited by ezrin, including guanine nucleotide exchange factors (GEFs) like

Dbl[172]. PTPσ could potentially regulate the activation of these effectors by targeting pTyr

residues that serve as SH2 binding sites, maintaining a dephosphorylated state that would prevent

recruitment of factors necessary to activate downstream signaling pathways. Interestingly,

several of these signaling pathways regulated by ezrin are important for cell adhesion. Rac1 is a

potent regulator of actin cytoskeletal dynamics and has been shown to regulate adherens junction

assembly through E-cadherin[143]. In addition, Src is a known mediator of cell adhesion and

adherens junction remodeling[170]. PTPσ may be indirectly modulating the stability of the

apical junction complex through regulating the activation of upstream effectors of epithelial cell

adhesion.

Proposed Mechanism: PTPσ regulation of adherens junction proteins We propose that PTPσ acts at the adherens junction to regulate the tyrosine

phosphorylation of AJC-associated proteins to maintain junction stability. Under normal

conditions (Figure 15A), PTPσ and associated RTKs work in opposition to modulate tyrosine

phosphorylation levels of AJC proteins according to the changing intestinal environment and the

needs of the cell. In the PTPσ(-/-) mouse (Figure 15B), loss of PTPσ regulation leads to an

74

accumulation of tyrosine phosphorylation on adherens junction proteins as the activity of the

RTKs are no longer counteracted by phosphatase activity. Key elements at the adherens

junction, including E-cadherin, β-catenin, and signal mediators such as ezrin, become hyper-

tyrosine phosphorylated, leading to a destabilization of the junction as cadherin-catenin

interactions dissociate and/or adherens junction remodeling pathways become dysregulated.

Consequently, as the intercellular interactions breakdown, paracellular permeability increases,

leading to the observed changes in TER found in the PTPσ(-/-) mice. This increased

permeability may lead to increased inflammation as pathogens in the intestinal lumen have

increased access to the lamina propria, causing an abnormal immune system response.

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Figure 15: Proposed mechanism for PTPσ mediated regulation of the adherens junction. A) PTPσ localizes to the adherens junction and works in concert with RTKs to modulate the tyrosine phosphorylation of AJC proteins. B) In the PTPσ(-/-) mouse, PTPσ is not expressed therefore the activity of the RTK is not counteracted, leading to an accumulation of tyrosine phosphorylation of AJC associated proteins. Increased tyrosine phosphorylation destabilizes cadherin-catenin interactions, leading to complex disassembly. In addition, tyrosine phosphorylation leads to the up-regulation of adherens junction remodeling pathways through modulators such as ezrin increasing the instability of the junction. A combination of these effects leads to reduced intercellular adhesion, increasing paracellular permeability. p120 – p120-catenin; β-cat – β-catenin; α-cat – α-catenin; Vcl – vinculin; FMN – formin; orange helix – F-actin

76

Figure 17: Proposed mechanism for PTPσ regulation of adherens junction stability

A

B

77

Implications for PTPσ in Paneth cell function The X-gal staining pattern observed in the small bowel of the PTPσ(+/-) mouse (Figure

6E) demonstrates strong staining at the base of the crypts, a region known to contain secretory

Paneth cells. The reduced lysozyme immunostaining observed in the neonatal (Figure 12B) and

adult (Figure 13A) PTPσ(-/-) mice suggest that PTPσ may play a role in the regulation of Paneth

cell function. Paneth cells contribute to intestinal barrier defence through the secretion of anti-

microbial factors to mediate bacterial clearance. Paneth cell dysfunction has been associated

with inflammation and reduced microbial clearance. Interestingly, reduced bacterial clearance

and increased bacterial colonization in the intestinal crypts (following inoculation by C.

rodentium) has been observed in the PTPσ(-/-) mouse previously by our group[13]. PTPσ could

potentially regulate processes associated with Paneth cell lysozyme production or secretion.

Furthermore, autophagy is known to play an important role in mediating Paneth cell granule

formation, which is necessary for its secretory function. Given the recently identified role for

PTPσ in the regulation of autophagy, it may be possible that loss of PTPσ is affecting the

maturation of secretory granules in the PTPσ(-/-) Paneth cell. Although the underlying effect is

unknown, the evidence suggests that PTPσ may influence Paneth cell function, although it is a

role that will require further investigation.

PTPσ in IBD pathogenesis In the context of IBD pathogenesis, it is possible that the defective barrier integrity

observed in the PTPσ(-/-) mouse intestine may contribute to the development of intestinal

enteropathies by increasing the exposure of the lamina propria to luminal pathogens. A ‘leaky’

intestinal epithelium has been previously implicated in IBD[128, 173]. Interestingly, barrier

defects have been observed in IBD patients[174] but the underlying genetic basis for these

effects were not well characterized. Our work presented here suggests a novel function for PTPσ

78

as a positive regulator of epithelial barrier integrity, which may help to explain the underlying

genetic risk in IBD associated with defects in permeability.

Future Directions

1) Further characterize the intestinal barrier defect in the PTPσ(-/-) mouse

The barrier defect observed in the PTPσ(-/-) mouse requires further characterization to

understand the underlying defects causing the destabilization of the epithelial monolayer.

Biochemical analysis of binding affinities between E-cadherin and its various catenin adapters in

the PTPσ(-/-) mouse would provide more evidence for the direct regulation of the cadherin-

catenin complex by PTPσ. If the members of the complex have reduced binding affinities

correlated with increased tyrosine phosphorylation, it would support the hypothesis that PTPσ is

a positive regulator of junction stability. The role and function of the tight junctions has also not

been evaluated in the PTPσ(-/-) mouse. Tjp-2 (ZO-2), an important mediator of tight junction

anchorage to the actin cytoskeleton, was identified in the MS screen as well as other modulators

of TJ formation. Given that tight junctions play an important role in mediating paracellular

transport, it would be important to characterize the stability of this junction in the PTPσ(-/-)

mouse.

2) Investigate the intestinal ligands of PTPσ

To date, the intestinal ligands for PTPσ have not been characterized. In order to fully

understand the extracellular signals that modulate PTPσ phosphatase activity, it will be necessary

to characterize which ligands bind to the ectodomain in the intestine. In addition to determining

the intestinal ligands, binding experiments can be conducted with PTPσ ectodomain mutants that

mimic the alternatively spliced variant implicated in UC pathogenesis (PTPσΔmeB, ie

79

PTPσΔIg3). Binding affinities could be measured to determine if the splice variant alters ligand

binding or the ability of the receptor to dimerize. This information will contribute to our

understanding of how this splice variant leads to IBD pathogenesis.

3) Evaluate the effect of PTPσ regulation on ezrin function

The effect of PTPσ regulation on ezrin function is not clear. Preliminary evidence does

suggest that PTPσ regulation may regulate ezrin localization, however, this effect will need

further validation using mutational analysis of tyrosine residues implicated in ezrin function.

First, it will be necessary to determine which specific phosphotyrosine residues in ezrin are

targeted by PTPσ. Mass spectrometry can be performed on intestinal epithelial cells both naïve

and transfected with PTPσ-targeted siRNA. Comparison of the tyrosine phosphorylation profile

of ezrin under these different conditions will provide evidence as to which residues are targeted.

Once these residues have been determined, site-directed ezrin mutants, with tyrosine mutated to

phospho-mimetic (Glu) or phospho-ablated (Phe) residues, can be generated. These mutants

will provide deeper understanding of how tyrosine phosphorylation affects ezrin function and

how this, in turn, influences downstream signalling which could affect epithelial cell function.

As a corollary to the mutagenesis, the signalling of ezrin can also be evaluated through

analysis of the activation of downstream effectors molecules such as Src or Rac1. This will

determine if adherens junction remodeling pathways are being activated due to loss of signal

attenuation by PTPσ.

4) Validate and characterize other putative substrates identified in MS screen

Several other potent AJC effector proteins were identified in the MS screen and their

dysregulation could contribute to the phenotype observed in the PTPσ(-/-) mouse. To determine

which of these putative substrates are real in vivo PTPσ targets, substrate trapping and

80

dephosphorylation experiments need to be repeated. Validated interactors should be examined

using immunofluorescence microscopy and other functional experiments to determine if their

function is altered in the PTPσ(-/-) mouse. Identification of more intestinal substrates for PTPσ

will provide a clearer picture of the downstream regulatory targets of PTPσ and how these may

influence intestinal cell function.

5) Explore the role of PTPσ in other immune cells

Evidence from the lacZ staining identifies X-gal positive cells present in the lamina

propria region of the PTPσ(+/-) intestine. As this region is known to contain lymphocytes,

which mediate immune function, it is possible that PTPσ may play a role in adaptive immunity.

PTPσ expression in T-cell lineages has been previously described[175]. Defective T-cell

function has been linked to inflammation and to IBD specifically. Furthermore, specific

cytokines, namely IL-6[169] and IL-10[176], have been implicated in the regulation of epithelial

barrier defence through the modulation of barrier permeability[177] Evaluating the secretion of

pro-inflammatory cytokines by T-cells in the PTPσ(-/-) mouse intestine by ELISA and

characterizing the subsets of T-cell populations present in the mice by FACS sorting would help

to determine if PTPσ plays a role in modulating T-cell mediated immune function.

6) Explore the role of PTPσ in Paneth cell function

The reduced lysozyme immunostaining observed in the PTPσ(-/-) mouse suggests that

Paneth cell function may be influenced by loss of PTPσ. To determine if this effect is due to

direct PTPσ regulation, the function of Paneth cells can be evaluated in the PTPσ(-/-) mouse

using functional assays and immunofluorescence microscopy. Expression levels of key

antimicrobial factors can be evaluated by Western blotting, while the role of autophagy in the

81

Paneth cells can be characterized by identifying the presence of autophagic markers by

immunofluorescence.

7) Generate tissue specific, catalytically-inactive and ΔIg3 PTPσ mouse mutants

The PTPσ(-/-) mouse line used for the experiments outlined in this report is a whole-

tissue knockout. This system cannot separate which effects are intestinal-specific and which are

caused by dysfunctions in other organs influencing intestinal function through mediators such as

hormones or cytokines. Given the range of processes affected by loss of PTPσ function, the

phenotype observed in the mice may be the result of a combination of these effects. To discern

to what degree intestinal PTPσ function contributes to the observed colitis phenotype, a tissue-

specific knock-out mouse generated using the loxP-cre recombinase system crossed against an

intestinal-specific Villin-Cre mouse would isolate the knock-out to the intestine to better

understand the phenotype. In the context of IBD pathogenesis, the effect of the Ig3 deletion

observed in UC patients on the function of the PTPσ has not been characterized. In order to

determine if this alternative splicing affects PTPσ signaling, an ΔIg3 PTPσ knock-in mouse

model can be generated. These mice would be more representative of the putative phenotype

expected in the UC patients as this model would reflect the specific disease associated variant,

isolating the effects caused by the polymorphism from the phenotype associated with the

complete PTPσ knock-out.

Another in vivo model that would be informative would be to generate a catalytically

inactive PTPσ mutant (PTPσ-CI) by mutating the catalytically essential cysteine residue within

the full-length protein. This would help to differentiate which effects are caused by loss of the

CAM-like function of the ectodomain of PTPσ versus its intracellular phosphatase activity.

82

Summary The intestinal barrier integrity of the PTPσ(-/-) mouse was disrupted as observed through

both ex vivo and in vivo assays, suggesting that PTPσ plays a role in modulating barrier defence.

Tandem mass spectrometry identified thirty-seven hyper-tyrosine phosphorylated proteins in the

PTPσ(-/-) intestine, a large proportion of which are known to regulate the formation and

maintenance of the apical junction complex, a key mediator of cellular adhesion. One of the

identified hits, the cytosketelal linker protein ezrin, was demonstrated to be a direct PTPσ

substrate and its cellular localization was found to be disrupted in the PTPσ(-/-) mouse small

bowel under DSS conditions, suggesting it is an in vivo PTPσ substrate. Paneth cell function

also appeared to be altered in the PTPσ(-/-) small bowel. Taken together, these results suggest

that PTPσ regulates the tyrosine phosphorylation of apical junction complex proteins, which in

turn modulates the permeability of the epithelial monolayer.

Conclusion The impaired barrier function observed in the PTPσ(-/-) mouse implicates PTPσ as

a positive regulator of barrier integrity. The hyper-tyrosine phosphorylated proteins

identified in the mass spectrometry screen suggest that PTPσ may target apical junction

complex proteins. My results also suggest that PTPσ may play a role in Paneth cell

function.

83

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