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Integrin-Linked Kinase 1 (ILKl) is necessary for myogenic differentiation in rat L6

myoblasts

Mathew Gordon Miller

A thesis submitted in conformity with the requirernents for the degree Master of Science, Graduate Department of Laboratory Medicine and Pathobiology, University of Toronto

@Copyrightby Mathew Gordon m e r 2000

191

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Integrin Linked Kinase 1 (ILK1) is necessary for myogenic differentiation in rat L6 myoblasts MSc 2000 Mathew Gordon Miller Department of Laboratory Medicine and Pathobiology, University of Toronto

Abstract

Primary myogenesis is easily modeled in vitro using myogenic ce11 lines such as C2C 12

and L6. This process is regulated by extracellular stimuli and intracellular signalling pathways. Through inhibitor studies, phosphatidylinositol3-kinase (PI3-K) and protein

kinase B (PKB) have been shown to be intracellular signalling molecules downstream of

growth factor and ECM signals necessary for myogenesis in vitro. Integrin-linked kinase

1 (ILKI) is a serine-threonine kinase shown to transduce ECM and growth factor signals

through the PD-Kand PKB. The data in this thesis show that inhibition of L K l signalling by transfection w t a dominant-negative mutant ILK(E359K) blocks ih rnorphological and biochemical differentiation of L6 rat myoblasts. These data suggest

ILKl is a required component in a myogenic differentiation pathway involving PD-K. Further expenments will focus on further characterization of, and experirnentation with.

the stable ce11 lines generated in this study, in order to dissect the differentiation and survival pathways in myogenic cells.

Table of Contents

Page Introduction 1 .O Skeletal Myogenesis in vivo and in vitro 2.0 Regulation of in vitro myogenesis 2.1 Myogenic Regdatory Factors 2.2 Growth Factor Signals 2.3 Extracellular Matrix (ECWntegrin signals 2.4 Natural and Engineered Gene Deletions 2.5 ECMIGrowth Factor Cross-Talk 3 .O Integrin-Linked Kinase (ILK 1) and Myogenesis 3.1 ILKl Structure and Function 3.2 ILKl Signalling in Myogenic Pathways

Experimental Procedures Results Discussion Literature Cited

List of Tables and Figures

Page

Figure 1. ILK l signalling pathways schematic. Figure 2. ILKl kinase activity is stirnulated in L6 myoblasts by ECM proteins relevant to myogenesis.

Figure 3. Regulation of ILKl activity in L6 myoblasts by the myogenic factor insuiin

Figure 4. Dominant negative ILK 1 mutant blocks morphological and biochemical differentiation of L6 rnyoblasts.

Figure 5. Sarcoplasmic reticulum [ C a " ] ~ ~ ~ a induction typicai of myogenic se differentiation in L6 is completely eliminated by transfection with ILK(E3 59K), and is amplified by transfection with wtILK1. Table 1 Ce11 cycle andysis of L6 myoblasts (vector control and ILK : transfectmts) under growth (GM), difFerentiating (DM), semm-free (SF), and serum-free plus lOOnM insulin (INS).

Figure 6. Insulin induces Ser473 phosphorylation of PKB in ILK(E359K) expressing L6.

List of Abbreviations

a-MEM Alpha Minimum Essential Media

ATP adenosine triphosphate

ADP adenosine diphosphate

bHLH basic Helix-Loop-Helix

cDNA complementary deoxyribonucleic acid

CEM Chick Bmbrysnis M y o b l a s ~

CHO Chinese Hamster Ovary

CMMD Congenital Merosin-Deficient Muscular Dystrophy

DAPI 4' ,6-Diamidine-2'-phenylindole dihdryochloride

DM Differentiation Media

DNA deoxyribonucleic acid

ECM Extracellular Matrix EDTA Ethylenediaminetetracetic Acid

EGF Epidermal Growth Factor

FACS Fluorescence Activated Ce11 Sorting

F Focal Adhesion Kinase M

FBS Fetai Bovine S e m FGF Fibroblast Growth Factor FN Fibronectin

GM Growth Media HEK Hurnan Embryonic Kidney

Id Inhibitor of Differentiation

IEC Intestinal Epithelial CeII

IGF Insulin-like Growth Factor ILK 1 Integrin-Linked Kinase One

IRS-1 Insulin Receptor Substrate One

LN Laminin

MAPK Mitogen-Activated Protein Kinase

MBP Myelia Basic Protein

MEF2 Myogenic Enhancing Factor 2

MN Merosin (LN-2)

MRF Myogenic Regdatory Factor

MyoD Myogenic Factor D

NP40 Nonidet P-40

PARP Poly (ADP-ribose) polymerase PCR Polymerase Chain Reaction PDGF Platelet-Derived Growth Factor PH Pleckstrin Homology PI3-K Phosphatidylinositol3-kinase

PIP3 Phopshatidylinositol Triphosphate PKB Protein K n s B (Akt) iae

PVDF Polyvinyldiene Fluoride

RNA Ribonucleic Acid

UT-PCR Reverse-Trascriptase Polymerase Chain Reaction

Ser Serine

SDS-PAGE Sodium Dodecylsulfare Polyacrylamide Gel Electrophoresis

TGF-B Transfomihg Growth Factor Beta

TNF Tumour Necrosis Factor

TUNEL Terminal Deoxynucleotidyl Transferase-mediateddUTP-X Nick End Labelling

wt Wild-Type

"This crowd has gone deadiy silent, a Cinderella story outta nowhere. Former greenrkeeper and noiv about to becorne the Masters champion. Cinderella Boy. "

-Car1 Spackler

1 would like to thank my supervisor Dr. Greg Hannigan for providing an excellent lab in which to complete this work. I would also like to acknowledge the work of Dr. Herman

Yeger, Dr. Dwayne Barber, and Dr. Harry Elsholtz on my advisory cornmittee. 1 was supported by an NSERC PGSA scholarship, and a Hospital for Sick Children ResTraComp scholarship. Thanks especidly to Chungyee Leung-Hagesteijn and Izabela Nmszewicz for constant help at the bench. I could not have completed this degree without the love and support of Jen Oblender.

introduction

1.0 Skeletai Myogenesis in vivo and in vitro

In vertebrates, ail skeletal muscle is derived from somitic cell precursors (Currie and lngham

1998). Somites are epithelial spheres of mesodemal cells that arise along the antero-posterior avis along side the neural tube. During development, the more ventral somitic cells lose their epithelial morphology and give rise to loosely connected mesenchymal cells known as the sclerotome. These cells are precurson of the ribs, vertebrae, and intervertebral discs (Kalcheirn et

al. 1999). The remainder of the somite exists as an epithelial structure consisting of the

dermomyotome (adjacent to the neunl tube) and myotome (more distal). Cells of the myotome rnigrate out to peripheral targets and give rise to ail muscles, while cells of the demomyotome xa migrate lateraily and form the body wall and limb musculature (Brand-Saben and Christ 1999, Emerson, Jr. 1993).

Inductive cues fiom surrounding tissues determine the fate of these somitic cells during the above processes. These eues dso signal the determination of these ceils as skeletal muscle precurson,

and, once the5 peripheral targets have been reached, the differentiation of these precursor

myoblasts into mature muscle fibers (Olson 1992). At their peripheral targets, these determined, somite-denved cells start expressing a group of muscle-speci£ic regdatory genes and may now be identined as myoblasts (Sabourin and Rudnicki 2000). These myoblasts next exit the cell cycle,

and begin to fuse and form multinucleate myotubes; a process referred to as primary myogenesis

(Cossu et al. 1996). Next, ushg the prirnary myonibes as scaffolding, a distinct population of secondary myoblasts slip under the basement membrane of the primary myonibes, fuse with each other, and form secondary myotubes (Olson 1992, Sabourin and Rudnicki 2000). Following these early events, significant reorganization must occur to achieve the organism's finai muscle structure. Secondary muscle fibres Form an independent basement membrane fiom that of the primary fibres, and the muscles split, become innervated, achieve their fuial pattern and grow (Gullberg et al. 1998).

One of the earliest steps in the conversion of myoblasts to muscle fibres is the elongation and fusion of myoblasts, to form multinucleated myotubes, and the induction of muscle-specific regulatory genes (Olson and Klein 1994). This carly myoblast fusion event is modeled in the mouse C2C 12 and rat L6 myoblast lines, among others, where differentiation is induced by s e m

withdrawal, or ce11 treatment with insulin or insulin-like growth factors (IGFs) (Stockdale 1992.

YaEe 1968).

Typically, myogenic differentiation is assessed in these mode1 systems

morphologically and biochemically. MorphoIogically, the presence of long, rnd~ucleate myotubes indicates myoblast differentiation in vitro. Biochemicai markers of terminal myogenic differentiation can include the myogenic regulatory factors myogenin and MyoD, the structural protein desmin, muscle-specific functional proteins such as the sarcoplasmic reticulurn [ C a r ~ T ~ a expression and creatine kinase activity, among othen (Mitsumoto et al. 1993). se These models have been u&ed fusion event. extensively to study the mechanisms that regdate this prirnary

2.0 Regulation of in viiro skeletal myogenesis

2.1 Myogenic Regulatory Factors

Myogenic regdatory factors (MRFs) are members of the basic heliu-loopheliu family of transcription factors. The MRF subfarnily includes MyoD, Myf-5, myogenin, and MRF4 (Olson and Klein 1994). These proteins contain a conserved basic DNA-binding domain necessary for sequence-specific DNA binding and a heliu-loopheliu motif required for heterodirnerization (Rudnicki and Jaenisch 1995).

MRFs heterodimerize with a farnily of

ubiquitous bHLH proteins. known as E proteins, and bind DNA at sites known as E boxes. E boxes are found in the promoters of many skeletal muscle-specific genes and rnediate gene activation in a MRF-dependent rnanner (Olson and Klein 1994). This family of transcription factors is unique in their ability to induce myogenic differentiation when transfected into nonmyogenic cells such as fibroblasts. In addition to activating genes associated with myogenesis, MRFs can auto- and cross-activate one anothers' expression in transfected cells (Borycki and Emerson 1997, Buonanno and Rosenthal 1996). These autoregulatory interactions may be responsible for arnplifying the expression of myogenic bKLH proteins above a threshold required for terminal differentiation or for conferring stability to the myogenic program. For example, the promoter for the m o w myogenin gene contains an E-box which rnay serve as a target for

regdation by other MRFs, or by rnyogenin itself (Nabeshima et al. 1993, Arnold and Winter 1998).

Recentiy, additional complexity has been noted in this family of transcription factors. Ju et al. (1999) have demonstrated that MyoR, another transcription factor of the bHLH family, serves

as a negative regulator of myogenic differentiation. This protein is developmentally regulated in

mouse: it is expressed frorn embryonic day 10.5 to 16.5, but drastically down-regulated after this tirne, during the penod of secondary muscle development. In ce11 culture MyoR expression is dom-regulated during myogenesis, and blocks the ability of MyoD to activate myogenesis. The regdation of myogenesis by the bHLH family of transcription factors involves both positive and negative regulators. The most well-charactenzed of these negative regulators is the Id (inhibitor of differentiation) subfarnily of bHLH proteins. Id proteins do not contain DNA binding domains and thus interfere with the heterodirnerization, and subsequent DNA binding, of MRFs by forming inactive heterodimers wt MRFs. Overexpression of Id proteins in Sol8 myoblasts ih

and marnmary epithelial cells inhibits differentiation, and Id proteins are downregulated in

response to myogenic differentiation signals (Melnikova et al. 1999, Desprez et ai. 1995).

MRFs are expressed in a temporal pattern through muscle development. In vitro, most myogenic

cetl lines express MyoD a d o r Myf-5 in the proliferative, myoblastic state.

During

differentiation, bHLH factors present in determined myoblasts initiate a pathway which leads to

the activation of the MEF3 (non bHLH) family of transcription factors (Lassar and Munsterberg 1994).

MEF2, in tum, directly interacts with MRFs and is necessary to activate the

transcription of myogenin, as well as other muscle-specifk genes (Sabourin and Rudnicki 2000).

Myogenin itself activates MEFZ function as well, creating a positive feedback loop enswing

myogenin expression is maintained in differentiated muscle (Naya and Olson 1999). Upon the

initiation of terminal differentiation myogenin andor MRF4 are expressed in myobiasts which

have exited the ceil cycle. Thus, in cell culture systems, such as the myogenic L6 and C2C12

lines, myogenin and MRF4 are considered markers of terminai differentiation (Gu et al. 1993).

Gene targetting studies have demonstrated that MRFs also regulate myogenesis in vivo. Mice lacking Myb and MyoD are completeiy without skeletal myoblasts and myofibres, apparently evidence for functional redundancy between the two factors. However, more recently, it has been proposed that MyoD and MyR regulate distinct lineages of differentiating muscle cells in

vivo. Myf5 is thought to regulate trunk muscle development, while MyoD would determine

early limb and branchial arch muscle (Kablar et al. 1997). Other work has demonstrated that myogenin and Myf5 are functionally interchangable in determining myogenic lineage and regulating rib formation in vivo, but that certain functions canied out by Myf5 c m o t be c d e d out by ectopically expressed myogenin (Wang and Jaenisch 1997). This may indicate that some aspects of myogenic regulation may be unique to a given MRF and are perhaps due to either differences in functional protein domains, or in their temporal and spacial expression in vivo. The importance of MRFs in the regulation of in vitro and in vivo myogenesis validates the use of well-characterized cell culture systems, such as rat L6 myoblasts, in the study of myogenic differentiation.

n Clearly, MRFs play vital roles in the regulation of skeletai rnyogenesis i vivo and in in vitro ceii

culture systems comrnody used to study these regdatory pathways. The extracellular stimuli

which elicit these nuclear responses have also been extensively studied using the weii

characterized i viko systerns available. n

2.2 Growth Factor Signals

The pmfound effect of peptide growth factors on the regulation of in vitro myogenesis has been

cleariy demonstrated. These growth factors c m be divided into two functiond groups with respect to their influence on the differentiation of myoblasts to fom myotubes. One group of soluble factors which includes fibroblast growth factor (FGF), transforming growth factor beta (TGF-B), and platelet deived growth factor (PDGF), inhibit myogenic differentiation in vitro. A second group of growth factors, consisting of insulin, insuiin-like growth factor one (IGF-I), and insulin-like growth factor two (IGF-II), are potent inducers of myogenesis i vino (Olson 1992). n

FGF and TGF-p are the two growth factors which most potently inhibit myogenesis in vitro.

Both FGF and TGF-f3 prevent myoblast differentiation through a mechanism independent of ce1

proliferation, and although it has not been determined if these growth factors regulate myogenesis

in vivo, they are both expressed at the correct place and time to perhaps regdate this process in

the developing embryo (Olson 1993, Allen and Boxhom 1989). These factors not oniy inhibit morphological differentiation, but also the expression and activity of biochemical markers of myogenesis. This would suggest that extracellular factors bind their appropriate receptors, and one or more signai transduction pathways carry this information to the nucleus, where they enact

specific effects on MRF funchon. Recently, it was s h o w that the inhibitory effect TGF-b has

on myogenesis can be attributed to the growth factor's ability to induce the export of the MEF2

transcription factor fiom the nucleus to the cy-toplasrn. This translocation prevents MEF2 fiorn

participating in an active transcriptional complex with MRFs, thus blocking the expression of

muscle-specific gene products (De Angelis et al. 1998) .

Not only has FGF been s h o w to inhibit myogenesis in vitro, but in addition, FGF is

downregulated with differentiation, and myogenesis can be induced by the expression of antisense FGF RNA (Groux-Muscatelli

et

al. 1990, Moore et a(. 1991). While the specific

signalling pathways which lead from the interaction of FGF and it receptor to the biological effects on differentiation have not been elucidated, recent work has shown that FGF receptor availability plays a critical role in myogenesis. In Sol8 muscle cells, myogenic differentiation can be induced by the expression of a truncated, dominant-negative FGF receptor, and conversely, constitutive expression of a full-length FGF receptor increased myogenesis in this system. Previous evidence had s h o w that receptors for both FGF and epidermal growth factor (EGF)are permanently lost during myogenic differentiation in vitro (Scata et al. 1999). It is also important to note that although FGF and TGF-P have similm effects on myogenesis, it is quite likely that

the signalling pathways which transduce these signais? at least at the eariier stages, are discrete,

because the FGF receptor is a tyrosine kinase, while the TGF-B receptor is a serine/threonine

kinase.

Other peptide growth factors are also able to inhibit myogenesis in vitro. Both platelet derived growth factor (PDGF) and tumour necrosis factor (TNF)inhibit the differentiation of myoblasts

in vitro (Szalay et al. 1997, Tidball and Spencer 1993). While each of these factors increases

myoblast proliferation, and inhibits differentiation, the specific signalhg pathways which transduce these signals have yet to be uncovered (Layne and Farmer 1999, Florhi et al. 1996).

Another distinct group of growth factors which includes insulin, IGF-1, and [GF-II, has drastically opposite effects on myogenesis as the group of peptide growth factors mentioned above. Insulin, IGF-I and IGF-II, aii are potent inducen of morphological and biochernical differentiation of myogenic cells in culture (Turo and Flonni 1982, Florini et al. 1996). While the specific nuclear events which mediate this induction still remain unknown, several recent studies have elucidated parts of the signalling pathway downstream of the binding of insuiin, or IGF-I or

II, with their respective receptors, and subsequent receptor tyrosine kinase activation that lead to

myogenic differentiation.

Through the use of both soluble inhibitor and dominant-negative transfection strategies, severai

groups have recently dernonstrated that phosphatidylinositol-3-kinase (PI3-K) is a downstream effector of insulin and IGF signais necessary for rnyogenesis in vitro. Kaliman et ai. (1996) first showed the ability of the PI3-K inhibitors LY294002 or worûnannin to block the differentiation of L6 rat myobiasts. Treatment with these inhibitors i the cdture media prevented n

morphologicai differentiation of the myoblasts as welI as the expression of severai marken of biochemical differentiation at the mRNA level, including myogenin, the p21 cdk inhibitor, and the

GLUT4 glucose transporter.

This treatment also prevented the differentiation of 10T112

fibroblasts transfected with MyoD. Transfection with MRFs such as MyoD is sufficient to induce myogenic differentiation in a varirty of non-myogenic cell h e s denved From each of the three germ layers (Olson 1993).

The above results From Kaliman et al. ( 1996) have been fûrther validated by P.K.Vogt's group

(Jiang et al. 1998) who e?camined the role of PI3-K in pnmary chick embyronic myoblast differentiation. Their work showed that transfection with a constitutively active PI3-K p l l Oa subunit increased the size of differentiated myotubes as well as elevated levels of several musclespecific proteins including MyoD, myosin heavy chah, and desrnin. Conversely, transfection

with a dominant negative mutant of the PI3-K p85 subunit (Ap85). or treatment with the

LY294002,interfered with morphological and biochemical differentiation in these cells.

More recent work by Kaliman et al. (1998) has provided additional evidence supporting the importance of PI3-K signals in myogenesis in vitro. Transfection of L6 myoblasts with a dominant negative p85 regdatory subunit of PI3-K blocked IGF induced myotube formation, myogenin gene expression and GLUT4 glucose transporter induction. T i effect was rnirnicked hs

by incubation with the LY294002 PD-K inhibitor. Taken together, these data provide strong

evidence that PI3-K transduces signds necessary for myogenesis in vitro, from such stimuli as

insulin and IGFs.

Protein kinase B (PKB)has been implicated as a downstrearn effector of PI3-K necessary for myogenic differentiation in vitro. A well established target of PI3-K signds, and mediator of ceil

survival signais, PKB is a serine-threonine kinase required for myogenesis in chicken embryo

o myoblasts (CEM) in v i ~ (Jiang et al. 1999). Ectopic expression of a constitutively active PKB

dramatically enhances myotube formation and expression of muscle specific proteins in CEM. Interestingly, this constinitively active PEU3 also rescues CEM from the blocks to morphological

and biochernical myogenesis imposed by incubation with the PI3-K inhibitor LY294002.

Ectopically expressing a wild-type PKB in this system induces a partial reversal of LY294002induced myogenic inhibition. Also, expression of a dominant negative PKB variant in these celis inhibits myotube formation and the expression of muscle-specific genes.

Other studies on P K B have also indicated it may be involved in the regdation of rnyogenesis in

vitro. Calera and Pilch (1998) have correlated an increase in activity and expression of the PKB

isoform Akt2 w t myogenic differentiation of Sol8 muscle ceiis. When mouse Sol8 cells are ih differentiated by ce1 confluence, serum withcirawd. or by exposure to insulin or IGF-1, AktZ protein levels rise dramatically, and undergo an increase in activity. Treatment of these ceiis with

LY294002 prevented this PKB induction, providing m e r evidence for its position downstream

of PI3-K.

More recentiy, another group has reproduced Calera and PiIch's (1998) data, showing P K B expression and activity are correlated with differentiation in C2C12 muscle ceUs (Fujio

el

al.

1999).

They aiso showed that by blocking the ceU cycle exit necessary for myogenic

differentiation, they were able to block PKB induction. Importantly, PKB was also shown to tramduce ceii survival signals in addition to participating in the regulation of myogenic diflerentiation. Transfection with two different dominant negative PM3 variants each increased apoptosis under serum-deprived conditions, as measured by Hoechst 33342 staining of condensed nuclei and TUNEL assay. Transfection with wild-type P U 3 partially rescued these

cells from the increase in apoptosis induced by serum withdrawal. This study shows that the

PKB may act in both differentiation and sunival pathways in myogenic cells. Taken together,

these data suggest that PEU3 can substitute for PI3-K in the regulation of myogenesis in vitro, and that PKB may be a downstrearn target of PI3-K essential for myogenesis. The data also provide

a strong case for P ' participation in both myogenic differentiation and survival pathways in ms

vitro.

To surnmarize: independent data fiom several in vitro systems have shown that growth factors

provide important regulatory signals for myogenesis. A large group of peptide growth factors including FGF and TGF-p inhibit myogenesis through largely unknown rnechanisms, while conversely, insulin and IGFs are potent of inducers of myoblast differentiation in vitro. Recent

in vitro studies have implicated PI3-K and PKB as important signal transduction molecules in a

myogenic pathway originating from the insulin and IGF receptors.

PKB signals have k e n

implicated in the regdation of celI survival in myogenic cells, in addition to king essential for their Merentiation.

2.3 Extracellular Matrix/Integrin Signals

In addition to growth factor signals, myogenesis is also regulated by signals provided by the extracellular matrix (ECM) (Gullberg et al. 1998). Integrins are the primary group of receptors for ECM proteins such as larninins, which are major components of muscle basement membranes

(Yamada 1997, Clark and Brugge 1995, Giancotti and Ruoslahti 1999). ECM promotes myotube

formation and survival through interactions with integrins, which transduce differentiation and anti-apoptotic signals (Sûeuli 1999). In vitro studies have revealed that ECM and i n t e g ~ repertoires change with the differentiation state of myogenic cells (Bozyczko et al. 1989,

Gullberg et al. 1998). In vitro, plating of myogenic cells on fibronectin stimulates myoblast

adhesion and proliferation, while retarding myotube formation. Plating of these cells on larninin-l enhances cell adhesion, m g a i n proliferation, and myotube formation (Vachon et al. 1996). irto, These effects indicate protein-specific effects on myogenesis generated through specific ECMintegrin interactions.

During myogenesis, myogenic cells undergo a major change in integrin complernent that is

accompanied by a change in the composition of the surrounding ECM. The ECM produced by

myoblasts is composed mainly of fibronectin, and the main integrins expressed are u5bl, the classic fibronectin receptor (Boettiger et al. 1995). However, differentiated myoblasts possess a basement membrane rich in laminin-1 and the muscle basement membrane-specific laminin

variant, merosin (iaminin-2). More specifically, 1arninin-l expression occurs during the early,

transitional, phase of myogenesis while merosin expression is strongly correlated wt myogenin ih

13

expression, and thus terminal differentiation (Vachon et al. 1996). Aiso during differentiation, the integrin complement of myogenic cells changes, as ct$l is replaced by a laminin receptor, u7B 1 (Song et al. 1992).

It is also interesting to note that changes in RNA splicing patterns affect the changing integrin repertoire during myogenesis. During in viîro differentiation, the cytoplasmic domain of the pl integin chah exhibits changes in its RNA splicing pattern from a

p 1A

form to a

p 1D

form

(Belkin and Retta 1998). In vifro,these 1D integnns localize to Z-bands and muscle attachment sites, posing important questions about their specific role in muscle biology. a 7 integrin also changes RNA splicing patterns during in vitro myogenic differentiation. a 7 integrin cytoplasmic domain splicing changes from the a7B form to the muscle-specific a7A fonn, and there is increased heterodimerization between a7A and

1D integrins in differentiated myotubes

7 (Schober et al. 2000). The extracellular domain of the a [email protected]

subunit aiso undergoes

XI alternative splicing in differentiated muscle cells, g e n e r a ~ g and X 2 isoforms which have

7 different affinities for laminin- l (Ziober et al. 1997). Both the the X1 and X2 variants of the a

integrin subunit are detected in adult cardiac muscle, lung, and undifferentiated myoblasts.

However, only the X2 form is found in adult skeletai muscle, suggesting that the splicing events

leading to the differential expression of the X1 and X2 variants are aiso developmentaiiy regulated. These data fit the proposed mode1 in which the regdation of ligand binding of the

14 a7XlPl receptor allows the dynamic adhesion events required during myogenesis, while the a7X2B1 provides the stable adhesion necessary at myotendious junctions and costamers.

The impact of these alternative splicing events on the biology of myogenesis is not Fully

rnderstood. Howewx, BeLlr. a d Retta (1 998) hswe demonstrated bat B 1D integrin expression

tnggers growth arrest in C2C12 myoblasts and NIH 3T3 fibroblasts. transfected with Cells transiently

P 1D integrin exit the

ce11 cycle and exhibit an inhibition of mitogen activated

protein (MM) kinases. Clearly, the P ID integrin isoform may participate in the transduction of

n growth inhibitory s i g d s during i vitro myogenesis. It is also important to note that, as these

ih expression background, the observed transfections were performed on cells wt a P1A i n t e g ~

effects were not due to an absence of 1A integrin signals, but radier a dominant-positive effect of pi D-transduced active signais.

It is possible that changes in integrin repertoire rnay impinge on several events important to the biology of myogenic differentiation. Not o d y might the a7Pl integrins participate in the synthesis and organization of the muscle basement membrane, but a change in integrin repertoire

may affect associated intraceliuiar signaluig pathways, thereby aEecting the regdation of

myogenesis itself.

The most definitive in vitro work investigating the role of the ECM in the regdation of myogenic differentiation and suMva.1 was done ushg differentiation-deficient clonal ceil Iines, and has shown that different laminin variants have specific effects on the growth, differentiation and survival of myoblasts (Vachon et al. 1996). At the mRNA level, the expression of laminin-2 (merosin) correlates with the terminal differentiation of

C2C12

myoblasts,

and

rhabdomyosarcoma cells, as measured by the induction of myogenin expression. This gradua1 upregulation of merosin expression is accompanied by a gradual decrease in laminin4 expression. Additionallyo inhibition of myogenesis in these ceil lines by the addition of the anti-myogenic growth factor, TGF-p, blocks the expression of merosin. These data ciearly indicate that differentiai expression of larninin-l and -2 variants is a function of the myogenic differentiation program.

To M e r investigate the specific biological roles of merosin and laminin-1 in myogenesis, Engvall's group selected Fusion-deficient and myotube-unstable myoblast cell lines (Vachon er al.

1996). Cells which formed unstable, apoptotic, myotubes were found to contain hi& levels of

laminin-1, but were completely merosin deficient, providing circumstancial evidence for the role

of merosin in myotube stability. Fusion-deficient myoblasts were f o n d to be deficient in both

lamitiui-l and merosin, suggesting a dual ECM signal regulating myotube formation. They then

directly assessed the function of laminin-1 and merosin on myogenic diflerentiation. When the myotube-unstable cells were plated on laminin-1 or fibronectin, no effect on myotube stability

was obsemed, however, plating on merosin exhibitied a clear dose-dependent increase of myotube

stability. These unstable myotubes were M e r shown, by TUNEL assay and DAPI staining for nuclear condensation, to undergo apoptosis. This clearly implicates merosin as a swival signai in these myogenic cells. In the fusion-deficient cells, plating on 1amililn-l or merosin induced a dose-dependent increase in myotube formation while, as expected, plating on fibronectin showed no effect. To M e r examine the role of merosin in myotube stability, Engvall's group transfected both the differentiation-deficient and myotube-unstable myobiasts with cDNA coding for full-length human merosin. Merosin expression rescued these cells fiom both the differentiation-deficiency and the myotube-instability. confimiing a role for merosin in

the differentiation of myoblasts, and subsequently, stability of myotubes. Interestingly, more

recent work fiom this group has Iînked merosin with the expression of the Pl D integrin isoform (Vachon er al. 1997). Immunostaining of merosin-deficient cells from the above snidies showed

that these cells show a disrupted expression of a7B 1D i n t e g ~ and that this disruption is not ,

compensated for by other laminin-binding htegrins such as a6B1 or ct6P4.

The ngnalling

pathway(s) which aansduce these survival and differentiation signals, and the nuclear events which produce the associated biological effects, have yet to be uncovered.

2.4 Naturai and engheered gene deletions

Loss of function experiments by targetted deletion of integrin subunits and ECM proteins in rnice, and by antibody perturbation, also indicate that integrin-mediated signalling is critical for

myotube formation and maintenance, and that ECM exerts specific effects during myogenesis. Early in vitro experiments using function blocking p1 integrin antibodies indicated that loss of B 1 fiction inhibited myotube formation (Menko and Boettiger 1987). However, more recent in

vitro studies using

8 1 negative ES cells have shown that p l

negative cells can differentiate into

fo rm

muscle in embryoid bodies (Hirsch et oi 1 998). A h ,

p 1 negative satellite cells isolated

c b e r i c embryos are able to fuse and form myohibes. However. these data are difficult to interpret. Although able to fuset $1 negative embryoid bodies expenenced delayed differentiation and a large number of unfused myogenic cells were noted. This still may reflect a role for BI integrhs in the fusion process. In chirneric embryos of wild type cells and pl integrin negative cells, m w l e fibres form in which B1 integrin-negative myoblasts have fused, indicating that ce11 migration fiom the somite has occurred in the absence of $1 integrin (Hirsch et al. 1998). These

data must aiso be interpreted cautiously, however, as it is dificuit to evaluate the role of integrins

in this migration event, given the wild type ce11 background in chimenc embryos. Signais nom pl integrins have also been shown to be important in ceii survival pathways through a loss of function strategy. Blocking 1 integrin function by antibody perturbation induces apoptosis and

drastically reduces myoblast survivai i vitro, irnplicating integrin signai transduction in these n

processes (Menko and Boettiger 1987).

Loss of function experiments targetting genes for integrin subunits and ECM molecules have also provided valuable information on the role in integrin and ECM in rnyogenesis. Deficiencies of the

a2 subunit of LN-Z/merosin in humans cause congenital, merosin deficient muscular dystrophy (CMMD), which can be modelled by targetted disniption of the a2 merosin subunit in mice

t (Vachon e al. 1997, Kuang et al. 1998, Hayashi et al. 1998). C M M D may involve loss of

function of LN-2 in both muscle and nerve tissue, since muscle-directed restoration of LN-2 expression only partially restores muscle integrity (Kuang et a . 1998). Homozygous deletion of l the a7 integrin subunit gene in mice leads to a progressive muscular dystrophy, suggesting altered function of the myotendinous junctions. The a7 subunit is thus critical for dystrophindystroglycan independent regdation of muscle fibre-ECM interactions and, interestingly. patients with CMMD specifically exhibit altered expression and localization of a7B1 integrin in myofibres, which is not obsemed in dystrophin-deficient patients or mice (Mayer et al. 1997).

The role of integrins in muscle development is cornplex, involving more than a7p 1 LN receptors.

The a5Bl integrin is a major receptor for fibronectin, which is an important rnediator of

mesenchymal ce11 motility, survival and shape changes (Sastry et a . 1996, Gullberg et a . 1995). l l

a5 integrin -1- chimeric rnice exhibited dystrophic muscle, with h& concentrations of as-nul1 i

l cells in skeletal muscle (Taverna et a . 1998). Features of dystrophic muscle in these mutant rnice

included giant fibres, with vacuoli, increased numben of large nuclei per fibre, and muscle degenemtion in head, thorax and limbs. The a 5 4 myoblasts differentiated nomally in vitro, however adhesion to, and sunival on fibronectin were impaired, indicating specific ECM influences on muscle developrnent relating to integrin a subunit identity (Tavema et a. 1998). l

More recently, knock-out and knock-in experiments with the f3 1D integrin subunit have provided valuable information on its function in myogenesis, but have yet to reveal an essentid role played by this splicing variant in myogenic differentiation. In fi 1D -1- mice, skeletal muscle formation proceeds normally (Baudoin et al. 1998) . The main heterodirnerizing partner of B 1D integrin,

wt PIA integrin, arguing against an essential role for p l D in myogenesis in vivo. In a knock-in ih

experirnent,

A integrin was replaced with

PL D integrin during development, resulting in a lethal

phenotype. This effect was mainly due ta effects to the nervous system and a reduction in ceil migration during development. The unique role played by f3 1D integrin in myogenesis has yet to be uncovered.

Antibody and genetic perturbation experiments. and elegant work with differentiation and myotube stability-deficient mutants, have provided critical insights into the Unportance of integrin-ECM interactions in muscle formation. Aithough the contribution of specific integins to certain steps of myogenesis has not been completely clarified, gene ablation experiments have

shown a minor role for B 1 integrins during early steps of myogenesis. Engvail's work on the role

of fibronectin, laminin-1, and merosin during myogenesis show convincingly that ECM-integrin interactions are essential for differentiation and sumival during specific steps of muscle

7 development in viîro. Gene targetting experiments have also s h o w an essential role for a

integrins and the a-2 chah of merosin in the stability of myofibers. Clearly, like sipals fiom

extracellular growth factors, integrin-ECM interactions signal specific critical events during myogenesis. Recent work has presented evidence that cross-talk occurs between growth factor

and ECM signals, and that this exchange of information may be important in the regulation of

myogenesis.

2.5 ECMIGrowth Factor Cross Talk

The above sections detailing growth factor and ECM signdling contributions to myogenesis make

a strong case that both groups of extracellular stimuli influence the regulation of myogenesis. The mechanism by which myogenic cells integrate these signds is not clear, however a growhg body of work has sought to understand the relationship and interaction between growth factor and

ECM signals, using several other biological rnodels. The ability of cells to integrate, and respond

to. a wide range of signais and combinations of signais is essential for the adaptability and sumival of organisms.

Several studies with epithelial ceil models have provided a strong argument that, in that system, signals fiom the ECM and Eorn soluble growth factors are necessary for survival. In mammary

epithelial ceils, appropriate cell-ECM interactions wt a basement membrane mediated by ih

specific integrins in combination with the presence of soluble hormones such as insulin is required for ceIl sumival (Farrelly et al. 1999). Removal or substitution of the appropriate basement membrane, or hormone depnvation, results in apoptotic death of these cells. Through

the use of function blocking antibodies, it was determined that the epithelial cell-basement

t membrane interaction necessary for ce1 survival is mediated by the c6 and pl integrin subunits

in rnammaty epithelial cells. In these function blocking experiments, incubation with insulin,

which has been established to provide a survival signal in this system, does not rescue the cells from apoptosis. Also, culturing these cells on an appropriate basement membrane in the absence of survival signailing hormones such as insulin leads to apoptotic death of cells. In this mamrnary epithelial ce11 system, it has been clearly established that signais from both the ECM and soluble growth factors such as insulin are criticai for ceil survival (Farrelly et al. 1999). Other data indicates that this biological effect is regulated by cross-talk, as these two signals are intepted

through the cytoplasmic signalling cascade involving PI3-K and P K B (Lee and Juliano 2000). In

this sarne mammary epitheliai ceIl model, mvimal activation of the insulin receptor occurs when the cells are cuitured on the appropriate basement membrane, as opposed to collagen 1. This phenornenon is replicated downstrearn of the insuiin receptor, where maximal activation of PU-K

and PKB also o c c m when cells are plated on basernent membrane, as opposed to collagen 1.

This study provides some insight into the possible mechanisms of ECM-growth factor cross tak in survival pathways.

A very elegant study using chinese hamster ovary cells expressing insulin receptors (CHO-T),

has shown that insulin treatment enhances ce11 adhesion to fibronectin, and that treatment with a

function blocking anti-dB 1 integrin antibody abolishes this insulin effect. Activating the a B 1 S

integrins on these celis by plating on fibronectin markedly potentiates the ability of insulin to

enhance insulin receptor and insulin receptor-substrate 1 (IRS- tyrosine phosphory lation 1) (Guilherme et al. 1998). This integrin activation also potentiates PI3-K activity in response to

insulin in CHO-T. Incubation of these cells with PI3-K inhibitors abolishes both the insulin- and

fibronectin-induced effects observed.

These data clearly indicate that cross-talk between ECM and growth factor signalling pathways

likely plays an important role in biological systems which depend on signals from both integrin-

ECM interactions and growth factor signals for proper regulation. In vitro myogencsis is clearly

dependent on signals fiom ECM and growth factors such as insulin (Gullberg et al. 1998, Olson 1993). However. in rnyogenesis models, the downstream factors which may mediate this poiential cross talk are not well known. Studies kom other biological systems show that PI3-K

and P U 3 are important downstream mediaton of both ECM-integrin and insulin signalling, and

are involved in the integration of these separate signais (Lee and Juliano 2000). In myogeaic

systems, recent work has also implicated these signaihg molecules as important mediators of survival and differentiation pathways (Kaliman et a[. 1998, Fujio et a . 1999). Integrin-linked l kinase 1 (ILKI) is a serine-threonin protein kinase which has been shown to be involved in regulation of ECM and insulin signalling through the PI3-K/PKB pathway, and may play a role

in the regdation of myogenic differentiation (Hannigan et al. 1996, Dekommeme et al. 1998).

3.O Integrin-Linked Kinase (ILK1) and Myogenesis

3.1 ILK1 structure and fiinction

ILKl is a ubiquitously expressed cytoplasmic protein seruidthreonine kinase which is implicated

in integrin signal transduction partially due to its interaction with

p 1 integrin subunits (Hannigan

et al. 1996). The protein encoded by the ILKl gene, p591LK1 its homologues are expressed in and

hurnan, mouse, rat. Drosophila sp., and C. elegans cells (Hannigan el al. 1996, Li et al. 1997,

Dedhar et al. 1999) . The human and mouse ILKl genes have k e n mapped to chromosome

l l p 15.5-15.4 interval, and chromosome 7El, respectively (Li et al. 1997, Hanni-gan er al. 1997).

The hurnan ILKl gene is expressed most strongly in heart. skeletd muscle. and pancreas. as

measured by Northeni blot analysis. The ILKl protein is made up of multiple domains within

452 amino acids: four contiguous ankyrin repeats, a pleckstrin-homology (PH) domain, a protein

kinase catalytic domain containhg twelve subdomains. and an integrin binding site (Hannigan et al.

1996). The primary fünction of ankyrin repeats is to mediate protein-protein interactions. and it

has been demonstrated that ILKl ankyrin repeats mediate interaction with the adapter protein

PMCH (Tu et al. 1999). This association may provide a physicai Link between ILKl and

components of the insulin signailîng pathway, as ILKI, PMCH, and Nck-2 are found in temary irnmunocomplexes. Nck-2 is an adapter protein kvhich can bind insulin receptor substrate 1 (IM-

1) and, through PINCH may iink ILKl with the innilin signalling pathway via this association (Tu et ai. 1998). The PH domain of ILKl Likely binds the PI3-K phospholipid second messenger

phosphatidylinositol (3,4,5)P3 (PIP3)and indeed PIP, activates recombinant ILKl in vitro

24

(Delcornmenne et al. 1998). The integrin binding site of ILKl associates with $1 integrin subunits

in vivo, and in vitro studies indicate it may also bind 82 and B3 subunits.

ILKl complexes with

pl integrins and localizes with integrins to focal adhesion plaques, supporting its role in regulating ce11 responses to ECM (Li et al. 1999).

3 2 ILK 1 signalling in myogenic pathways

It has been shown. that in epithelial cetl models. ILKI participates in signalling pathways

emanating from growth factors and ECM, leading through PI3-K and PKB (see Fig. 1).

Interaction of epithelial cells with growth factors such as insulin, or wt ECM induces a rapid, ih transient increase in ILKl activity, and experiments employing dominant negative and

pharmacologie inhibitos of PI3-K have indicated that ILKl activation by these disparate

receptors is dependent on PI3-K activity (Delcomrne~e a/. 1998). Likewise, loss of the et

PTEN tumour suppressor gene which codes for a phosphatase antagonistic to PI3-K, has

recently been shown to elevate ILKl activity levels, in tum activating PKB, indicating that gain

of PI3-K activity stimulates ILK1-mediated signalhg (Persad et al. 2000). ILKl is thus

proposed to function downstream of PI3-K and upstrearn of PKB, in epithelial cells stimulated

by growth factors or matrix. As each of these factors has previously k e n s h o w to be an

important regulator of myogenesis i vitro, I investigated the role of ILKl in the in vitro n myogenesis of rat L6 myoblasts.

'==...; 'MEK

1

Cyctin 01

Mye

AP-1

Figure 1. Signalhg pathway diagram showing pathways activated by ECM and growth factors transduced through ILK 1. Integrin engagement and clustering, or growth factor receptor stimulation, resdts i the formation of signailing complexes including adapter proteins such as n Nck-2 and IRS-1, as well as kinase effectors such as ILKl and Shc. ILKl is activated by these stimuli via PI3-K, and regulates downstream molecules including PKB and GSK3 (Dedhar 2000).

The data presented in this thesis demonstrate that ILKl signals tue necessary for L6 in vitro

myogenesis. L K 1 activity k elevated by treatment of L6 cells with insuiin, and this elevation is

-

-

blocked by pre-treatment with wortrnannin and LY294002,inhibitors of PI3-K. ILK1 activity is

also rapidly, but transiently, stimulated by ECM substrates relevant to myogenesis: fibronectin, laminlli-1 and laminin-2 (merosin) in L6. ILKl activation kinetics Vary with each ECM

substrate. Stable L6 transfectants expressing a kinase-deficient variant of ILKl, which is shown to act in a dominant negative fashion (ILK(E359Q are unable to differentiate under conditions of serum withdrawal, and insulin treatment. In contrast, cultures of L6 cells expressing an active wild type ILK 1 transgene (wtILK 1) display increased numben of morphologically and biochernically differentiated ceils relative to control rnyoblast cultures, which exhibit a very low background of myotube formation. This result indicates that ILK l overexpression induces L6 cell cycle withdrawal, in contrast to its effects in epithelid cells, where it promotes S-phase transit. Although the ILK dominant negative blocks insulin-, and sem-withdrawal induced

myo blast differentiation, insulin-stimulated reanangement of the actin cytoskeleton, insulininduced rescue From apoptosis, and insulin-induced activating phosphorylation of P K B on ser473 is unimpaired, indicating a insulin receptor-distal convergence of ILKl and insulin signal transduction pathways. Taken together, these data indicate that ILKl is a critical mediator of

myoblast differentiation signals. apparently acting as a PI3-K effector in this signal transduction

pathway.

Experimental Procedures

Reagents- Media for cell culture were purchased fiom the media preparation facility of the

Ontano Cancer Institute (Toronto, Ontario, Canada). Fetal bovine senun (FBS), fibronectin

(FN), merosin (MN), laminin (LN), TRIZOL, Lipofectamine, antibiotic/antimycotic, G4 18,

0.25% trypsidEDTA and insulin (bovine, crystalline, zinc) were obtained From GibcoBRL

(Burlington, Ontario, Canada). Polyclonal anti-ILKl antibody and myelin basic protein were purchased fiom Upstate Biotechnologies Inc. (UBI, Lake Placid, New York), or altematively

MBP was a generous gift of Dr. Mario Moscarello (Hospital for Sick Children, Toronto, Ontario,

Canada). Anti-insulin receptor antibody ( A M ) and LY294002 were purchased fiom Calbiochem

(San Diego. California, USA).

Anti-PKBIAkt antibodies were from Transduction Labs

(Mississauga, Ontario, Canada). The anti-phospho-serine473 antibody was From New England Biolabs (Mississauga, Ontario. Canada). The FSD anti-myogenin monoclonal antibody was obtained boom the Developmental Studies Hybridoma Bank, University of Iowa. Anti-

sarcoplasmic reticulum [Ca"]~TPase antibody (A52) was obtained from Dr. Arnim Klip (Hospital for Sick Chiidren, Toronto, Ontario, Canada).

[ $ 2 ~ ]was purchased ~ ~ ~

fiom

Phamiacia (Mississauga, Ontario, Canada). PVDF Immobilon P transfer membranes were

obtained f?om Millipore (Mississauga, Ontario, Canada). Protein A-Sepharose beads,

wortmannin, bovine serum albumin (BSA), and monoclonal anti-b-actin antibody (AC-15) were puchased kom Sigma (Oakville, Ontario, Canada). Autoradiography and cherniluminescence

results were imaged on Kodak X-Omat film. AU tissue culture plastics (NUNC) were purchased fiom Life Technologies (Burlington, Ontario, Canada).

cDNA Vectors, Transfection, and Cell Culture-L6 rat skeletal myoblasts were maintained in a-

MEM supplemented with 10% FBS (vlv) and l % antibioticlantimycotic (vfv). For

differentiation, cells were grown to 80% confluency and then media was replaced with a-MEM supplemented wt 2% FBS (vh) and 1% antibiotic/antirnycotic (vh) (DM) and allowed to ih differentiate for 2-6 days, as indicated. cDNAs encoding a fidl length active (Hannigan et al..

1996), and a kinase-deficient dominant negative ILK(E359K) cDNA (Novak et of.. 1998,

Delcommeme et al.. 1998), were subcloned into pcDNA3.1 (Invitrogen, Carlsbad, California, USA). Expression vectors were iransfected into second passage L6 cells using Lipofectamine, and transfected cells were selected with 85OpghL G418 (determined to be lowest completely lethal concentration with parental L6). Stable clones were isolated by limiting dilution, and equal passages between 3 and 10 used for experiments. To assay insulin stimulation of ILKl activity cells were cultured under senim-f?ee conditions (a-MEM with antibiotic/antimycotic) overnight. To inhibit P13K activity, cells were pre-treated for 20 min with 200nM wortrnannin, or 20pM

LY294002,in s e m - f r e e medium. Cells were treated with 1OOnM insulin in serum-fiee medium

n for indicated times, and then lysed in situ i NP40 buffer for kinase assay as described above. To

assay ECM stimulus of ILKl activity, L6 cells were seeded into 6-well tissue culture dishes

coated with fibronech (FN), laminin (LN), merosin (MN) (Aat IOpg/ml) or bovine s e m

albumin (BSA) at 2.5mg/rnl as a negative control. Cells were then lysed i situ at indicated n timepoints, and assayed for ILK 1 kinase activity as described below.

RNA exînzction and RT-PCR- test for the presence of transgenic vanscripts in transfected cell To

lines, total RNA was isolated using the TRIZOL reagenf according to the manufacturer's specifications. Briefly, frozen ceIl peliets (1x10~celldtube, -80°C) were thawed into 1 mL

TRIZOL reagent and resuspended by pipetting. Suspensions were incubated for 5 minutes at

room temperature, and then 200 uL chloroform/tube was added. Each tube was then shaken for

15 minutes on a [nutator] at room temperature. and then incubated for 3 minutes at room

temperature. The tubes were then centrifuged at 15 000 rpm for 15 minutes at 4°C. The

aqueous (top) phase was removed from each tube, and 500

uL isopropanol was added to each.

The tubes were then mixed by inversion and incubated for 10 minutes at room tempenture. The tubes were then centrifuged 15 000 rpm for 15 minutes at 4°C. RNA pellets were then washed once with 1 mL 75% ethanol. and the pellet was spun down at 15 000 rpm for 15 minutes at

4°C and air dried. Each RNA pellet was then dissolved in 50 u DEPC-H20 for 10 minutes at L

58°C. RNA quality and quantity was checked by electrophoresis on 1.2% agarose-forrnaldehyde

gels, and by spectrophotometric analysis on a Ultraspec 3000 WNisible spectrophotometer

(Pharrnacia). cDNA was synthesized nom Zpg total l

A using the First Strand Synthesis kit

and oligo dt primen h m Clontech. The cDNA was subjected to polymerase chah reaction

(PCR) using the Taq PCR Master Kit from Qiagen, and the following primers: (myc/H.is

specific)

ATGATGACCGGTATGCATATT-3 ',

and

(ILK1

specific)

5'-

ATGGACGACATTTTCCACTCAG. PCR reactions were carried out using `?outhdown" PCR

with cycle times as follows: denaturation 94°C 10 min, 20 cycles:[denaturation 94°C 1 min,

a n n e h g 6YC-55OC 1 m n (touchdown O.S°C/cycle), elongation 72OC 2 min], 10 i cycles:[denaturation 94°C 1 min, annealing 55OC 1 min, elongation 72OC 2 min],elongation 77°C

10 min.

PCR prociucts were visuaiized on ethidiuni broiiUile-1% agarase gels sn a D IR LW

Transillurninator (DiaMed, Mississauga, Ontario, Canada).

Niîclear Sfuining o L6-To visualize nuciei, L6 cells grown on glas coverslips were h e d with f

70% ethanol and stained with hematoxylin. Excess stain was washed off with double distilled

water, and cells were visudized and photographed at 40x magnification under a light microscope.

Preparation o Total Membranes- Total L6 ceil membranes were prepared as previously described f (Mitsumoto et al. 1993). Briefly, cells were rinsed twice with cold homogenization buffer

(250mM sucrose, 20 mM HEPES pH 7.4, 5 rnM NaN3, and 2 rnM EGTA). Cell monolayers

were scraped into cold homogenization b a e r containhg 1u leupeptin, 1 uM pepstatin, 10 u M M

E-64, and 200 uM phenylmethylsulfonyl fluoride (PMSF), homogenized with 20 strokes

(Dounce type A homogenizer), and then centrifuged at 700g for 5 min at 4OC. The supernatant ikom this low-speed spin was then centrifùged for 1h at 1901000g to obtain the total membrane fraction used for western blot analyses.

Western blotthg- To assay protein expression levels, we harvested and Iysed L6 cells in Nonidet

P-40 (NP40) lysis bufEer (150m.M NaCI, 1% NP-40 (vlv), 0.5% sodium deoxycholate (wjv), 50mM HEPES pH 7.5, lpg/mL each aprotinin and leupeptin, SOpg/mL phenlmethylsulphonyl fluoride), or altematively, isolated total membrane fraction as shown above. Protein concentration was measured by the Bradford method using the BioRad protein assay kit. 1OOlg NP40 lysate

was separated on 12% SDS-PAGE gels and transferred to PVDF membrane (80V, 1.25hr).

PVDF membranes were blocked in 5% non-fat rnilk (wh) ovemight, and with primary antibody

O, at recommended dilutions (a-ILKI O.jp9/rnL, FSD 1 :L O a-PKB 1 500, a-ser473 1 :1000, A52

1 500, Ab-4 1:400, AC- 15 15000)

ILKl Immune Compler Kinase Assays- For analysis of ILKl kinase activity, cells were lysed in

NP40 buffier, supplemented with IrnM sodium orhovanadate and 5mM sodium fluoride as

phosphatase inhibitors.

Equal amounts of protein fiom these ceU lysates were

immunoprecipitated with a-ILK1 polyclonal antibody as previously described (Hannigan et al. 1996), and immune complexes were incubated at 3 0 ' ~for 30 minutes with myelin basic protein

(2.5 pgfreaction) and y [ 3 ' ~ ](SpCUreaction). Myelin basic protein (MBP) was used as an ~ ~ ~

exogenous substrate for ILK1. The reactions were stopped by addition of 4X concentrated SDSPAGE sample buffet (Laemmli 1970). Phosphorylated proteins were separated on 15% SDS-

PAGE gels. MBP bands were visualized by autoradiography with X-Ornat film. As a control for

equal ILKl amoag assayed samples, immune-complexes were electrophoresed, transferred to

PVDF membrane and probed with anti-ILK1 antibody as described above.

FACS Anaiysis- L6 cells were harvested with trypsin/EDTA, and equal numben of cells were

washed with PBS/2%FCS and fked in 70% ethanol on ice for 30 minutes. Ceils were washed in

PBS containing 3% (vlv) FCS, then in 70% ethanol, and stained with 0.1 mg/ml propidium

iodide/0.6% (vlv) NP40 with Zmg/rnL RNAse i the dark, at room temperature for 30 rninutes. n Cell suspensions were filtered through 85um Nitex mesh and kept at J*C in the dark, until ready for FACS analysis. Ce11 cycle analysis was performed at the Hospital for Sick Children Flow Cytometry Facility on 15 000 single propidium iodide stained cells, and data based on doublet discrimination was collected by a FACSCAN analyzer (BD Biosciences. San Jose, California) and

fined using the DNA analysis program Modfit LT 2.0 (Verity Software, Maine).

Results

ILKi kinase activity û modulafed in L6 cells by tzxtracellular matru and insulin

Laminin, merosin (laminin-3) and fibronectin have ail been implicated in regulating aspects of

myogenesis in vitro and in vivo. To investigate the potential role of ILKl in myogenic signalhg, I tested the ability of these ECM proteins important in L6 myogenesis to stimulate iLK1 kinase activity. ILKl kinase activity was robustly stimulated by each ECM protein (Fig. 2): however differences in activation kinetics in each case were observed. iLKl was activated on FN at 5 and

30 minutes, while LN induced a rapid transient activation, which was

mas at 5

minutes.

Conversely, MN induction of ILKl activity displayed relatively delayed kinetics, with maxird activation at 30 minutes. These results c o n h that ILKl activity i stunulated in L6 c e k s

* .--e

interacting with ECM proteins known to play a role in myogenic kifirentiation and survival.

5 min

30 min

Figure 2. ILKl kinase activity is stimulated in L6 myoblasts by ECM proteins relevant to myogenesis. L6 myoblasts were plated on fibronectin (FN), laminin (LN), or merosin (MN) (lOug/mL), or BSA control(2.5mg/m.L) for 5 and 30 minutes. ILKl kinase activity was measured by in vitro ILKl immune cornplex kinase assay ushg myelin basic protein (MBP) as exogenous substrate.

Insuiin is a potent differtntiating agent for myoblasts, and has k e n shown to activate ILKl in epithelial ceus, such as HEK 293. We therefore examined ILKl activity in insulin-treated L6 myoblasts. Confluent L6 cultures were maintained in GM or DM and cultured for up to 6 days. All celis were then sem-starved overnight in a-MEM. Cultures were pre-treated or not, with

ZOOnM wortmannin, then treated with lOOnM insulin for 0. lO or 20 minutes, after which they

were lysed and assayed in ILKl immune complex kinase assays. ILKl activity was stimulated at

10 minutes post-insulin treatment in both myoblasts and myotubes, with activity decreasing by 20 minutes (Fig. 3a). Wortrnannin pre-treatment dramatically reduced this response, indicating PI3-K dependence of ILKl activation by insulin, as has been documented in epitheliai cells.

Identical inhibition was obtained by pre-treating cells with another P I X inhibitor. LY294002 (data not shown). There was no difference between myoblasts and rnyotubes in the ILKl response to insulin, and in both populations ILKl stimulation was sensitive to PD-K inhibitors. Western blots of the immune complexes indicated equal arnounts of ILKl were assayed for kinase activity (data not shown). Induction of differentiation did not affect ILKl protein expression levels, as judged by western blotting of myoblast and myotube lysates (Fig. 3a). O r results u indicate that activation of ILK. by myogenic concentrations of insulin or by ECM, is dependent on PI3-K activity, consistent with previous fmdings in epithelial cells.

- Wod

+ wort

293

BSA

MT

MB

20uM LY2W002 1 0 û n ins (min) ~

-

oe

+

10

O

10

LGvector control wtILKI

I-

iLK(U59K)

I

I

Figure 3. Regdation of ILKl activity in L6 myoblasts by the myogenic factor insuiin a. [nsulinstimulated L6 myoblasts (10% FCS) and myotubes (2%FCS, 6 days) were assayed for ILKI cataiytic activity i h u n e cornplex kinase assays, using MBP as exogenous substrate. Cens n were treated for the indicated times with l O O n M insulin with or without 20 minute pre-treatment with the PI3-K inhibitor wortmannin. b. Kinasedeficient, dominant-negative ILKl mutant

(E359K) blocks insulin-induced ILK 1 activation. L6 myoblasts (ILK(E359K), and vector control) were assayed for ILKl catalytic activity by immune complex kinase assay on MBP as exogenous substrate. Cells were treated for the indicated times with 100nM insuiin w t or ih without pretreatrnent with the PI3-K inhibitor, 20uM LY294002. c. RT-PCR analysis of wtILK1, ILK(E359K), and empty vector control L6 cultures shows the presence of myc/HIS tagged transcripts in the ILK(E359K) transfected ceils, but not in control or wtILK 1 cultures. Total RNA was isolated by TRIZOL extraction? and cDNA was synthesized with oligo dt pnmers. This cDNA was subjected to PCR using an ILK1-specific 5' primer, and a mycMis specific 3' primer. The expected 1.4 kb product corresponds to the full-length ILKl coding

Expression o a dominant negative ILKl mutant biocks induction o L6 di~erenriationf f

In light of the Pt3K-dependent response of ILKl to myogenic signals, we next sought to directly examine the role of ILKl in myoblast differentiation. Stable L6 transfectants canying expression vectos encoding Full length active (wtiLK 1), or kinase-deficient (dominant negative) mutant (ILK(E359K) ILKl proteins were analyzed for ILKl effects on myoblast differentiation. The presence of mycNis tagged transcripts was confirmed in the ILK(E359K) transfected cultures by

RT-PCR(Fig. 3c). Treatment of ILK(E359K)cells (four independent clones) with insulin failed to

stimulate tLK1 kinase activity, although vector control transfectants showed normal, LY294002sensitive ILKl induction (Fig. 3b). This c o n f i s that the dominant negative effect of the

ILK(E359K) mutant extends to L6 cells. Most strikingly, the ILK(E359K) cultures showed a

complete block to fusion and myotube formation, under conditions of DM induction (Fig. 4a) or insulin (not shown) treatment. Vector controls showed normal levels of myotube formation when cdtured in DM, with a minimal background of myotube formation in GM, however under growth conditions ILKl wild-type transfectants (four independent clones) exhibited more extensive

fusion and myotube fornation than did the controI cultures (Fig. 4a). W conclude Eom the e

rnorphological experiments that ILKl kinase activity is required to signal L6 myoblast fusion and

myotube formation

.

,

.-

c

I ! J

L6 vector control

L6-ILK(E359K)

Figure 4. Dominant negaiive ILK 1 mutant blocks morphological and biochemical differentiation of L6 myoblasts. a. Stable transfectant clones expressing wtILK 1, ILK(E35 K ,or vector control 9)

were cdtured in 10% (GM) or 2% (DM) serum conditions. Four independent clones of each (one representative clone of each show: vector control, ILK(E359K), wtILKl were allowed to daerentiate as described, fixed, stained for nuclei with hematoxylin, and photographed (40~). No significant myotube formation was seen in ILK(E359K) transfected clones, and myotube formation above background was seen under growth conditions in wtIL K I transfected clones. b. Vector control and ILK(E359K) transfected L6 cells were grown for 48h after reaching 80% confluence under growth (GM-lO%FCS) or differentiation (DM-2%FCS) conditions. At 48h, celis were lysed and assayed for myogenin expression by western blot. Membranes were stripped and re-probed with ILK antibody to control for levels of p591LK1.

Biochemical markers of myoblast differentiation were next assayed in the ILK(E359K) and

wtiLKl cultures, to determine whether the ILK(E359K) effect on morphoIogic differentiation

reflected inhibition of ce11 fusion, or represented failure to initiate biochemicai differentiation. We therefore assayed ILK(E359K) L6 clones for the induction of muscle-speci fic gene expression. Myogenin is a marker of rnyoblast differentiation, and in L6 blasts induced in DM, myogenin

expression is also induced and peaks at 48 hours d e r induction. Myogenin levels remain low in

myoblasts (Fig. 4b). Sarcoplasmic reticulum [Ca"]~TPase is another well characterized biochernicai marker of differentiated L6 (Mitsumoto et al. 1993). At confluence, L6 cells were cultured in either GM, or induced to differentiate in DM. After 48 hours in culture cells were lysed and assayed by western blot for expression levels of myogenin and sarcoplasmic reticulum

[Ca"]~TPase. Consistent with the morphological block to differentiation? we found that

myogenin expression is cornpletely abolished in ILK(E359K) transfected cells (Fig. 4b). Conversely, in wtILKl transfected myotube cultures, myogenin expression is equivalent or slightly higher than the level seen in differentiated vector control or parental L6 cultures (Fig. 4). Sarcoplasmic reticulum [ C a r ~ T P a s eexpression was assayed in total membrane hctions

isolated h m control L6 and ILK(E359K) transfected cells (Fig. 5). While control L6 cells show a typical upregulation of sarcoplasmic reticulum [Ca*]~TPase upon differentiation, we could not detect sarcoplasmic reticulum [ C a q ~ T P a s e expression in L6 expressing ILK(E359K) under growth or differentiation conditions. The tvtiLK1 transfected L6 show a dramatic induction of [CaH]ATPase, above normal levels in both the myotube and myoblast populations. This induction correlates positively with the increased level of myotube formation seen in the wtILKl L6 cultures, and our results indicate that ILKl is a positive signal mediating both biochemical and morphological differentiation of L6. It is also interesting to note that when these total membrane (and the corresponding cytoplasmic) fractions were analysed for ILKl expression by western blot analysis, that ILKl is s h o w to associate with cellular membranes for the first tirne. This analysis also shows that, in wtILKl transfected cultures, that ILKl associates with cellular membranes more than in ILK(E359K), or control L6 cultures.

r

4

Fipre 5. Sarcoplasmic rekeutum [Caf*]~TPase induction typical of myogenin differentiation in L6 is completely eliminated by transfection with ILK(E359K), and is amplined by transfection with wtlLK1. w t n K l transfection results in increased ILKI i membrane preps (M) under n dBerentiating conditions (C-cytoplasmic fiaction). This ciifference in ILK 1 expression is not seen in whole ce11 lysates (data not shown). Vector control, ILK(E3 59K) and W L K l transfected L6 cells were differentiated for 6 days as described, and total membranes were analysed by western blot for sarcoplasmic reticdum [CaTATPase expression. bactin blo& arë shown to control for equal Ioading.

As ce11 cycle withdrawal is a prerequisite for myoblast differentiation, we e&ed

the effect of

the wtILKl and ILK(E359K) transgenes on L6 cell cycle profile, in order to c o n f i that

ILK(E359K) effects were not simply a consequence of withdrawal. L6 wtlLKl and ILK(E359K)

transfected cells stained with propidium iodide and analysed by FACS showed expression of these transgenes does not affect the L6 cell cycle profile under 10% serum conditions (Table 1). Next, we checked for the effect of each transgene on the ability of L6 cells to exit the cell cycle during the initiation of differentiation. These results with insulin stimulus, or serurn withdra~val, presented in Table I indicate that semm withdrawal (ie. serum-fiee treatment) induced a significant

(1 1%-19%) apoptotic response in the ILK(E359K) cultures, which was not observed in the

control or wtlLKl transfectant cultures under the sarne conditions. Treatment of the ILK(E359K) cells with Ulsulin resuited in a loss of the apoptotic fnction, indicating that insuiin provides a survival signai in the absence of obvious ILKl activation. Similar protective effects were seen with IGFl treatment, which also induces a rapid transient stimulation of ILKl activity in L6 control cultures (data not shown). The protective effect of insulin on the ILK(E359K) cells indicates that expression of the dominant negative ILKl protein does not impair insulin receptor function. Western blotting of transfectant cell lysates also indicated equivalent levels of insulin receptor expression among wtILK1, ILK(E359K) and control L6 cultures (data not shown). Also, we examined the ability of each ce11 line to form membrane d e s in response to insulin stimulus. L6 cells undergo a rearrangement of the actin cytoskeleton when stimulated with insulin that results

in the formation of membrane-localized bundles of actin filaments known as membrane m e s

(data not shown). wtILK1, ILK(E359K), and vector control L6 cells aii form typical membrane

niffles with insulin stimulus. These results indicate that insulin receptors are activated normdy

in the ILK1-deficient cells, and thus the Ioss of insulin-induced differentiation is a h c t i o n of perturbing ILK 1 signal transduction.

Table 1: Ce11 cycle analysis of L6 myoblasts (vector control and ILK transfectants) under growth (GM), differentiating (DM), sem-free (SF), and serum-free plus 100nM insulin (MS). Duplicate numbers are from two independent experiments. Nd. not done.

which itself has been shown to provide critical ILKi has been implicated in the activation of Pm,

survival and differentiation signals during myoblast differentiation. Full activation of PKB requires

phosphorylations on both Thr308 and Ser473. ILKl has been suggested to mediate Ser473 phosphorylation, in response to treatment of cells with growth factors or plating on ECM substrata. We examined insulin stimulation of PKB Ser473 phosphorylation in wtILKl and

ILK(E359K) L6 cultriles, in order to examine the possible inhibition of this event by the dominant

negative ILKl protein. Surprisingly, we found that Ser473 phosphorylation is stimulated to

essentidy equivalent degrees, in each of the wtiLK1- and ILK(E359K)-expressing lines (Fig. 6). Thus inhibition of ILKl activity by the dominant negative mutant does not significantiy inhibit

PKB activation, suggesting PKB activation is not sufficient for L6 myoblast differentiation. a

L6 vector ILKl(E359K) wtILK1

total PKB

I

ILKl

Figure 6. W i n induces Sa473 phosphorylation of' P-KB in IL~(E359~)-ex~ressin~ L6. Control (C) and insulin-induked ( cultures of L6 expressing ILK(E359K), or wtKK were Iysed I ) and assayed by western blotting for levels of Ser473 (phospho-PKB), as weiI as total PKB and ILKl ievels.

DISCUSSION

The data presented in this thesis show that L6 myoblast differentiation requires active ILKl. Transfection of L6 myoblasts with a kinase-inactive dominant negative ILKl mutant (E359K) blocks L6 myogenesis, as assesed by morphological and biochemicai markers, while transfection

with a wild-type ILKl cDNA hcreases L6 myogenesis under growth conditions and amplifies

the expression of sarcoplasmic reticulurn [Ca"]ATPase, a well-established marker of myogenic differentiation. Despite these drarnatic biological effects induced by altentions in ILKl

signaliing, the precise downstrearn mechanisms which mediate these biological effects are unknown. Previous data generated in studies of iLKl signahg in epitheliai cell models may provide insight into the sigialling role of ILKl in this in vifro myogenesis model, and into the possible mechanisms by which ILKl can act as a regulator of rnyogenesis in L6. ILKl has been shown to participate in a number of signalling pathways, including the PI3-WPKB pathway (Delcommeme et al. 1998, Lynch et al. 1999), which may play a role in the regulation of myogenesis in vitro. The data presented here aiso suggest complexity in the pathways regulating myogenic differentiation and myocyte m i v a l beyond a simple linear signaihg relationship between PI3-K, LU, and PKB.

In epithelial cells, previous work has demonstrated that ILKl stimulates S phase transit,

anchorage independent growth and tumorigenesis (Hannigan et al. 1996, Radeva er al. 1997).

Unlike many dominant acting oncogenes, ILK1 overexpression induces senun-dependent colony

growth in soft aga. These data suggest that receptor tyrosine kinases signal through ILKl to

stimulate integrin-independent growth. In fact, it has been s h o w in a variety of epithelial cells, that growth factors such as insulin transiently stimulate ILKl protein kinase activity (Delcornmeme et al. 1998). The data presented here also show that ILK1 protein kinase activity

c m be transiently stimulated by growth factors such as insulin in myogenic cells. However, in

these cells, different groups of growth factors can have drarnatically varying effects on myogenic cell biology. Peptide growth factors such as FGF and TGF-P are potent inhibitors of

myogenesis in vitro, while insuiin and IGFs hduce myogenic differentiation (Olson 1992). This additional complexity in the biology linked with growth factor signal transduction suggests that the integration of these, and other, extracellular stimuli. perhaps by signalling molecules such as ILKl, controls the net biologicd effect.

D t fiorn epithelial cells suggests ILKl regdates cell cycle transit through speicfic mechanisms. aa

Overexpression of ILKl in IEC 18 cells results i constitutiveiy elevated levels of cyclin Dllcdk4 n activity, and hyperphosphorylation of the retinoblastoma susceptibility gene product (pRB), leading to an increase in S-phase transit (Radeva et al. 1997). Integrin-mediated adhesion to ECM produces similar effects on cell cycle machinery as these seen in ILKl overexpressing IEC18, raising the possibility that ILKl signals play a role in the regulation of cell cycle proteins, perhaps through p27lcyclin D l ratio. This function may provide a plausible mechanism by which ILKl signals may regulate L6 myogenesis. The bHLH transcriptional apparatus critical for myogenesis has been showm to be intirnately linked to ceil cycle control, partially through the

S-phase inhibitors p2 1 and pRB (Zacksenhaus et al. 1996). Withdrawai fiom the cell cycle, and

into Go, is required for myoblasts to begh the differentiation process, and to express musclespecific genes. This link is best shown in mice with targetted deletions of p2 1

''

and p57K1P2,

where double p21/p57 nuil myoblasts proliferate and undergo apoptosis at increased rates, and

are unable to f o m myotubes (Zhang et al. 1999). This phenotype overlaps that seen in

myogenin 4- mice, suggesting that Go entry is cntical for myogenin expression, as myogenin is expressed independently of pZ 1.

Two alternative models, each consistent with current in vivo and in vitro data, have been

proposed to relate celi cycle arrest with MRF expression (Zhang et al. 1999). The first states that unknown differentiation signals activate MRF expression, while p3 1 and p57 are induced independently and bring about G1 arrest and pRB activation.

pRB and MRFs, such as

myogenin, wouid then be able to cooperate in the regulation of muscle-specific gene expression.

The second model states that p21 and/or p57 themselves provide a cntical differentiation signal in this myogenic model. However, ectopic expression of wtILKl or ILK(E359K) in L6 cells

does not have a significant effect on ceil growth or cycling, thus the observed block to myoblast differentiation in the ILK(E359K) cells indicates block of a critical differentiation signal, and is not simply the consequence of ce11 cycle withdrawal. It would be important to address these

proposed models relating celi cycle arrest and MRF expression using the L6 stably transfected lines generated here.

Initially, this issue could be tested in our model by assaying wtILKl and ILK(E359K) cultures

for cyclin D 1, p2 1, and p27 expression under growth and differentiation conditions. As FACS

analysis shows that the ceil cycle profile of L6 cells is not significantly affected by wtILKl or ILK(E359K) expression, one would expect the cyclin D l/p2 1(p27) ratio to be essentially unchangeci under each condition. However, if ILKl affects L6 ceii differentiation through a p21/p27 pathway that is independent of cell cycle regulation, the expression of these molecules may be inhibited by ILK(E359K) expression and induced by wtILKl expression. It would also

be interesting to overexpress p21 in the L6 ILK(E359K) transfectants, and to assay these double

transfectants for myotube formation. If p21 c rescue these cells from the myogenic block m imposed by ILK(E359K), it would suggest that ILK1-induced p21 expression is required for L6 myoblast differentiation. However, if these p21/ILK(E359K) double transfectants do not undergo myogenesis, it would suggest that Cdk inhibitors are not sficient independent

differentiation signals, and that [LKl may activate other factors required for differentiation. Taken together, data describing the role of cell cycle regulation and ILKl signalling in myogenesis M e r suggest that additional complexity must exist in myogenic signalling pathways to separate growth and differentiation signals.

In light of this cell-type specific variation of ILKl effects on ceil biology, it is also interesting to note that a srnall molecuie specific inhibitor of ILKl activity (KP-1) recently been developed has by Kinetek Pharmaceuticals. This inhibitor was identified through a high-throughput screen of proprietary compounds, as part of a large-scale effort to identifiy potential therapeutic agents which could be used to treat cancers which have arisen due to the overexpression, or increased activity, of a specific oncogenic protei. kinase. However, by showing that L K 1 activity can be necessary for differentiation of mesenchymal cells, these data raise the issue of the tissue-specifc

effects of protein kinase signalling cascades. Clearly if, on the one hand, ILK 1 activity can promote nimour formation in the epithelia of nude mice (Wu et al. 1998), while on the other hand, promote differentiation of L6 muscle cells, F a t care must be taken in the development of kinase-inhibitor based therapies in order to assure proper targetting of the therapeutic agent(+

Upon integrin engagement with ECM substrates, numerous ''outside in" intraceildar signahg cascades are initiated, leading to the stimulation of tyrosine phosphorylation, turnover of phosphoinositides, and the activation of the Ras-rnitogen activated protein kinase (MAPK) pathways via focal adhesion kinase (FM); each leading to changes in gene expression and ce11 biology (Aplin et al. 1999). Previous work with epithelial cells has shown that ILKl activity is stimulated by plating on fibronectin, with a rapid stimulation peaking at 3045 minutes, followed by a rapid decline back to basal levels by 60 minutes @elcornmenne et al. 1998). In contrast,

FAK is stimulated maxirnally by plating on fibronectin at 60 minutes, a time when ILKl signais

have diminished (Aplin et al. 1999). T i indicates the existence of severai distinct signailhg hs pathways, each initiated by integrin sigding fiom focal adhesions. Clearly, these distinct pathways may each play a role i the regdation of myogenesis. ILKl has also been shown to n regulate the composition of the ECM, an example of "inside-out" integrin signalling. Wu et ni.. (1998) demonstrated that ILKl overexpression in IEC 18 epithelial cells results in an upregulation of fibronectin mat* assembly, and that this effect is dependent upon the kinase activity of

ILKI, as transfection with ILK(E359K) does not produce this effect. My work with L6 cells

indicates that ILKl activity is modulated by ECM substrates in ceils of mesenchymal origin. Three ECM substrates relevant to myogenesis (fibronectin, laminin, and merosin) each activate

ILKl activity in L6 myoblasts with different activation kinetics. The next logicai step in this

work would be to determine the effects of ILK(E359K) and wtILKl transfection of ECM activation of ILKl kinase activity. The data 1 present here clearly show that ILK(E359K) transfection blocks ILKl activation by insulin, a potent myogenic growth factor. One would expect, then, that ILK(E359K) transfection would also block ILKl activation by ECM stimulus.

wtILKl L6 transfectants could also be assayed for ILKl kinase activation by larninin, merosin.

and fibronectin, and it would be interesting to see the effects of this ectopically expressed ILKl

on the magnitude and kinetics of ILKl activation by each of these ECM substrates relevant to

myogenesis. It would also be very informative to assay other ECM initiated events in these L6 transfectants such as membrane d i n g , as well as activation of relevant kinases such as FAK. Alterations in the normal pattern of activation of any of these phenornena by transfection with

ILK(E359K) or wtILK 1 would yield information regarding possible interaction with ILK 1

signailhg pathways. Recent work has suggested that ILKl regulates PKB activation through either direct phosphorylation on serJ73, or an indirect mechanism (Delcommeme et al. 1998, Lynch et al. 1999). However, FACS analysis data presented here show that in ILK(E359K) transfected cells, insulin-dependent ce11 swival and PKB phosphorylation on ser473 are in tact, perhaps indicating that other mechanisms are able to activate PKB in the absence of detectable

ILKl signals. If PKB activation by plating on fibronectin is unafEected by the ILK(E359K)

transgene as s h o w here in L6 myoblasts, the presence of an additional PKB activating mechanisrn in L6 would be supported.

It wouid also be interesting to investigate the composition of the ECM generated by the

ILK(E359K) and wtILK1 transfected cells. It is possible that these ILKl transfections may

affect myogenesis through an "inside-out" effect, as they have been shown to modulate ECM assembly in epithelial cells (Wu et al. 19981, and ECM composition has k e n shown to have dramatic effects on myoblast differentiation and myotube survival (Gdberg 1998). It would be reasonable to surmise that ILK(E359K) transfected myoblasts subjected to differentiation stimulus (e.g. senun withdrawal. insulin) would produce an ECM rich in fibronectin typical of undifferentiated myoblasts. However, if the composition of these differentiation-blocked celis is typical of myotubes (e.g. larninins replacing fibronectin as the predomhant ECM proteins) it would indicate that ILK(E359K) is blocking the intracellular transduction of these ECM signals via integrins, but that inside-out pathways regulating ECM composition are unaffected by Uihibiting ILKl activation. Given the prominent role in myogenesis played by ECM-integrin interactions, it will be very important to thoroughly investigate the role ILKl signals play in the myogenic differentiation and survival pathways initiated by the ECM.

Recent genetic evidence fiom work in the nematode C. elegans also supports a cntical role for 8 1 integridILK1 signais in muscle development. PAT-4 @aralysed, arrested elongation at two-fold)

C. elegans mutants overlap and rescue the PAT-3 phenotype, caused by a mutation in the C.

elegans 1 integrin homologue. Sequencing of these PAT-4 alleles identified two inactivating mutations in the C. elegans ILKl homologue. The PAT phenotype describes a specific

developmental defect, in which there is a defect in the assembly of focal adhesion-like structures

at muscle attachment sites of the nematode body wall (Dedhar et al 1999). These data provide

evidence that the link between muscle development and integrin/ILKl signds may be evolutionarily ancient.

The work presented here demonstrates that ILKl c m be activated by insulin and ECM signals in cells of mesenchymal origin, and M e r that stimulation of ILKl by i s l n or ECM in L6 nui myoblasts is blocked by pretreatment of cells with two well-characterized inhibitors of PI3K,

wortmannin and LY294002. PI3K activation may involve interaction of PIP3 with ILKI, since

this lipid has been s h o w to activate ILKI kinase activity in vitro, and since ILKl has a putative

PH domain. Although there is a potential Y d PI3K interaction motif in the N-terminal region

of ILKl (Delcommeme et al. 1998), it seems unlikely that this motif functions in mediating the direct association of Pl3K with ILKI, since ILKI is not detectably tyrosine phosphorylated af'ter insulin treatment of L6 cells (MM and GH, unpublished data). Another way to approach the question of the PI3-WILKl relationship would be to stably transfect L6 cells with a cDNA coding for a constitutively active ILKl mutant (Lynch et al. 1999). Not only would one expect these cells to fonn myotubes under growth conditions, and to express muscle-specific markers such as sarcoplasrnic reticulurn [Ca*]~TPase and myogenin at enhanced levels, but this mode1

wouid allow the M e r investigation of the PI3-WILK1 link. If a criticai L6 differentiation signal

is transduced fiom PD-Kthrough ILK1, in these ceUs expressing a constitutively active ILKl ,

the addition of PIS-K inhibitors such as wortmannin and LY294002 shouid be unable to block

myogenesis as they do in control ceils. Altematively, a cDNA coding for a dominant-negative

m t n of the PI3-K p85 regdatory subunit (Kaliman et al. 1998) could be CO-transfectedwith uat

the constitutively active ILKl mutant to determine if ILKl signals c m compensate for the

52

absence of the PI3-K signal critical for myogenesis. Overail, this model would be very usefui in leaming more about the specific roles and relationship of ILKl and PI3-K signals in myogenic

signalling pathways.

In this study, L6 myoblasts transfected with expression vecton coding for wtILK1, and a dominant-negative ILKl variant have proved to be a very powerful model in determinhg the role of ILKl in differentiation and survival pathways regulating myogenesis. However, certain

limitations exist which have prevented the posing of certain key questions regarding ILKl regulation of myotube survival. Data in this thesis show that transfection with ILK(E359K) blocks myogenic differentiation in L6. Thus, we are unable to determine the role of ILKI signais in myotubes through our dominant-negative strategy. However, technologies do exist that would aliow us to address these questions using the well charactenzed L6 model we have k e n working with. By using the ecdysone-inducible expression vector system in the development of stable ce11 lines with L6, we would be able to effectiveiy turn the expression of ILKl, or ILKI mutants, on or offat specific times during myogenic differentiation. This system would be set up by

CO-

transfection of L6 cells with an expression vector conditionally coding for ILKI, or an ILKl mutant, and another expression vector constitutively coding for the cytoplasmic receptor for the synthetic insect hormone ecdysone. Adding ecdysone to the culture media results in hormonereceptor binding, and the binding of this complex to the promoter of the other expression vector, which is othenvise "silent", leading to very tight regulation of hansgene expression. The addition of ecdysone to L6 cells does not affect differentiation (data not shown). Thus, using this system,

we wouid be able to differentiate L6 ceus, and then induce the expression of ILK(E359K),

wtILK1, or a constitutively active ILKI, and examine any effects on myotube survival using well

established assays for apoptosis such as propidium iodide staining with FACS analysis, TUNEL assay, or by Western analysis for PARP cleavage. An aiternate approach to this problem would

be to transfect L6 cells with cDNAs coding for ILK1, or ILKl variants, under the control of a

myogenin prornoter sequence. This would result in transgene expression only upon the onset of terminal differentiation, and thus allow the assesment of the role of each transgene on the surivival of differentiated L6 myotubes. Either of these approaches may be helpful i addressing n the role of ILK 1 signals in myotube survival.

One of the advantages of using epitope-tagging expression vectors is that the expression of the transgene of interest can be easily assayed by any number of well-established methods. However, this process can be technically difficult in stable transfectants created with the pcDNA3.1 myc/His tag vector used in this study.

I tried several methods to detect the

expression of the wtILKl and ILK(E359K) transgenes in the stable L6 lines described above. Several different approaches were taken to detect the myc and His epitope tags in L6 iysate including Western blotting for the tags, CO-irnmunoprecipitation the tags and ILK 1, isolation of of His-tagged proteins using the Ni-NTA affuiity column, as well as an altemate approach assayhg cuitured ceiIs by immunocytochemistry for each tag. Unfortunately, none of these strategies yielded d e f ~ t i v reçults showing the expression of the transgenes in these stable cell lines. RTe

PCR analysis of LK(E359K) transfected cells using ILK1-specific, and mycMs tag specific

primers did indicate the presence of ILK(E359K) transcripts in the L6 clone tested (Fig. 3c).

However, 1 could not detect the presence of transgenic ILKl transcripts in the wtILKl clone tested. It is likely that this technical problem codd be due to darnage to the myc/His primer binding site in the particdar wiILK1 clone assayed by RT-PCR. Altematively, other wtILKl clones could be tested using the primers generated for the earlier assay, or perhaps another myc/His specific primer could be generated to overcome this technical hurdle.

The immune-complex kinase assay used in this study has been quite usefixi in the description of

the ability of various stimuli to affect ILKl sign;illing. However, the precise composition of

these immune complexes, generated through L6 total ceil lysate treatment with anti-ILKI

polyclonal antibodies, has not been determined. This raises the possibility that other kinases andior phosphatases rnay be immunoprecipitated dong with IL K 1, and perhaps interfere wi th

our assessment of ILKl kinase activity at any given time.

We plan to analyse the precise

composition of ILKl immune complexes using mass spectrometry.

The data descrîbing the

composition of these complexes may validate, or othenvise shed light on, the immune-complex kinase assay results generated to date.

Recent work has suggested a possible physical link between insulin signahg pathways and ILKI, perhaps providing a mechanism for integrin-growth factor cross talk. PiNCH, a LIM domain protein that interacts w t the N-terminal a m - i i k e repeats of [ L U , can be ih

CO-

immunoprecipitated with ILK1, niggesting their interaction in vivo (Tu et al. 1999). This relationship between ILK1 and PINCH has also been validated using the C. eiegans genetic mode1

descnied above. Unc-97 is the C. elegans homologue for PMCH, and CO-localizeswt the ih

PAT-3 integrin subunit (homologue of the p l integrin subunit).

Unc-97 RNA interference

experiments generate a PAT phenotype, suggesting that in C. elegans, ILKl and PINCH regulate

matris assembly at muscle a t t a c h e n t points during embryogenesis (Dedhar et al. 1999). PMCH

and ILKl are found in ternary complexes with another adaptor protein, Nck2. As NcU is capable of interacting with the insulin receptor substrate, IRS-1,this complex codd describe a mechanism for linking ILK1 to activated insulin receptor (Tu et al. 1998). Whatever the

molecular mechanism, in both epitheliai and mesenchpal ceil lineages ILKl activation is regulated by a subset of stimuli that activate PI3K activity, however ILKl activation prornotes drastically different outcornes in the two lineages. Our results in L6 cells suggest that committed myoblasts require PI3K activation of ILKl for fusion and terminal differentiation. It is possible that the link between ILKl and the insulin signalhg pathway is mediated via molecular interactions involving the PINCH and Nck-2 adapter proteins.

Other data presented in this thesis show that the ILK(E359K) cell cultures exhibit a significant

apoptotic index (1 1 19%) under s e m fiee conditions, indicating that ILKl signalhg is required for some aspects of myoblast survivai. In this, the L6 behave similady to HEK 293 epithelial cells expressing the dominant negative ILK(E359K) protein. S e m or insulin treatrnent of L6

-

ILK(E359K) cultures blocks this apoptotic response, suggestuig that insulin signals nirvivai

through an aitemate pathway in LKLdeficient cells. The insulin-stimulated phosphorylation of PKB at Ser473 is robust in the ILK(E359K) cells, and this activation is likely to play a role in the protective effect of insuiin in these cells. PKB has been shown to be essential for differentiaton

of primary chick embryo myoblasts in vitro (Jiang et al. 1999), however o u . data suggest that

PKB activation (indicated by ser473 phosphorylation) i the absence of ILKl signals is n

insuficient for L6 myoblast differentiation. While it is possible that a low level of insulinstimulated ILK 1 activity in the ILK(E3 59K) cells allows some ILK 1mediated survival andlor differentiation signaling to PKB,this seems likely i n ~ ~ c i eto account for the robust induction nt of Ser473 phosphorylation that is seen in these cells. Clearly then, coordinated activation of

ILKl and PKB in response to myogenic signais may be required to complete the differentiation

program, however in the absence of ILKl activity, PKB is still capable of transducing protective signals. Indeed. ectopic expression of a dominant-negative P K B was f o n d to increase, and wild type P K B decrease the apoptotic index in differentiating C2C12 myoblasts, indicating PEU3 is a positive mediator of myoblast survival (Fujio et al. 1999). An important question in this light is whether ILKl is required for integrin-rnediated myotube survival. The development of L6 lines conditionally expressing ILKl and ILK(E359K), as mentioned above, will be valuable tools in addressing this question experimentally.

These data show that ILKl signals are necessary for L6 myogenesis in vitro. Transfection with cDNA coding for ILK(E359K) blocks L6 morphological and biochemical myogenesis, while

wtILKl cDNA enhances myogenesis under growth conditions.

ILKl kinase activity is

stimulated by ECM substrates and growth factors shown to be relevant to myogenesis, and this rapid, transient activation is abrogated by pre-incubation with PI3-K soluble inhibiton (Delcommeme et al. 1998). These hdings place ILKl as a downstream effector of PI3-K signals necessary for L6 rnyogenesis. Contrary to data generated in epithelial cells (Radeva et al.

1997), propidiurn iodide stainiag and FACS analysis of the stable lines generated here indicate

that wtILKl or ILK(E359K) do not affect L6 ceU cycle profile, suggesting that ILKl transduces

a differentiation signal independent of ce11 cycle withdrawal. These data also show that insulin

acts to protect ILK(E359K) transfected cultures, which undergo a significant degree of apoptosis under senun-fiee conditions, From programmed ceil death. Supporting evidence of this

phenornenon cornes from the robust phosphorylation of PKB or ser473 in ILK(E359K) cells treated with insulin. Taken together, these data indicate that PKB signals are not suffi~cientfor L6 myogenesis in the absence of ILKl signals, but may transduce survival signals through an

ILKl independent pathway. The L6 stable lines generated in this study will provide a very

useful tool for the dissection of myogenic differentiation and survival pathway in the fùture.

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