A novel mechanism of sequestering fibroblast growth factor 2 by glypican in lipid rafts, allowing skeletal muscle differentiation

Total number of characters 39998 1 2 3

A NOVEL MECHANISM OF SEQUESTERING FGF-2 BY GLYPICAN IN 4

LIPID RAFTS, ALLOWING SKELETAL MUSCLE DIFFERENTIATION 5 6

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Jaime Gutiérrez and Enrique Brandan 12

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Centro de Regulación Celular y Patología (CRCP), Centro de Regeneración y 16

Envejecimiento (CARE), Departamento de Biología Celular y Molecular, MIFAB, 17

Pontificia Universidad Católica de Chile, Santiago, Chile. 18

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Running title: Glypican in raft domains sequesters FGF-2 27

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Author correspondence: Enrique Brandan, Departamento de Biología Celular y 30

Molecular (Department of Cellular and Molecular Biology), Facultad de Ciencias 31

Biológicas (Faculty of Biological Sciences), P. Universidad Católica de Chile, Casilla 32

(P.O. Box) 114-D, Santiago, Chile. Fax 56 2 635 5395. E-MAIL: [email protected] 33

Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Mol. Cell. Biol. doi:10.1128/MCB.01164-09 MCB Accepts, published online ahead of print on 25 January 2010

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ABSTRACT 34

Heparan sulfate proteoglycans (HSPGs) are critical modulators of growth factor 35

activities. Skeletal muscle differentiation is strongly inhibited by fibroblast growth 36

factor type-2 (FGF-2). We have shown that HSPGs present at the plasma membrane are 37

expressed in myoblasts and are downregulated during muscle differentiation. An 38

exception is glypican-1, which is present throughout the myogenic process. Myoblasts 39

that do not express glypican-1 exhibit a defective differentiation, with an increase in the 40

receptor binding of FGF-2, concomitant to an increased signaling. Glypican-1 deficient 41

myoblasts show a decreased expression of myogenin, the master gene that controls 42

myogenesis, myosin and the myoblast fusion index. These defects were reverted by 43

expression of rat glypican-1. Glypican-1 is the only HSPG localized in lipid raft 44

domains in myoblasts, resulting in the sequestration of FGF-2 away from FGF-2 45

receptors (FGFRs) located in non-raft domains. A chimeric glypican-1, containing 46

syndecan-1 transmembrane and cytoplasmic domains, located in non-raft domains 47

interacting with FGFR-IV and enhanced FGF-2 dependent signaling. Thus glypican-1 48

acts as a positive regulator of muscle differentiation by sequestering FGF-2 in lipid rafts 49

and preventing its binding and dependent signaling. 50

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Key words: Skeletal muscle differentiation / heparan sulfate proteoglycans / glypican-1 59

/ FGF-2 / raft membrane domains / FGF-2 mediated signaling. 60

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INTRODUCTION 61

Heparan sulfate proteoglycans (HSPGs), key components of cell surfaces and 62

extracellular matrix (ECM), can influence cell growth and differentiation processes by 63

interacting with a large number of macromolecules. One of the most recognized 64

functions of HSPGs is the ability to modulate different growth factor activities. In this 65

context, cell-surface HSPGs bind soluble ligands, increasing their local concentration 66

and modulating ligand-receptor encounters (5). Fibroblast growth factor type-2 (FGF-2) 67

and hepatocyte growth factor (HGF) signaling are markedly enhanced by HSPGs. In 68

particular, FGF-2 completely depends on heparan sulfate to transduce an intracellular 69

signal through its receptors (FGFRs) (49, 66, 87), through the formation of the ternary 70

complex HSPG-FGF-2-FGFR (62). However, if HSPGs are localized at the ECM they 71

can decrease FGF-2 signaling by sequestering it away from the transducing receptors 72

(9). 73

Skeletal muscle formation and regeneration is a complex and regulated process 74

that involves activation, proliferation and differentiation of a muscle precursor, 75

implicating the participation of heparan binding growth factors, such as FGF-2 (13), 76

HGF (2) and transforming growth factor type-β (TGF-β) (50). Skeletal muscle 77

differentiation is regulated by the expression of specific combinations of muscle 78

regulatory transcription factors. Among them, a family of basic helix-loop-helix 79

transcription factors, called muscle regulatory factors (MRFs), is critical for muscle 80

differentiation (20, 73). The activity of MRFs, particularly myogenin, the master gene 81

involved in skeletal muscle differentiation, is highly depressed in the presence of FGF-82

2, HGF or TGF-ß (2, 13, 50). 83

The role of HSPGs in skeletal muscle physiology, as well as in the skeletal 84

muscle differentiation process, has been previously revised (41). In mature skeletal 85

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muscle tissue, HSPGs also act as co-receptors for the asymmetric form of acetyl-86

cholinesterase, increasing its concentration at the neuromuscular junction (10, 63). 87

Inhibition of proteoglycan sulfation in C2C12 cultures (52, 58), a satellite cell line 88

derived from regenerating adult mouse skeletal muscle undergoing in vitro terminal 89

myogenic differentiation, or from intact myofibers (19), affects the proper progression 90

of the in vitro myogenic program. Syndecans and glypicans are the two families of 91

HSPGs that localize to the plasma membrane. Syndecans are bound to the plasma 92

membrane through a highly conserved transmembrane domain, and are composed of 93

four separate genes in mammals (5, 24, 65), whereas glypicans are bound to the plasma 94

membrane by a glycosyl-phosphatidylinositol (GPI) linkage corresponding to six 95

separate genes (30, 32). 96

We have shown that during C2C12 myogenesis, the expression of all syndecan 97

forms are downregulated (33, 37, 44, 55), whereas the expression of glypican-1, which 98

is the only glypican expressed in myoblasts, remains constant throughout the process (8, 99

37). This differential expression may reflect different functions or macromolecular 100

specificity during myogenesis. Syndecans have been reported to modulate FGF-2 101

activity during in vitro myogenesis (33, 43, 66) and to participate in cell-cell and cell-102

matrix adhesion in development and adult wound repair (65). It has been reported that 103

syndecan-3 and syndecan-4 are expressed during embryonic limb skeletal muscle 104

formation by developing myocytes (19, 55), and that the expression continues in adult 105

muscle tissue restricted to satellite cells (19). Knock out mice for syndecan-3 exhibit a 106

novel form of muscular dystrophy while syndecan-4(-/-) satellite cells fail to 107

reconstitute damaged muscle, which reveals the importance of these macromolecules in 108

the skeletal muscle differentiation process (23). 109

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Little is known about the localization of HSPGs to specific plasma membrane 111

sub-domains. In this context, it can be speculated that glypicans might be associated 112

with lipid raft domains, which corresponds to sphingolipid and cholesterol-rich domains 113

forming phase-separated “lipid rafts” in the membrane (7, 12). Lipid raft domains might 114

be involved in signal transduction processes through specific receptors and proteins 115

anchored by GPI (11, 45). Since all the syndecans are downregulated during 116

myogenesis (33, 37, 44, 55), it is believed that their absence facilitates the silencing of 117

FGF-2 mediated signaling, helping the process of skeletal muscle differentiation and 118

allowing the expression of myogenin. The presence of glypican-1 on myoblast surfaces 119

during the entire process of skeletal muscle differentiation is puzzling, since it is also 120

able to form the ternary complex HSPG-FGF-2-FGFR when glypican-1 and FGFR-I are 121

overexpressed in the same cell (79). This paradoxical situation leads us to study 122

glypican-1 role in the modulation of FGF-2 signaling during the process of skeletal 123

muscle differentiation. We hypothesized that glypican-1, located in lipid raft domains, 124

sequesters FGF-2 avoiding the interaction with its transducing receptors. This, in turn, 125

allows the expression of myogenin, subsequent myoblast fusion and expression of late 126

muscle differentiation markers. In this study, we evaluated glypican-1 localization at the 127

plasma membrane related to FGFRs, and its direct participation in the modulation of 128

FGF-2 activity. Our results strongly suggest that glypican-1 localizes to lipid raft 129

domains where it interacts with FGF-2, sequestering it away from the FGFRs, which 130

prevents signaling and results in a strong positive effect on skeletal muscle 131

differentiation. 132

6

MATERIALS AND METHODS 133

1- Cell culture 134

The mouse skeletal muscle cell line C2C12 (ATCC) (85) was grown and differentiation 135

induced (43). Myoblasts were treated with FGF-2, HGF, TGF-β1 or platelet derived 136

growth factor (PDGF) (R&D, Minneapolis, MN, USA); heparin and methyl-beta-137

cyclodextrin (MβCD) Sigma Chemical, St. Louis, MO, USA); and phosphatidylinositol-138

specific phospholipase C (PI-PLC) Invitrogen, Carlsbad, CA, USA) (14), as indicated in 139

the corresponding figures. MβCD (1mM) or PI-PLC (0.5 U/ml) treatments were done in 140

PBS, containing 0.1 mM CaCl2 and 1 mM MgCl (PBS Ca+2

/Mg+2

), for 1 h at 37 °C in a 141

humidifying chamber. The heparin wash involved the incubation of cells in PBS 142

Ca+2

/Mg+2

, containing 10 µg/ml of heparin, and gentle agitation for 10 min at 4 °C. This 143

was repeated twice. Heparitinase (Hase) myoblast treatment (Seikagaku, Tokyo, Japan) 144

was undertaken as described (37). For the phosphorylation experiments, the cells were 145

serum starved for 6 h and then treated for the indicated times. 146

For the inactivation of FGF-2, 1 or 10 µg/ml of the soluble form of the FGFR-I 147

(rhFGF R1a(IIIc)/Fc Chimera, R&D, Minneapolis, MN, USA) or 1, 5 or 10 ug/ml of a 148

blocking antibody for FGF-2 (anti-FGF-2 neutralizing antibody Millipore #05-117, 149

Bedford. USA) were exogenously added, and their inhibitory effects were analyzed by 150

the FGF-2 dependent activation of ERK 1/2. In both cases the indicated concentrations 151

of FGF-2 were pre-incubated for 30 min at 37° C. In the case of the soluble receptor 0.1 152

µg/ml of heparin was added. The differentiation medium (2% of horse serum) was pre-153

incubated with 10 µg/ml soluble FGFR-I or 10 µg/ml of the neutralizing antibody for 30 154

min at 37° C, prior to being added to the cells. The FGF-2 neutralizing antibody was 155

replaced daily. 156

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2- Transient transfection and generation of stable clones: 159

The myogenin reporter plasmid pMyo-Luc (68), shRNA for glypican-1 and its 160

corresponding control (shCtrl), the pEGFP-N1, pRL-Sv40, the pcDNA3.0 empty vector 161

(all from Invitrogen, Carlsbad, CA, USA ) and the pcDNA3.0- rat-glypican-1 (courtesy 162

of Dr. Ralph D. Sanderson, Department of Pathology, University of Alabama at 163

Birmingham, Birmingham, Alabama USA) were transfected using Lipofectamine Plus 164

transfection reagent (Invitrogen, Carlsbad, CA, USA), according to the supplier’s 165

protocol. For the reporter experiments, the cells were plated in 24-well plates. 48 h after 166

transfection, the cells were induced to differentiate for 30 h in the presence of FGF-2. 167

The samples were subsequently assayed for dual luciferase activity (Promega, Madison, 168

WI, USA). 169

shRNA expression vectors were constructed and packaged into recombinant 170

lentiviruses, using the BLOCK-iT Lentiviral RNAi Expression System (Invitrogen, 171

Carlsbad, CA, USA), according to manufacturer’s instructions. The target sequence for 172

the shRNA against mouse glypican-1 (shGly) (NM_016696) was: 5’-173

GACCATCCGCCAGCAGATTATGC-3’. The pU6-shGly was used in transient 174

transfection experiments. From this plasmid we generated the pLenti6-U6-shGly vector, 175

which was co-transfected with the packaging plasmid mixture into 293FT cells to 176

produce recombinant lentiviruses. The conditioned medium of the lentivirus producing 177

cells was harvested and titrated using C2C12 myoblast cells. Transductions were 178

performed at the multiplicity of infection of 50. The cells were maintained in normal 179

growth medium for 48 h after transduction, and supplemented with 30 ug/ml blasticidin 180

(selection antibiotic). The target gene knockdown in transient transfections and in 181

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selected clones was confirmed by Western blot analysis. A stable cell line expressing a 182

scrambled sequence was made as the control. 183

The chimeric HSPG containing the extracellular domain of rat glypican-1 and 184

the cytoplasmatic domain of mouse syndecan-1 (GlySyn) was donated by Dr. Ralph D. 185

Sanderson (Department of Pathology, University of Alabama at Birmingham, 186

Birmingham, Alabama, USA) (46, 86), and to which a FLAG epitope in the amino 187

terminal of extracellular domain was incorporated. 188

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3- Isolation of lipid rafts 190

Lipid rafts were prepared as described previously (91), with some modifications. All of 191

the buffers and instruments used in the procedure described below were at 4°C. Briefly, 192

C2C12 myoblasts from a 150 mm dish, either control or treated, were collected in cold 193

PBS and resuspended in 400 ul of lysis buffer, containing 25 mM MES (2-[N-194

morpholino] ethanesulfonic acid), pH 6.5, 150 mM NaCl, with a mixture of protease 195

inhibitors and 1 mM PMSF supplemented with 1% Triton X-100, 1% Lubrol, or 1% 196

Brij 35, as indicated in each case. Cells were homogenized by passing them through a 197

21 gauge needle three times, incubated for 20 min on ice, and then homogenized with 198

ten strokes of a loose-fitting Dounce homogenizer. Homogenates were mixed with 400 199

ul of 90% sucrose (final concentration, 45%), loaded at the bottom of a Sorvall 4 ml 200

centrifuge tube, and overlaid with 1.6 ml of 35% sucrose and 1.6 ml of 5% sucrose, 201

both in the lysis buffer (80). The samples were centrifuged at 45,000 rpm for 18 h at 202

4°C in an AH-650 rotor. Twelve fractions (330 ul each) were collected from top to 203

bottom and designated as fractions 1–12. 204

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4- SDS-PAGE, Western blot, slot blot and co-immunoprecipitation: 206

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Aliquots from each gradient fraction were separated on 8% SDS-PAGE (Mini Protean 207

II; Bio-Rad, Richmond CA, USA), and electrotransferred onto Immobilon membranes 208

(Millipore, Bedford. USA). Western blots were probed using various primary 209

antibodies: rabbit anti-mouse FGFR-I (1:500) (Cell Signaling, Danvers, MA, USA); 210

Biotinylated anti-mouse FGFR-IV (1:500) (R&D, Minneapolis, MN, USA); rabbit anti-211

caveolin-1 (Cav-1) (1:500) (Santa Cruz biotechnology, Santa Cruz, CA, USA); anti-212

glypican-1 M95 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse 213

anti- Na+/K

+ATPase (1:1,000) (Upstate biotechnology, Lake Placid, NY, USA). To 214

reveal the distribution of the ganglioside GM1 in the gradients, 30 µl of each fraction 215

was spotted onto nitrocellulose membranes using a Bio-Rad Slot Blot apparatus, and 216

probed with horseradish peroxidase-coupled CTX (1:10,000) (Sigma-Aldrich, MO, 217

USA) (39). 218

To identify HSPG core proteins, samples containing equivalent amounts of 219

protein were incubated with Hase and Chondroitinase ABC (Seikagaku, Tokyo, Japan) 220

(79), and analyzed by western blot the anti-∆-heparan sulfate monoclonal antibody 221

(anti-stub) (Seikagaku, Tokyo, Japan), as described previously (17, 37, 55), and 222

visualized by ECL. 223

For immunoblot analysis, myoblasts were lysed in 50 mM Tris-HCl, pH 7.4, 0.1 224

M NaCl, 0.5% Triton X-100 with a mixture of protease inhibitors and 1 mM 225

phenylmethylsulfonyl fluoride. For analysis of phosphorylated proteins, cell extracts 226

were prepared in RIPA buffer (58). Aliquots with equivalent amounts of proteins were 227

subjected to SDS-PAGE gel electrophoresis in 8% polyacrylamide gels, 228

electrophoretically transferred onto immobilon membranes (Millipore, Bedford. USA), 229

and probed with rabbit anti-phospho ERK 1/2 (1:1000); mouse anti-FLAG 1:5000 230

(Stratagene, La Jolla, CA, USA) ; rabbit anti ERK 1/2 (1:1000); rabbit anti-phospho 231

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AKT (1:1000) (Calbiochem, La Jolla, CA, USA); mouse anti-α-tubulin (1:5000); mouse 232

anti-myosin (1:5,000) (Sigma-Aldrich, MO, USA); mouse anti-glyceraldehyde-3-233

phosphate-dehydrogenase (GADPH, 1:2000) (Chemicon, Temecula, CA, USA); rabbit 234

anti-myogenin (1:500) and mouse anti-caveolin-3 (1:1,000) (Santa Cruz Biotechnology, 235

Santa Cruz, CA, USA). 236

All immunoreactions were visualized by enhanced chemiluminescence (Pierce, 237

IL, USA), using a ChemiDoc-It HR 410 imaging system (Upland, CA, USA). 238

For the co-immunoprecipitation experiment WT and glypican-1 deficient 239

myoblast (C6) were transiently transfected as indicated in the Figure. 48 h after, the 240

cells were incubated whit DMEM 0.1% BSA supplemented with FGF-2 (20 ng/ml) for 241

3 hours on ice with gentle agitation. The cells were lysed in TS buffer (Tris 20mM, 242

pH7.4, NaCl 150mM, MgCl2 1mM, CaCl2 1mM), 0.1% Triton X-100 and pre-cleared by 243

centrifugation. The extracts were immunoprecipitated for 3 hours at 4°C using 10 ug of 244

a mouse anti-FLAG antibody or 10 ug of a rabbit-anti mouse syndecan-4 (nS4ED, 245

kindly donated by Dr. Alan C. Rapraeger, Department of Pathology and Laboratory 246

Medicine, University of Wisconsin, Madison, USA), electrophoresed and analyzed by 247

western blot with rabbit-anti FGFR-IV, rabbit-anti glypican-1, as described above or 248

with the rabbit-anti mouse syndecan-4 (1:1000 from a 1mg/ml stock solution). 249

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5- Immunofluorescence microscopy 251

Cells to be immunostained were grown on coverslips. The medium was removed and 252

the coverslips rinsed with PBS. Cells were fixed with 3% paraformaldehyde for 15 min 253

at 4°C, incubated for 30 min in PBS containing 3% BSA and incubated with the 254

primary antibody for 1 h in the same buffer. Rabbit anti-glypican-1 (1:300) (courtesy of 255

Dr. David Carey, Sigfried and Janet Weis Center for Research, Danville, PA, USA.); 256

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mouse anti-FLAG 1:1000; mouse anti-laminin 1:50 (Telios Pharmaceuticals, San 257

Diego, CA) and mouse anti-fibronectin (1:300) were used, as indicated in the 258

corresponding figures. 259

For the detection of myosin the cells were permeabilized with PBS containing 260

0.05% Triton X-100 (2 min at 4 °C), incubated for 30 min in PBS 3% BSA, and 261

subsequently with the primary antibody mouse anti-myosin (1:300) (Sigma-Aldrich, 262

MO, USA). Cells were rinsed with PBS 3% BSA and further incubated for 1 h with 263

Alexa 488 conjugated antibodies (1/1,000) or Alexa 568 conjugated antibodies 264

(1/1,000) (Invitrogen, Carlsbad, CA, USA). 265

Nuclear staining was done using 0.1 µg/ml of Hoechst 33258 in PBS for 10 min 266

(58). After rinsing, the coverslips were mounted and viewed under a Nikon Diaphot 267

inverted microscope equipped for epifluorescence. 268

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6- FGF-2 affinity labeling and crosslinking assay. 270

Carrier-free FGF-2 was radiolabeled with Na[125

I] using chloramine-T. Binding and 271

crosslinking of 125

I-FGF-2 to cell surfaces were assayed (27). In some experiments, the 272

cells were pre-treated with Hase (37) or competed with 200 molar excess of cold 273

FGF-2. 274

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7- Protein determination. 276

Protein was determined with the bicinchoninic acid protein assay kit (Pierce) with BSA 277

as standard. 278

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RESULTS 279

Glypican-1 is required for successful skeletal muscle differentiation. 280

To evaluate the role of glypican-1 during myogenesis, we studied the effect of 281

its absence in this process. By lentiviral infection of a the short-hairpin RNA (shRNA) 282

specific for mouse glypican-1 in C2C12 myoblasts, we produced a stable clone (C6) 283

that expressed low levels of glypican-1, as determined by Western blot analysis of the 284

corresponding HSPG core protein after Hase digestion (Fig. 1A). The core proteins of 285

any proteoglycan that is substituted with heparan sulfate can be traced using the mAb 286

3G10 (anti-stub) (79) that recognizes a neo-epitope generated after the Hase treatment. 287

shRNA-mediated knockdown of glypican-1 has no effect in the protein levels of other 288

HSPGs, since the infection with a lentiviral vector to express a scramble shRNA 289

(shCtrl) in a stable manner results in HSPG core protein levels that are equivalent to 290

those present in WT extracts (Fig. 1A). Figure 1B shows the kinetics expression of 291

myogenin, the master gene that controls skeletal muscle differentiation, and the late 292

skeletal muscle differentiation markers, myosin and caveolin-3 (cav-3) (34) in WT, 293

shCtrl and C6 myoblasts under differentiation conditions. The latter showed a 294

diminished expression of myogenin concomitant with a significant reduction in myosin 295

and cav-3 by day 5 of differentiation, compared to WT and shCtrl . 296

Immunofluorescence analysis (Fig. 1C) confirmed that myoblasts (left panels) not 297

expressing glypican-1 are unable to form myosin-expressing myotubes after 5 days of 298

differentiation (Myotubes D5, right panels). 299

Figure 2A shows that muscle differentiation is modulated specifically by 300

glypican-1 since the deleterious effect over the expression of muscle specific proteins in 301

the C6 myoblasts was restored after re-expressing this HSPG by transient transfection 302

with rat glypican-1, which is not recognized by the shGly, expressing myosin and cav-3 303

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similar to the WT levels. This Figure also shows that the transiently transfected WT 304

myoblasts with the shGly express very low levels of myosin and cav-3, as in the clone 305

C6. In this sense, the lack of myotube formation observed in the C6 clone (as shown in 306

Fig. 1C) is glypican-1 specific too, since it can be partially restored by re-expressing rat 307

glypican-1, as indicated in Figure 2B. In concordance, myoblasts transiently transfected 308

with the shGly considerably diminished the amount of myotube formation (Fig. 2B). 309

Figure 2C shows the level of glypican-1 core protein in myoblasts under each 310

experimental condition, determined by the anti-stub antibody after Hase treatment. 311

Figure 2D shows the glypican-1 levels of WT, C6 and C6 re-expressing glypican-1 312

myoblasts determined by an antibody specific against glypican-1. 313

The sums of these results indicate that glypican-1 is critical for skeletal muscle 314

differentiation, evaluated by the induction of myogenin, myosin, caveolin-3 and 315

myotube formation. 316

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The binding of FGF-2 to its receptors and its consequent signaling is augmented in 318

glypican-1 deficient myoblasts. 319

HSPGs are essential for FGFR activation by FGF-2, acting as co-receptors of 320

this muscle differentiation inhibitory growth factor. It is possible that glypican-1, 321

contrary to syndecans, might be sequestering FGF-2 away from its transducing 322

receptors. To determine this, we evaluated the binding of radiolabeled [125

I]-FGF-2 to 323

FGFRs through affinity labeling experiments, in the presence or absence of glypican-1. 324

Figure 3A shows that the crosslinking of [125

I]-FGF-2 to FGFR-I and –IV increased in 325

C6 myoblasts, as well as in wild-type myoblasts transiently transfected with the shGly. 326

Importantly, the levels of FGFRs were unaffected by glypican-1 silencing (Fig. 3B). 327

The observed binding of FGF-2 to its receptors is specific since it is totally competed by 328

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an excess of cold FGF-2 and dependent of HSPGs since it is abolished when the cells 329

were pre-treated with Hase (87) (Fig. 3C). 330

Subsequently, we evaluated if such increased binding of FGF-2 would result in 331

an augmented FGF-2-dependent signaling. The extent of phosphorylation of the 332

extracellular regulated kinase 1/2 (phospho ERK 1/2) in response to this growth factor 333

was determined (83). Figure 4A left, shows that C6 myoblasts required lower FGF-2 334

concentrations to induce phospho ERK 1/2, compared to WT myoblasts. Figure 4A 335

right shows quantification of two independent experiments. This increased response to 336

FGF-2 in the absence of glypican-1 is specific, since re-expression of rat glypican-1 in 337

C6 myoblasts revert such heightened sensitivity to FGF-2, as shown in Figure 4B left. A 338

quantification of this experiment is shown in Figure 4B (right panel). Similar results 339

were obtained after inducing the phosphorylation of AKT by FGF-2 (26) (data not 340

shown). Then we asked if the absence of glypican-1 could alter the cellular response to 341

other heparin-binding growth factors such as TGF-β1 (48, 67, 76), PDGF (35, 59, 69), 342

or HGF (3, 18, 47). Figure 4C shows that the extent of phosphorylation of Smad-2 in 343

response to TGF-β1, or the phosphorylation of ERK 1/2 in response to PDGF, were 344

unaltered in the C6 glypican-1 deficient myoblast respect to the WT myoblast. 345

Interestingly, the induction of phospho ERK 1/2 in response to HGF was diminished in 346

the glypican-1 deficient myoblast. These results suggest that glypican-1 is not involved 347

with the regulation of TGF-β and PDGF signaling, but they do not exclude the 348

possibility that other signaling pathways, such as HGF, could be regulated directly or 349

indirectly by glypican-1. 350

Since the FGF-2-dependent inhibition of myogenin expression depends on the 351

activation of the MAPK pathway (83), we determined the inhibitory effect of FGF-2 on 352

the expression of myogenin. Figure 5A (upper panel) shows the inhibitory effect of 353

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FGF-2 on the activity of pMyo-Luc, which is a reporter plasmid containing the 354

promoter region of myogenin coupled to the luciferase gene (68). Exposure of the cells 355

to FGF-2 resulted in significant inhibition of pMyo-Luc activity. However, C6 cells 356

showed a marked shift in the dose-response curve from an IC50 2.0 ng/ml for wild-type 357

myoblasts, to 0.5 ng/ml for non-expressing glypican-1 myoblasts. Re-expression of rat 358

glypican-1 in C6 myoblasts shifted the FGF-2 sensitivity to values closer to WT (~1.3 359

ng/ml). Similar results were obtained when the expression of glypican-1 in wild-type 360

myoblasts was diminished by transient transfection of the shGly (Fig. 5B). A shift in the 361

dose-response curve was observed from an IC50 2.0 ng/ml for wild-type cells to 0.6 362

ng/ml for myoblasts transfected with shGly. When the wild-type myoblasts were 363

transfected with the scrambler shRNA, no effect in the IC50 was observed (Fig. 5B). 364

The above result suggests that the altered muscle differentiation process 365

observed in the glypican-1-deficient myoblasts could be explained by an augmented 366

sensitivity to the inhibitory signaling of FGF-2. To probe this, we decided to block the 367

FGF-2 present in the differentiation medium, which is produced by the myoblast itself 368

(data not shown) (43), through its inactivation with a soluble form of the FGFR-I or by 369

the use of a neutralizing antibody against FGF-2. Figure 5C and 5E shows that the 370

phospho ERK1/2 levels induced by exogenously-added FGF-2 in WT and C6 myoblasts 371

was diminished in the presence of the soluble receptor or the neutralizing antibody, in a 372

dose dependent manner. Figure 5D shows that the myogenin and myosin levels of WT 373

myoblasts after 2 or 4 days in differentiation media are slightly augmented in the 374

presence of the soluble FGFR-I. However, in the C6 myoblasts the presence of the 375

soluble receptor importantly restores the diminished levels. Similar results were 376

obtained with the neutralizing antibody against FGF-2. Under these conditions, 377

myogenin and myosin levels were augmented in the WT and C6 myoblasts respectively, 378

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when the corresponding cells were treated with the neutralizing antibody as indicated in 379

Figure 5F. 380

Altogether, these results clearly indicate that glypican-1 inhibits the binding of 381

FGF-2 to its transducing receptors thus diminishing the FGF-2-dependent signaling, and 382

that the blockage of endogenous FGF-2 increased the expression of myogenic markers 383

in glypican-1 deficient myoblasts. These results indicate that glypican-1 inhibits FGF-2-384

dependent signaling in myoblasts, modulating the muscle differentiation process. 385

386

Glypican-1 is the only HSPG localized in myoblast lipid raft domains, binding 387

FGF-2 and not co-localizing with FGFRs. 388

The previous results demonstrate that glypican-1, contrary to the syndecans that 389

act like FGF-2 co-receptors, negatively regulate FGF-2-dependent signaling. To act like 390

a co-receptor, HSPGs require a spatial and structural condition to allow the formation of 391

the signaling ternary complex HSPG-FGF2-FGFR (54, 64, 87). In this sense, it is 392

strictly necessary that the HSPGs physically interact with the FGFRs on the plasma 393

membrane. Hence, we decided to evaluate the distribution of HSPGs in myoblast 394

plasma membrane domains and to compare their distribution with the FGFRs. For this, 395

myoblasts were solubilized in Triton X-100 and fractionated in sucrose density 396

gradients. Figure 6A shows that only glypican-1 was enriched in low-density fractions 397

together with classical markers of raft domains, such as GM-1 and caveolin-1, 398

suggesting that glypican-1 localizes in lipid raft domains. On the other hand, all the 399

members of the syndecan family, and some glypican-1, co-migrated at high density 400

fractions together with the sodium potassium ATPase (Na+/K

+ATPase), which is a non-401

raft domain marker. Figure 6B (upper panel) shows, by indirect 402

immunocytolocalization analysis, that glypican-1 presents a punctuated pattern on the 403

17

cell surface, suggesting its association with membrane microdomains (arrows), as well 404

as glypican-1 localized at the ECM with a fibrillar pattern, which is a typical feature of 405

ECM proteins (arrowheads) (8, 58). When lipid rafts were disrupted by MβCD 406

treatment (60), glypican-1 and caveolin-1 were mostly displaced from lipid raft to non-407

raft domains (Fig. 6C). Under this condition, the punctuated staining of glypican-1 408

changed to a more even staining on myoblast cell surfaces (Fig. 6B, middle panel). 409

When myoblasts were incubated with PI-PLC to remove the plasma membrane-410

associated glypican-1 (8), only a remaining stain associated to the ECM was observed 411

(Fig. 6B, lower panel). Then, we asked if the transiently transfected rat glypican-1in 412

the C6 clone was associated with lipid raft domains. For this, C6 myoblasts transfected 413

with rat glypican-1 containing a FLAG epitope, or the empty vector as control, were 414

fractionated as shown in Figure 6A. The fractions were harvested in three groups; I 415

(fractions 1-4), II (fractions 5-8) and III (fractions 9-12), and the distribution of rat 416

glypican-1was determined with an anti-FLAG antibody. Figure 6D (left panel) reveals 417

that rat glypican-1 is mainly associated with the lipid raft domains (group II) as well as 418

some fractionated in non-raft domains (group III). Figure 6D (right panel) shows that 419

rat glypican-1 expressed in C6 myoblasts exhibited a punctuated distribution pattern on 420

the cell surface, which was disrupted after MβCD treatment. All these results strongly 421

suggest that glypican-1 associates with membrane microdomains. 422

If glypican-1 present in lipid rafts regulates the binding and signaling of FGF-2, 423

it must interact with the ligand. WT and C6 myoblasts were incubated with [125

I]-FGF-424

2, then solubilized with Triton X-100 and fractionated in sucrose density gradients. 425

Figure 7A (left panel), shows that [125

I]-FGF-2 co-fractionated in raft and non-raft 426

domains, which is probably evidence of binding to heparan sulfate chains present in 427

glypican-1 and syndecan members respectively. To determine that binding of [125

I]-428

18

FGF-2 to raft domains was indeed to glypican-1, C6 myoblasts were incubated with the 429

radioactive ligand and fractionated like the WT cells. As expected, no [125

I]-FGF-2 430

migrated in raft domains, whereas all detectable [125

I]-FGF-2 migrated together with the 431

non-raft domain markers (Fig. 7A, right panel). These results clearly indicate that 432

glypican-1 is the only HSPG associated with lipid raft domains in myoblast cell 433

surfaces, where it binds and concentrates FGF-2. 434

Next, we determined the plasma membrane localization of the transducing 435

FGFRs. Western blot analysis of fractionated WT myoblasts showed that FGFR-I and 436

FGFR-IV, the main FGF-2 receptors expressed in skeletal muscles (42), were found 437

only in non-raft domains, co-fractionating with syndecans (Fig. 7B). To determine if the 438

plasma membrane FGFRs were located in non-raft membrane microdomains 439

specifically, we analyzed the distribution of the affinity-crosslinked FGFRs to [125

I]-440

FGF-2 followed by Triton X-100 solubilization and sucrose density fractionation. 441

Figure 7C shows that both receptors bound to FGF-2 fractionated only at the higher 442

density fractions, together with non-raft markers. 443

These data indicate that the plasma membrane FGFRs, as the FGF-2 co-444

receptors syndecans, are only present in non-raft domains, where they can interact and 445

facilitate FGF-2 signaling. 446

Since glypican-1 localized in raft domains would be responsible for the 447

sequestering of FGF-2, we expressed a chimeric form of a HSPG containing the 448

extracellular domain of rat glypican-1 and the transmembrane and cytoplasmic domains 449

of mouse syndecan-1 containing a FLAG epitope (F-GlySyn). C6 myoblasts were 450

transfected with F-GlySyn, lysed and subjected to sucrose density fractionation. Figure 451

8A shows that this chimeric HSPG revealed by an anti-FLAG immunoblot migrated 452

only at high-density fractions. The signaling mediated by FGF-2 in C6 myoblasts 453

19

expressing F-GlySyn form was evaluated. Figure 8B shows in F-GlySyn transfected 454

myoblast, that FGF-2 induce phospho ERK 1/2 to levels even higher than observed in 455

the mock transfected or in the glypican-1rescued C6 myoblasts. Consistently, the 456

induction of myogenin and myosin diminished when the chimeric HSPG form was 457

expressed compared to control transfected or glypican-1 rescued C6 myoblasts, as 458

shown in Figure 8C. These results suggest that the F-GlySyn form, present in non-raft 459

domains, would be acting as a presenter of FGF-2 to its transducing receptors. If so, F-460

GlySyn should interacts with the FGFRs. Figure 8D shows that by co-461

immunoprecipitation experiments with anti-FLAG antibodies, that FGFR-IV was co-462

immunoprecipitated with F-GlySyn. As expected, rat glypican-1 containing a FLAG 463

epitope as well as mock myoblasts did not co-immunoprecipitate any FGFR-IV. As 464

positive control syndecan-4 was co-immunopreciptated with FGFR-IV (Fig. 8D). The 465

same Figure shows the expression of rat glypican-1 and the quimeric F-GlySyn 466

determined by immunoreactivity with the anti glypican-1 antibody. Finally the presence 467

of syndecan-4 co-immunoprecipitated from control C& myoblasts is shown. The above 468

results clearly indicate that glypican-1 modulates muscle differentiation processes most 469

likely by sequestering FGF-2 in lipid raft domains, avoiding interaction of the ligand 470

with its receptors. 471

472

Glypican-1, present on the plasma membrane, is required for successful skeletal 473

muscle differentiation, independent from the ECM. 474

Previously it has been shown that glypican-1 is present on the plasma membrane 475

and on the ECM (8). Glypican-1 that is present in non-raft domains (Fig. 6A), as 476

evidenced by its core protein after Hase treatment, likely corresponds to ECM-477

associated glypican-1 since most of it disappears after a cell surface heparin wash. This 478

20

competes for the heparan sulfate chain binding sites without affecting the levels of 479

glypican-1 in the lighter fractions associated to raft domains (Fig. 9A). The heparin 480

wash effectively removes most of the ECM-associated glypican-1, since its fibrillar 481

pattern over the cell disappears after the wash, without affecting the cell-associated 482

glypican-1, as shown by immunofluorescence analysis in Figure 9B. To confirm that 483

part of glypican-1 is associated to the ECM, we determined the co-localization of 484

glypican-1 and the ECM protein laminin. Figure 9C shows that a fraction of glypican-1 485

co-localizes with laminin. All these results suggest that the glypican-1 that fractionates 486

in non-raft domains corresponds to an ECM-associated HSPG. 487

Since glypican-1 was found to be associated with lipid raft domains and also 488

present at the ECM, we raised the question of whether myoblast glypican-1 associated 489

with the plasma membrane, or with the ECM, is required for its negative role over the 490

FGF-2-dependent signaling and skeletal muscle differentiation. Figure 10 shows that in 491

myoblasts transiently transfected with a scrambled shRNA (shCtrl) and an expression 492

vector for E-GFP, myogenin expression was detected in the nuclei (Fig. 10A, D and G). 493

Consequently these cells were also able to fuse with forming myotubes (Fig. 10I). In 494

contrast, WT myoblasts transiently co-transfected with the shGly and an expression 495

vector for E-GFP were unable to express nucleus-associated myogenin after 2 days 496

under differentiation conditions (arrows, Fig. 10B, E and H), confirming that in the 497

absence of glypican-1 this early step in the muscle differentiation process is abolished. 498

These glypican-1 deficient cells, after 4 days under differentiation conditions, were 499

unable to fuse with forming myotubes that express myosin (Fig. 10C, F). Interestingly, 500

these occurred despite the fact that the glypican-1 negative E-GFP-expressing 501

myoblasts (green) were surrounded by glypican-1 present at the ECM network, as 502

evidenced by specific glypican-1 immunostaining (arrowheads). Figure 10J shows 503

21

quantification of this experiment. The left panel indicates that almost 40% of the nuclei 504

of control transfected E-GFP-expressing myoblasts were positive for myogenin, 505

whereas in the shGly-transfected myoblasts, this value was less than 5%. The right 506

panel shows that almost 35% of the control transfected myoblasts were able to fuse with 507

myosin-expressing myotubes, revealed by the co-expression of myosin and E-GFP. On 508

the contrary, less than 10% of the shGly transfected myoblasts were able to fuse with 509

myosin-expressing myotubes. These results strongly suggest that glypican-1 present on 510

the plasma membrane in lipid raft domains is required for successful skeletal muscle 511

differentiation, independent from the ECM-associated glypican-1. 512

22

DISCUSSION 513

In this paper we have shown that glypican-1 is required for a proper skeletal 514

muscle differentiation process. Myoblasts with low levels of glypican-1, either by 515

transient transfection with shGly or a stable clone that constitutively expresses this 516

shRNA (C6 myoblast clone), show low levels of myogenin and myosin with a 517

diminished fusion index when compared to WT myoblasts after having been induced to 518

differentiate. Another marker of skeletal muscle differentiation, namely caveolin-3 (34), 519

presents the same behavior. These defective consequences are glypican-1 dependent, 520

since re-expression of glypican-1 in the C6 myoblasts with rat glypican-1 restores 521

myosin and caveolin-3 expression, as well as myotube formation. The muscle 522

differentiation process depends on the expression levels of glypican-1, since other 523

clones, which express intermediate levels of glypican-1 compared to WT and C6 524

myoblasts, express medium levels of myosin when induced to differentiate (data not 525

shown). 526

It has been well established that FGF-2 a strong myogenesis inhibitor (13, 43, 527

58, 78), diminish the expression of the master gene myogenin (70, 71). Thus, its 528

signaling must be finely controlled. Skeletal muscles mainly express FGFR-I and 529

FGFR-IV (42), which both have a high affinity for FGF-2 (57). FGFs are normally 530

present in muscle tissue and appear to be released upon injury and are expressed at 531

higher levels during regeneration (20, 22, 75). Both receptors have different expression 532

patterns during the muscular differentiation process. FGFR-I is temporally unchanged 533

during the initial days and diminishes later, but it is still present during muscle 534

differentiation (40, 42, 61). In contrast, FGFR-IV is upregulated during this process (42, 535

88), and has been proposed to be essential for muscle regeneration (90). Since the 536

signaling of FGF-2 through its receptor depends on the presence of HSPGs (66, 87), 537

23

regulation by these FGF-2 co-receptors seems to attenuate FGF-2-dependent signaling, 538

thus allowing myogenesis. In different systems, it has been shown that syndecans (6, 25, 539

31, 33, 43, 84, 89) and glypicans (53, 77, 81) have the ability to bind FGF-2, 540

modulating its binding and signaling. 541

We, among others, have shown that HSPGs are essential for FGF-2-mediated 542

signaling in skeletal muscle cells (17, 21, 33, 43, 56, 66). It has been demonstrated that 543

syndecan-1 and-3 are directly involved in this phenomenon, acting as co-receptors of 544

FGF-2 in myoblasts (33, 43). This is crucial, since myoblasts that do not express 545

HSPGs, or are deficient in some of its forms, present an effected process of skeletal 546

muscle formation. 547

The expression of all syndecans is downregulated during the skeletal muscle 548

differentiation process (33, 37, 44) suggesting that might be associated with a 549

diminished sensitivity to the inhibitory effect of FGF-2. In contrast, the expression level 550

of glypican-1 is constant through this process, hence being the main HSPG present 551

during myogenesis (8, 37). 552

Our results unequivocally demonstrate that glypican-1 is required for terminal 553

myogenesis, which raises the question of how glypican-1 regulates the FGF-2-554

dependent signaling during the muscle differentiation process. Our experimental 555

evidence indicates that in the absence of glypican-1, the binding of FGF-2 to its 556

receptors augments, increasing the activation of the MAPK ERK 1/2 and PI3K/AKT 557

pathways and the FGF-2-dependent inhibition of myogenin expression. These effects 558

were directly associated with the absence of glypican-1, since rat glypican-1 re-559

expression restored FGF-2-dependent signaling near to WT levels. The blockage of 560

FGF-2 activity present in the differentiation medium using a soluble form of the FGFR-561

I or a neutralizing antibody against FGF-2, partially restored the altered muscle 562

24

differentiation process in the glypican-1 deficient myoblast, suggesting that the 563

deleterious effect of the absence of glypican-1 over the myogenesis is a consequence of 564

an increased sensitivity to FGF-2. 565

The formation of the ternary signaling complex involving HSPG, FGF2 and 566

FGFR (72) requires that these three components physically interact on the plasma 567

membrane. We show that in myoblasts glypican-1 is the only HSPG found associated 568

with lipid raft membrane domains, away from all the syndecans and FGFRs. The 569

localization in raft domains is sustained by low-density fractionation in the sucrose 570

gradients and co-fractionation with specific lipid raft domain markers. Glypican-1 571

shows a punctuated appearance, which typically characterizes lipid raft domain 572

localization, and disappears after MβCD treatment (4). This pattern corresponds to 573

plasma membrane glypican-1, since totally disappears after treatment with PI-PLC. 574

Glypican-1 localization in raft membrane domains is reinforced by the total abolishment 575

of staining in glypican-1-deficient cells, and the re-appearance of the punctuated 576

staining after re-expression of rat glypican-1. In contrast, all the syndecan forms and 577

FGFRs co-fractionated in non-lipid raft domains, determined by co-fractionation of 578

specific markers at high-density sucrose fractions. Other authors have suggested that 579

clustering of syndecans 1 and 4 with antibodies, or after treatment with FGF-2, induces 580

a re-localization of part of this HSPG from non-raft to raft microdomains in lymphoid 581

and epithelial cells respectively (51, 82). We did not observe any change in the 582

distribution of HSPGs when myoblasts were treated with FGF-2 (data not shown). This 583

indicates that glypican-1 remains the only HSPG associated with lipid rafts under our 584

experimental conditions. 585

These results suggest that gypican-1 could be sequestering FGF-2 in lipid rafts, 586

away from its transducing receptors. Our experiments indicate that FGF-2 binds and co-587

25

migrates with glypican-1 in myoblast isolated membrane raft domains, since no FGF-2 588

was found in lipid rafts of myoblasts deficient for glypican-1. FGF-2 bound to FGFRs 589

located at the cell surface fractionated in non-raft membrane domain fractions, as 590

determined by crosslink assays. Furthermore glypican-1 does not interact with FGFR-591

IV determined by co-immunoprecipitation experiments. This suggests that the FGF-2-592

FGFR complex is formed and maintained in a different spatial localization than 593

glypican-1. The notion that co-localization of HSPG with FGFRs is critical for FGF-2 594

signaling is reinforced by the experiments expressing the F-GlySyn chimeric form. 595

Despite containing a glypican-1 ectodomain, this HSPG was expressed in non-raft 596

domains most likely as a consequence of the presence of syndecan cytoplasmic and 597

transmembrane domains on its structure. This chimeric form increased FGF-2 598

dependent signaling, interacting at least with FGFR-IV. 599

Since glypican-1 is endogenously processed to a soluble form that is 600

incorporated to the ECM (8) co-localizing with laminin, the possibility that glypican-1 601

present in the ECM is sequestering FGF-2 cannot be excluded. Glypican-1 deficient 602

myoblasts did not express myogenin nor fused with control myoblasts, which expressed 603

myogenin and later formed elongated myotubes. These processes occurred in an ECM 604

enriched with glypican-1, synthesized and processed by the control myoblast. This 605

suggests that glypican-1 present in raft membrane domains is the required form for a 606

proper muscle differentiation processes, and is probably responsible for the inhibitory 607

effect on FGF-2 bio-availability. 608

Other functions for glypican-1, besides the inhibitory effect on FGF-2 609

availability, cannot be excluded. HSPGs interact with several ECM constituents (5) and 610

glypican-1 deficient myoblasts present a diminished capability to fuse and form 611

elongated myotubes. This might reflect the possibility of other functions of glypican-1. 612

26

Since HSPG can bind several ligands (28), such as Wnt (15), bone morphogenic protein 613

(BMP), FGF (53, 77, 81), sonic hedgehog (16, 29), distinct members of TGFβ (48, 67), 614

PDGF (35, 36, 59, 69) and HGF (3, 47), it is highly possible that glypican-1 might have 615

other functions in the raft membrane domain. We tested the signaling response to 616

TGFβ-1, PDGF and HGF in the presence or absence of glypican-1. We did not detect 617

any differences between glypican-1 deficient and WT myoblasts to TGF-β and PDGF, 618

suggesting that the response to these growth factors does not depend on glypican-1. 619

However, we detected a decrease in signaling response to HGF in glypican-1 deficient 620

compared to WT myoblats, this might indicate that glypican-1 directly or indirectly is 621

involved in the signaling response to HGF, nevertheless the localization of c-Met, the 622

receptor for HGF, in membrane microdomains is controversial (74). It is worth 623

mentioning that in myoblasts, it has been shown that BMP receptor type II (BMP-RII) is 624

located in lipid raft membrane domains (38) and that the BMP-RII and the BMP-RIA 625

are upregulated during myogenesis (1), although there is no functional evidence for this 626

co-localization. 627

In summary, we have shown that glypican-1 located in membrane raft domains 628

diminishes the bio-availability of FGF-2, sequestering this growth factor away from its 629

transducing receptors. As a consequence, a decrease in FGF-2-dependent signaling 630

occurs, allowing skeletal muscle differentiation to succeed. This novel mechanism of 631

sequestering FGF-2 in lipid rafts, together with the downregulation of the syndecans 632

which co-reside with FGFRs, might be essential to assure successful skeletal muscle 633

differentiation during development and muscle regeneration. 634

27

ACKNOWLEDGEMENTS 635

The authors are indebted to Dr. David J. Carey (Siegfried and Janet Weiss 636

Center for Research, Danville, PA, USA) and to Dr. Ralph D. Sanderson (University of 637

Alabama at Birmingham, Alabama, USA), for providing anti glypican-1 antibody and 638

pcDNA 3.0-rat-glypican-1, respectively. We thank Drs. Juan Larraín and Hugo Olguín 639

(P. Universidad Católica de Chile) for offering encouragement and helpful suggestions. 640

This study was supported by research grants from FONDAP-Biomedicine # 13980001, 641

CARE PFB 12/2007 and the Muscular Dystrophy Association # 89419, as well as the 642

doctoral fellowship granted to Jaime A. Gutierrez by CONYCYT, Chile. 643

28

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927

37

FIGURE LEGENDS 928

929

FIGURE 1: Glypican-1 is required for a successful muscular differentiation 930

process. A: C2C12 myoblasts (WT) were infected with a lentiviral vector to generate a 931

stable clone that expresses a shRNA control (shCtrl) or a shRNA specific for mouse 932

glypican-1 (C6). Glypican-1 levels were determined by Western blot analysis using 933

anti-stub antibodies that recognizes a neo-epitope generated in the heparan sulfate 934

chains after digestion with Hase, enabling the core proteins of any HSPG to be 935

visualized. Syn-3, syn-1, syn-2 and syn-4 represent syndecan-3, -1, -2 and -4, 936

respectively. B: WT, shCtrl and C6 myoblasts were induced to differentiate for 0, 2, 4 937

and 6 days in the differentiation medium (Days DM). Cell extracts were analyzed by 938

Western blot for myogenin, myosin and caveolin-3. Tubulin levels are indicated as a 939

loading control. In A and B, the molecular weights are indicated in kD. C: In a parallel 940

experiment WT and C6 myoblasts were fixed and analyzed by Phase contrast or indirect 941

immunofluorescence for glypican-1 (red) and myosin (green) after 5 days of 942

differentiation (Myotubes D5). Nuclei were stained with Hoechst (blue). 943

944

FIGURE 2: Re-expression of glypican-1 restores the impaired muscular 945

differentiation observed in glypican-1 deficient myoblasts. A: WT and C6 myoblasts 946

were transiently transfected with shGly or with rat glypican-1 (Gly), respectively. 48 h 947

after transfection, the myoblasts were induced to differentiate into myotubes for 4 days 948

(Myotubes D4). The extracts were analyzed by immunoblot analysis for the late muscle 949

differentiation markers, myosin and caveolin-3 (Cav-3). GAPDH levels were used as a 950

loading control. B: Phase contrast images of each experimental condition of A, at day 4 951

of differentiation. C: The glypican-1 protein levels of the myoblast transfected as in A 952

were determined after 48 h by immunoblot analysis by the anti-stub, as described in the 953

38

legend of Figure 1A, D: The glypican-1 protein levels of the myoblast transfected as in 954

A were determined after 48 h by immunoblot analysis with a glypican-1 specific 955

antibody. In A, C and D the molecular weights are indicated in kD. 956

957

FIGURE 3: The binding of FGF-2 to its receptors is augmented in glypican-1-958

deficient myoblasts. A: FGF-2 cell surface receptors of WT myoblasts transiently 959

transfected with or without shCtrl and shGly, and C6 myoblasts transiently transfected 960

with or without rat glypican-1 (C6-Gly), were affinity crosslinked to [125

I]-FGF-2 at 961

4ºC. Cell extracts were separated on SDS-PAGE, and then exposed to a phosphorimager 962

(left). On the right, the gel was stained with Coomasie blue as a loading control. B: The 963

same extracts of A, on the left, were analyzed by Western blot to determine the total 964

protein levels of the FGFR-I and FGFR-IV. GAPDH levels were used as a loading 965

control. C: Myoblasts were treated with or without Hase, then FGFRs were affinity 966

crosslinked to [125

I]-FGF-2 at 4ºC in the presence or absence of an excess of cold FGF-967

2. On the right, the gel was stained with Coomasie blue as a loading control. In A, B 968

and C the molecular weight standards are indicated in kD. 969

970

FIGURE 4: Myoblasts deficient in glypican-1 are more sensitive to FGF-2, but not 971

to other heparin binding growth factors. A: WT and C6 myoblasts were treated with 972

the indicated concentration of FGF-2 for 5 min. Cell extracts were analyzed for phospho 973

ERK1/2 by immunoblot analysis. The levels of total ERK 1/2 were used as a loading 974

control. On the right, a quantification of two independent experiments is shown. B: C6 975

myoblasts were transiently transfected with or without rat glypican-1 (C6-Gly), and 48 976

h after transfection the cells were treated as in A. A quantification of this experiment is 977

shown on the right. C: WT and C6 myoblasts were treated with the indicated 978

39

concentrations of TGFβ-1, PDGF for 15 min or HGF for 5 min. The levels of phospho-979

Smad 2 or phospho ERK1/2 were determined by immunoblot analysis. GAPDH or total 980

ERK 1/2 levels were used as a loading control. In A, B and C the molecular weight 981

standards are indicated in kD. 982

983

FIGURE 5: The absence of glypican-1 induces an increase in the FGF-2-dependent 984

inhibition of the muscular differentiation process. A: WT and the C6 myoblasts were 985

transiently co-transfected with pMyo-luc, the transfection control plasmid (pRL-SV40), 986

an empty pcDNA3.1 plasmid as control, or rat glypican-1 to re-express glypican-1 in 987

the C6 myoblasts (C6-Gly). 48 h after transfection, the cells were induced to 988

differentiate for 30 h in the presence of FGF-2 at the indicated concentrations. The 989

values (pMyo-Luc/pRL-SV40 activity) are expressed as percentages of reporter activity 990

in the absence of FGF-2 for each experimental condition. B: WT myoblasts were co-991

transfected with the reporter system described in A, and a shCtrl or shGly. After 30 h in 992

the differentiation medium, pMyo-Luc and pRL-SV40 activities were determined and 993

expressed as in A. The values shown in A and B are the results obtained from three 994

independent experiments performed in triplicate and correspond to the mean and 995

standard deviation. C and E: WT or C6 myoblasts were treated with FGF-2 pre-996

incubated with or without increasing concentrations of a soluble form of the FGFR-I 997

(FGFR (S)) or a neutralizing antibody against FGF-2 (anti-FGF2), respectively. The 998

phospho and total ERK1/2 levels were determined by immunoblot analysis. D and F: 999

WT and C6 myoblasts were induced to differentiate in the presence or absence of the 1000

FGFR (S) or the anti-FGF2, respectively. The anti-FGF2 was replaced daily. Myosin 1001

and myogenin levels were analyzed by immunoblot after 2 or 4 days. Tubulin levels 1002

40

were used as a loading control in both cases. In all figures the molecular weights are 1003

indicated in kD. 1004

1005

FIGURE 6: Glypican-1 is the only HSPG associated with lipid rafts. A: C2C12 1006

myoblasts were lysed and then fractionated in sucrose density gradients (5-45%). The 1007

twelve fractions collected were analyzed by immunoblot for HSPGs, as explained in the 1008

legend of Figure 1A, as well as the lipid raft membrane protein markers, GM-1 1009

(ganglioside GM-1) and Cav-1. Na+/K

+ATPase (ATPase), were used as a non-lipid raft 1010

domain marker. On the left, the molecular weight standards are indicated in kD. B: 1011

Indirect immunocytolocalization analysis for glypican-1 (red) in C2C12 myoblasts 1012

treated with or without MβCD or PI-PLC. The nuclei were stained with Hoechst (blue). 1013

The arrows indicate the punctuated pattern of glypican-1 on the cell surface, and the 1014

arrowheads point at the ECM associated glypican-1. C: C2C12 myoblasts were treated 1015

with or without MβCD, and then fractionated as in A. The fractions were analyzed for 1016

glypican-1 and caveolin-1 distribution by Western blot analysis. D: On the left, C6 1017

clone myoblasts were transiently transfected with rat glypican-1 containing a FLAG 1018

epitope in its amino terminal (F-Gly) or the empty vector. After 48 h, the cells were 1019

fractionated as in A. The twelve fractions were pooled into three groups; I (fractions 1-1020

4), II (fractions 5-8) and III (fractions 9-12), and analyzed for the distribution of rat 1021

glypican-1 using an anti-FLAG antibody. On the right, in a parallel experiment, C6 1022

myoblasts transfected with F-Gly were treated with or without MβCD, then fixed and 1023

analyzed by immunofluorescence for FLAG epitope. Syn-3, syn-1, syn-2 and syn-4 1024

represent syndecan-3, -1, -2 and -4, respectively. 1025

1026

41

FIGURE 7: Glypican-1 concentrates FGF-2 in lipid raft microdomains that 1027

exclude FGF-2 signaling receptors. A: WT and C6 myoblasts were incubated with 1028

[125

I]-FGF-2 for 3 h at 4 ºC, and then fractionated as in Figure 1. The fractions were 1029

analyzed for HSPGs by anti-stub, as in Figure 1A, or exposed to a phosphorimager to 1030

detect the distribution of [125

I]-FGF-2. B: C2C12 myoblasts were fractionated as 1031

explained in Figure 7A. The fractions were analyzed for the distribution of FGFR-I and 1032

FGFR-IV, as well as membrane distribution markers Cav-1, GM-1 and Na+/K

+ATPase 1033

(ATPase). C: FGF-2 cell surface receptors in C2C12 myoblasts were affinity 1034

crosslinked to [125

I]-FGF-2 at 4ºC. The cells were lysed and fractionated, as explained 1035

in Figure 7A. Aliquots of each fraction were separated on SDS-PAGE (4-10%). The gel 1036

was dried and exposed to a phosphorimager (upper panel), or analyzed by Western 1037

immunoblot for Cav-1, GM-1, and ATPase. [125

I]-FGF2-FGFR-I and [125

I]-FGF2-1038

FGFR-IV correspond to [125

I]-FGF2 crosslinked to FGFR-I or FGFR-IV, respectively. 1039

In A, B and C the molecular weight standards are indicated in kD. 1040

1041

FIGURE 8: The expression of glypican-1 outside of the lipid raft acts like a FGF-2 1042

co-receptor. A: C6 myoblasts were transiently transfected with a chimeric HSPG (F-1043

GlySyn) composed of the extracellular domain of rat glypican-1 and the transmembrane 1044

and cytosolic domain of mouse syndecan-1, containing a FLAG epitope. After 48 h the 1045

cells were lysed and fractionated as in Figure 6A. The distribution of F-GlySyn was 1046

evaluated by anti-FLAG analysis. B: C6 myoblasts were transfected with or without rat 1047

glypican (F-Gly) or F-GlySyn and treated with increasing concentrations of FGF-2. The 1048

phospho ERK 1/2, total ERK1/2 and tubulin levels were determined by immunoblot 1049

analysis. C: C6 myoblasts were transfected with or without rat glypican (F-Gly) or F-1050

GlySyn and induced to differentiate for 2 (D2) or 4 (D4) days to determine the 1051

42

myogenin, myosin and tubulin levels by immunoblot. D: WT or C6 myoblasts were 1052

transiently transfected as in C. 48 h after. The cells were lysed and the extracts 1053

immunoprecipitated with an anti-FLAG antibody or an anti mouse syndecan-4. The 1054

immunoprecipitates were analyzed by western blot for the presence of FGFR-IV, F-Gly 1055

and F-GlySyn with an anti-anti glypican-1 or syndecan-4. The molecular weight 1056

standards are shown in kD. 1057

1058

FIGURE 9: Glypican-1 is present in the plasma membrane lipid rafts and in the 1059

extracellular matrix. A: WT myoblasts were fractionated as shown in Figure 7, after 1060

being washed with heparin in PBS Ca2+

/Mg2+

(Heparin Wash) or PBS Ca2+

/Mg2+

alone 1061

(control). The fractions were analyzed for HSPG core proteins and caveolin-1 1062

distribution. B. Indirect immunofluorescence for glypican-1 (red) of cells treated as in 1063

A. C: Indirect immunofluorescence for glypican-1 (red) and laminin (green) in WT 1064

myoblasts. 1065

1066

FIGURE 10: Glypican-1 is required on the plasma membrane for a proper 1067

muscular differentiation process, independent from extracellular matrix-1068

associated glypican-1. C2C12 myoblasts were transiently co-transfected with the 1069

scrambler shRNA (shCtrl) and a plasmid containing the sequence for E-GFP in A, D 1070

and I, or the shGly in B, E, C and F. 48 h after transfection, the myoblasts were induced 1071

to differentiate for 2 (A, B, D and E) or 4 days (C, F and I). The cells were fixed and 1072

analyzed by immunofluorescence for glypican-1 (red) in A, B and C; myogenin (red) in 1073

D and E; or myosin (red) in F and I. G and H are the same images as D and E, 1074

respectively without the E-GFP signal, in order to better visualize myogenin nuclear 1075

staining. The arrowheads indicate the ECM-associated glypican-1. The arrows indicate 1076

43

the nucleus of transfected cells. J: On the left is a quantification of the co-transfected 1077

myoblasts (shCtrl/E-GFP or shGly/E-GFP) containing myogenin positive nuclei, 1078

compared to the total co-transfected cells (E-GFP positive) after 2 days of 1079

differentiation of ten random fields. On the right is a quantification of the E-GFP-1080

myosin positive myotubes compared the total of E-GFP expressing cells, after 4 days of 1081

differentiation. The data correspond to the mean +/- SE of ten random fields. 1082

A Bk D k D 0 2 4 6 0 2 4 6 0 2 4 6W T s h C t r l C 6 M y o g e n i nD a y s D Mg l y p i c a n � 1s y n � 3s y n � 1 1 1 88 5 C a v 33 6 M y o g e n i nM y o s i n2 0 5s y n � 2s y n � 4 4 73 6 5 52 0 T u b u l i nC a v � 3CW T M y o t u b e s D 5M y o b l a s t sC 6 M y o s i n G l y p i c a n1 0 0 u m 5 0 u mG l y p i c a n

F I G U R E 1

BA C t r l s h G l yW T C 6 W T C 6M y o b l a s t s M y o t u b e s D 4 W TC t r l G l yM y o s i nC a v \ 3 2 02 0 5k Dk D C 6 1 0 0

"u mG A P D H 3 6k DC DW T C 6M y o b l a s t s C 6M y o b l a s t sg l y p i c a ns y n \ 3s y n \ 1s y n \ 21 1 88 54 7k D 1 1 88 5k Dg l y p i c a n \ 1s y n \ 4y4 73 6 4 73 6

F I G U R E 2

BA B F G F R � I VF G F R � IA 1 1 81 1 8k DG A P D HCF G F R � IF G F R � I V 1 7 01 3 01 7 01 3 0 H a s eC o l d F G F � 2k D k D k D3 69 57 25 54 39 57 25 54 3 F G F R � IF G F R � I V 1 1 88 54 7 1 1 88 54 72 03 42 03 41 2 5 I ¯ F G F ¯ 2 C o o m a s i e 1 2 5 I ¯ F G F ¯ 2 C o o m a s i e2 04 73 62 6 2 04 73 62 6F I G U R E 3

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F I G U R E 7

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 4 5 %5 % S u c r o s e1 7 0 F í G l y S y nA k D a B C 6 F û G l y S y nF û G l yM o c k P h o p h o4 4k DC 69 57 25 54 31 3 01 7 0 F � G l y S y n P h o p h oE R K 1 / 2T o t a lE R K 1 / 2T u b u l i n4 44 2k D4 44 2G M � 1C 6 DC T u b u l i nC 6M y o g e n i nT u b u l i n5 5k D a3 6 I P P � F L A Gk DI P P � S y n 4 W B : F G F R � I V1 1 8D 2D 0 M y o s i n2 0 5k D a W B : G l y p i c a n � 1W B : S y n � 43 67 24 7T u b u l i n5 5 W B : S y n � 43 6D 4D 0 F I G U R E 8

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