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Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells

Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect... Background: Via the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF. Methods: C2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed. Results: Glypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration. Conclusions: Glypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains. Keywords: Glypican-1, Heparan sulfate proteoglycans, Hepatocyte growth factor, HGF-mediated signaling, Raft membrane domains, Skeletal muscle Background muscle fibers [2]. HGF was originally identified as a scatter The process of skeletal muscle regeneration is initiated factor because of its ability to increase the motility of sev- immediately after injury by the release of growth factors eral normal and neoplastic cells [4,5]. The requirement of and cytokines from injured muscles, blood vessels, infil- HGF for migration of muscle precursor cells during mouse trating inflammatory cells and extracellular matrix (ECM) muscle development has been established by the genetic reservoirs. These factors include basic fibroblast growth ablation of HGF or the HGF receptor (Met). In both cases, factor 2 (FGF-2) and hepatocyte growth factor (HGF) the result was the absence of hindlimb muscles, which are [1-3]. The factors promote the activation, proliferation, mi- formed by muscle precursor cells that migrate from the gration and survival of satellite cells (SCs), which are the dermomyotome [6-8]. In vitro studies have shown that muscle stem cells responsible for the formation of new HGF not only induces the proliferation and migration of myogenic cells but that it also delays muscle differentiation by inhibiting the expression of MyoD and myogenin, two * Correspondence: ebrandan@bio.puc.cl master myogenic regulatory transcription factors [3,9,10]. Centro de Regulación Celular y Patología (CRCP), Centro de Regeneración y The expression of HGF and Met are downregulated during Envejecimiento (CARE), Departamento de Biología Celular y Molecular, MIFAB, Pontificia Universidad Católica de Chile, Santiago, Chile © 2014 Gutiérrez et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 2 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 myogenesis, which is consistent with attenuation of myo- was clearly decreased in the absence of glypican-1, suggest- genic inhibitory signaling of HGF [11-13]. Therefore, HGF ing that glypican-1 was a positive regulator of HGF signal- plays key role during myogenesis, regulating the prolifera- ing [38]. tion, migration and subsequent differentiation of muscle Because HGF and Met are found in lipid raft domains precursor cells. [17-20], we hypothesize that glypican-1 in these domains Upon HGF binding, Met is activated by dimerization is necessary to stabilize the HGF-Met signaling complex. with subsequent trans-phosphorylation of four tyrosine In the present study, we report that the presence of residues which act as docking motifs for signaling media- glypican-1 in lipid rafts was required for maximum HGF- tors, including mitogen-activated protein kinase (MAPK), dependent signaling, localizing and stabilizing HGF and extracellular signal-regulated kinases 1 and 2 (ERK1/2) Met in its phosphorylated or activated state (phospho- and phosphoinositide 3-kinase protein kinase B (AKT), Met). We also show that glypican-1, phospho-Met and among others [14-16]. HGF interact, indicating that they form part of a signaling It has been proposed that HGF and Met form a complex complex in lipid rafts. Finally, we show that glypican-1 is in lipid rafts, which are sphingolipid- and cholesterol-rich required for myoblast migration induced by HGF in vitro domains that form phase-separated lipid rafts in the mem- and in vivo, demonstrating the requirement of glypican-1 brane. In these domains, Met is stabilized by HGF to in- expression and HGF for processes such as muscle stem duce its activation [17-20]. cell therapy, where the migration of myoblasts must be Another important component of the HFG-Met sig- enhanced. naling is the ubiquitous transmembrane glycoprotein CD44, the major receptor for hyaluronic acid [21,22]. In Methods different cell types, the activation of the MET receptor Cell culture by HGF depends on the presence of some isoforms of The mouse skeletal muscle cell line C2C12 (American CD44 [21]. As proposed, HGF, Met and CD44 would form Type Culture Collection, Manassas, VA, USA) [40] and its a complex in lipid raft membrane domains, which cor- derived clone deficient in glypican-1 expression [38] were responds to sphingolipid- and cholesterol-rich domains grown as previously described [31,38]. Myoblasts were forming phase-separated lipid rafts in the membrane, treated with HGF (R&D Systems, Minneapolis, MN, USA) where Met would be stabilized by HGF inducing its acti- as indicated in each experiment. Methyl-β-cyclodextrin vation [17,18]. (MβCD) (Sigma-Aldrich, St Louis, MO, USA) treatment HGF also binds to heparin, heparan sulfate (HS) and at 1 or 10 mM concentrations were performed as previ- dermatan sulfate [23-27]. Heparan sulfate proteoglycans ously described [38]. For the phosphorylation experiments (HSPGs), key components of the cell surface and the of Met, ERK1/2 and AKT, the cells were serum-starved ECM, regulate many processes related to cell growth for 6 hours and then treated for the indicated times. and differentiation. Cell-surface HSPGs bind soluble li- gands, increasing their local concentration and modulating Transient transfection and generation of stable clones ligand–receptor interactions [28]. For example, HSPG is The pcDNA3.0 empty vector (Invitrogen, Carlsbad, CA, required for FGF-2-dependent signaling through its recep- USA) and pcDNA3.0 vectors containing rat glypican-1 tors (FGFRs) [29-32], forming the ternary complex HSPG- and chimeric HSPG comprising the extracellular domain FGF-2-FGFR [33]. However, the exact role of HSPG in of rat glypican-1 were fused to the transmembrane and HGF signaling is poorly understood. In vitro assays have cytoplasmic domains of mouse syndecan-1 containing a shown that heparin increases the mitogenic effect of HGF FLAG epitope in their amino-terminal F-Gly and F- and facilitates its oligomerization, inducing Met dimeri- GlySyn, respectively [38]. Transfection were carried out zation and activation [34]. Previously, we showed that using Lipofectamine and PLUS reagents (Invitrogen) ac- myoblast migration induced by HGF was strongly inhib- cording to the supplier’s protocol. ited if the cells were depleted of HS chains, indicating that at least the myoblast cell response to HGF depended on Isolation of lipid rafts HS [23]. Lipid rafts were prepared as described previously, with We have also previously shown that myoblasts express some modifications [38]. All of the buffers and instru- different membrane-bound HSPGs, the four transmem- ments used in the procedure described below were used brane syndecans and glypican-1, which corresponds to a at 4°C. Briefly, C2C12 myoblasts from a 150-mm dish were glycosylphosphatidylinositol-anchored HSPG [31,32,35-39]. lysed in 400 μl of lysis buffer (25 mM 2-(N-morpholino) Glypican-1 is the only HSPG located in lipid raft microdo- ethanesulfonic acid, pH 6.5, 150 mM NaCl, with a mixture mains, which sequester FGF-2 to avoid its interaction with of protease inhibitors and 1 mM phenylmethanesulfonyl FGFRs. Thus, glypican-1-deficient cells exhibit enhanced fluoride supplemented with 1% Triton X-100). Cells were sensitivity to FGF-2. In contrast, HGF-dependent signaling incubated for 20 minutes on ice, then homogenized with Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 3 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 ten strokes of a loose-fitting Dounce homogenizer. Ho- For coimmunoprecipitation experiments, wild-type and mogenates were mixed with 400 μl of 90% sucrose (45% glypican-1-deficient myoblasts (C6) were transiently trans- final concentration), loaded at the bottom of a Sorvall 4-ml fected as indicated in the figure legends. At 48 hours after centrifuge tube (Thermo Scientific, Asheville, NC, USA) transfection, cells were serum-starved for 4 hours, then ei- and overlaid with 1.6 ml of 35% sucrose and 1.6 ml of 5% ther treated or not treated with 20 ng/ml [ I]HGF in sucrose, both in the lysis buffer without Triton X-100. The Dulbecco’s modified Eagle’s medium (DMEM) 0.1% bo- samples were centrifuged at 45,000 rpm for 18 hours at vine serum albumin (BSA) for 5 minutes. The cell extracts 4°C in an AH-650 rotor. Twelve fractions (330 μleach) in RIPA buffer plus phosphatase inhibitors were incu- were collected from top to bottom and designated as bated with anti-FLAG M2 Affinity Gel (Sigma-Aldrich) fractions 1 to 12. Only the last ten fraction were ana- for 3 hours at 4°C. The beads were sequentially washed lyzed, because the low-density lipid raft–enriched frac- in RIPA buffer, then in heparitinase reaction buffer tions started at fraction 5 in several previous assays that (20 mM Tris, 150 mM NaCl, 1 mM MgCl ,1mM Ca we performed. Cl , pH 7.4). The beads were then treated with hepariti- nase and chondroitinase ABC for 3 hours at 37°C. The SDS-PAGE, Western blot and coimmunoprecipitation assays bound material was eluted with protein loading buffer Aliquots from the last ten fractions of the different sucrose and assessed by Western blot analysis for total Met, density fractionations were separated on 8% SDS-PAGE phospho-Met and glypican-1 or exposed to a phosphori- gels (Mini-PROTEAN II; Bio-Rad Laboratories, Hercules, mager to detect [ I]HGF. CA, USA) and electrophoretically transferred to Immobilon membranes (EMD Millipore, Bedford, MA, USA). Western Biotin labeling and precipitation of biotin-labeled proteins blots were probed using the following primary antibodies: Biotin labeling was conducted as previously described rabbit anti-mouse Met (1:200) (Santa Cruz Biotechnology, [43]. Equal amounts of protein (100 μg) obtained from Santa Cruz, CA, USA), rabbit anti-phospho-Met at Tyr previously biotinylated cell extracts were precipitated for 1234 and Tyr 1235 (1:1,000) (Cell Signaling Technology, 2 hours at 4°C using streptavidin agarose resin (Thermo Danvers, MA, USA), rabbit anti-caveolin-1 (1:500) (Santa Fisher Scientific, Rockford, IL, USA). The bound material Cruz Biotechnology), rabbit anti-glypican-1 M-95 (1:500) was eluted with protein loading buffer and assessed by + + (Santa Cruz Biotechnology), mouse anti-Na /K -ATPase Western blot analysis for total Met as described above. (1:1,000) (Upstate Biotechnology, Lake Placid, NY, USA) and rat anti-CD44 (1:500) (BD Pharmingen, San Jose, CA, Transwell migration assays USA). Migration assays were conducted using 24-well, 8-μm-pore To identify glypican-1, samples containing equivalent transwell systems (EMD Millipore). C2C12 and C6 myo- amounts of protein were treated with heparitinase and blasts were seeded onto the upper part of the chamber at a chondroitinase ABC (United States Biological, Swampscott, density of 100,000 cells per well in 300 μlofserum-free MA, USA) as previously described [39,41] prior to SDS- media. The lower chamber was loaded with 500 μlof PAGE and Western blot analysis using anti-glypican-1 M- serum-free media with or without 20 ng/ml HGF or 10% 95 antibody. fetal bovine serum (FBS) (data not shown). The cells were For analysis of phosphorylated proteins, cell extracts allowed to migrate for 8 hours. Migration was assessed by were prepared in radioimmunoprecipitation assay (RIPA) removing the cells on the upper side of the transwell with buffer in the presence of phosphatase inhibitors as previ- a cotton swab, then staining the remaining cells with crys- ously described [38,42]. Aliquots with equivalent amounts tal violet, and solubilizing the cells in 1% Triton X-100 to of protein were subjected to SDS-PAGE in 8% polyacryl- measure the absorbance of the Triton X-100 solution at amide gels, electrophoretically transferred to Immobilon 595 nm [44]. membranes (EMD Millipore) and probed with the fol- lowing antibodies: rabbit anti-phospho-ERK1/2 (1:1,000), In vivo myoblast migration assay mouse anti-FLAG (1:5,000) (Stratagene, La Jolla, CA, USA), Myoblasts were labeled with the vital dialkylcarbocyanine rabbit anti-ERK1/2 (1:1,000), rabbit anti-phospho-AKT dye DiI (red fluorescence) according to the supplier’s (1:1,000) (Calbiochem, San Diego, CA, USA), mouse anti- protocol (Sigma-Aldrich). Aliquots containing 500 × 10 α-tubulin (1:5,000) (Sigma-Aldrich), mouse anti-myosin myoblasts were resuspended in 30 μl of physiological (1:5,000) (Sigma-Aldrich) and mouse anti-glyceraldehyde serum and kept on ice. Immediately before grafting, 1 μl 3-phosphate dehydrogenase (1:2,000) (Chemicon Inter- of physiological serum containing or not containing 10 ng national, Temecula, CA, USA). All immunoreactions were of carrier-free HGF was added to myoblast. Three-month- visualized by enhanced chemiluminescence (Pierce Bio- old C57BL/10 mice were used as hosts, and cells were technology, Rockford, IL, USA) using a ChemiDoc-It 410 slowly injected longitudinally in both tibialis anterior (TA) high-resolution imaging system (UVP, Upland, CA, USA). muscles of mice under isoflurane gas anesthesia. Cells Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 4 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 treated or not treated with HGF were injected into the Statistical analyses contralateral TA muscles. After 7 days, the TA muscles The number of replicates is indicated in the figure leg- were snap-frozen in isopentane before being entirely cut ends for each experiment. Data are presented as the in transversal 7-μm cross-sections. Muscle cross-sections mean ± standard deviation. Statistical significance was as- were visualized under a Nikon Diaphot inverted micro- sessed using two-way analysis of variance and a Bonferroni scope (Nikon Instruments, Melville, NY, USA) equipped multiple-comparisons posttest. Differences were consid- for epifluorescence. Concentric rings disposed 200 μmfrom ered statistically significant at P < 0.05. each other were superimposed on the selected muscle cross-section photographs. The total number of migrating Results myoblasts was determined by counting the labeled cells Myoblasts require glypican-1 expression for proper that had migrated more than 200 μm from the injection site hepatocyte growth factor signaling (which was determined by the border of the more intense To evaluate the role of glypican-1 in the myoblast response fluorescence) [45]. The percentage of cells that reached to HGF, C2C12 myoblasts and the derived clone C6, which more than 600 μm over the total migrating myoblast was expresses low levels of HSPG [38], were treated with in- quantified. These percentages were used to compare the creasing concentrations of HGF. Phosphorylation of the migration of myoblasts between the different conditions. Met receptor (phospho-Met) and the second messengers All mice had free access to water and a chow diet until they AKT (phospho-AKT) and ERK1/2 (phospho-ERK1/2) in were studied. All protocols were conducted in strict accord- response to HGF were analyzed by Western immunoblot- ance with the formal approval of the Animal Ethics Com- ting. Figure 1A shows that the phosphorylation levels of mittee of the Pontificia Universidad Católica de Chile. Met, AKT and ERK1/2 increased in a HGF concentration– dependent manner. However, glypican-1-deficient myo- blasts required higher concentrations of HGF to induce Hepatocyte growth factor affinity labeling and binding assay phosphorylation of the same proteins. The diminished re- Carrier-free HFG was radiolabeled with Na I using the sponse to HGF in the absence of glypican-1 was specific, chloramine T method as previously described for FGF-2 because glypican-1 reexpression resulted in the rescue of [38]. The biological activity of the radiolabeled HGF was HGF sensitivity. The same figure comparing wild-type determined by its ability to induce phosphorylation of (WT), glypican-1-deficient and glypican-1-overexpressing ERK1/2 compared to unlabeled HGF as described above. myoblasts also shows that the total levels of Met, AKT and The binding of [ I]HGF to cell surfaces was performed ERK1/2 were not affected by the different conditions of as described previously with some modifications [46]. glypican-1 expression. Quantification values from three in- Briefly, subconfluent myoblasts were incubated for 2 hours dependent experiments are shown in Figure 1B. Figure 1C at 4°C in DMEM containing 0.2% BSA, 25 mM 2-[4-(2- shows that expression levels of Met present at the cell hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), surface were unaltered by the presence or absence of pH 7.4, and 10 ng/ml [ I]HGF. To determine nonspecific glypican-1, as determined by labeling of the extracellu- binding, parallel cultures were incubated under the same lar proteins with biotin followed by precipitation with conditions with the addition of a 200-fold excess of un- streptavidin-agarose and detection with a specific anti- labeled HGF. After several washes in binding buffer and Met receptor antibody using Western blots. once with phosphate-buffered saline to remove unbound Becausemuscleprecursor cellsmigrate in response to ligand, the cells were sequentially washed twice with 2 M HGF during skeletal muscle development and regeneration, NaCl in 20 mM HEPES, pH 7.4, for 5 minutes (low affinity we decided to evaluate the role of glypican-1 in HGF- binding) and twice with 2 M NaCl in 20 mM NaAc, dependent migration. Figures 2A and 2B show that HGF pH 4.0, for 5 minutes (high-affinity binding) [47-49]. The induces the migration of WT myoblasts tenfold. In con- cells were extracted, and the protein content was deter- trast, less than twofold induction was found in glypican-1- mined as indicated below. The amount of radioactivity deficient myoblasts. In the absence of HGF, WT and present in the low- and high-affinity washes and cell ex- glypican-1-deficient myoblast migration was essentially the tracts was determined using a γ scintillation counter. The same. Together, these results suggest that glypican-1 is re- counts per minute (cpm) values were corrected for the quired for a proper myoblast response to HGF, as deter- protein content in the cell extracts. mined by activation of HGF-dependent signaling and myoblast migration. Protein determination Protein content in cell extracts was determined with a Met is localized and activated in lipid rafts by a HGF- and bicinchoninic acid protein assay kit (Pierce Biotechnology) glypican-1-dependent mechanism with BSA used as the standard according to the supplier’s We have shown that glypican-1 was the only HSPG as- protocol. sociated with lipid raft microdomains in myoblasts [38]. Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 5 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 1 (See legend on next page.) Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 6 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 (See figure on previous page.) Figure 1 Myoblasts require glypican-1 expression for proper hepatocyte growth factor signaling. (A) Wild-type (WT) C2C12 myoblasts and C6 myoblasts (glypican-1-deficient clone) transiently transfected with rat glypican-1 (C6-Gly), were serum-starved for 6 hours and then treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. The cell extracts were analyzed by immunoblotting for total HGF receptor (Met) levels, phospho-Met (Tyr 1235/1349), phospho- and total AKT levels, phospho- and total levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2), glypican-1 core protein (after heparitinase treatment), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin. Total Met, AKT and ERK1/2 were used as loading control of its respective phosphorylated forms. GADPH and tubulin were used as loading control of glypican-1 expression levels. The Western blot images are representative of three independent experiments. (B) Quantitation of phospho-Met, phospho-AKT and phospho-ERK1/2 from three independent experiments is shown. Values are expressed as mean ± standard deviation. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. *P < 0.05, **P < 0.01. (C) Cell surface proteins of WT and C6 myoblasts labeled with EZ-Link Sulfo-NHS-Biotin (Pierce Biotechnology) as described in Methods. Aliquots of the cell extracts containing equal amounts of protein were precipitated with streptavidin-sepharose beads. The bound material was analyzed by Western blot immunoblotting against total Met. Aliquots of each assay obtained prior to the precipitation were analyzed by Western blot immunoassay for total Met, with tubulin used as the input control. Molecular weight standards are shown at left. The results presented in Figures 1 and 2 suggest that to disrupt lipid raft structure. WT myoblasts pretreated glypican-1 acts as a positive regulator of HGF signaling. with MβDC at two different concentrations (1 nM and 10 Therefore, we studied the association of Met with lipid raft nM) were stimulated with increasing concentrations of membrane domains and the possible role of glypican-1 HGF for 5 minutes. Figure 4A shows that total Met levels and HFG in this localization. To accomplish this objective, did not change significantly after treatment, but the HGF- WT and glypican-1-deficient myoblasts were either un- dependent activation of AKT and ERK was diminished in treated or treated with 10 ng/ml HGF, then fractionated in myoblasts with disrupted lipid raft domains. In addition, sucrose density gradients. Figure 3 shows that in untreated Figure 4B shows that both Met and caveolin 1 were relo- WT myoblasts (control), Met fractionated in lipid rafts calized from lipid rafts to non-lipid-raft fractions after (fractions 5, 6 and 7) and non-lipid-raft fractions (fractions MβCD treatment. 10, 11 and 12) to almost the same extent. In contrast, in The results of the present study indicate that Met, glypican-1-deficient myoblasts, almost all Met fractionated phospho-Met and glypican-1 colocalized in lipid raft do- in the non-lipid-raft fractions. In both WT and glypican-1- mains of the plasma membrane. Moreover, glypican-1 deficient myoblasts, the basal phosphorylation level of Met expression and lipid raft integrity were required to sus- (as shown in Figure 1) was exclusively present in non- tain the HGF-dependent signaling. Next, we evaluated lipid-raft fractions. The distributions of caveolin 1 and so- whether glypican-1 per se or its presence in lipid raft do- + + dium/potassium ATPase (Na /K -ATPase) were used as mains was required to sustain the HGF signaling mediated lipid raft and non-lipid-raft markers, respectively. These by the Met receptor. A chimeric form of HSPG containing results suggest that glypican-1 is required to distribute part the extracellular domain of rat glypican-1 and the trans- of the total HGF receptor to lipid raft domains. After the membrane and cytoplasmic domains of mouse syndecan- treatment with HGF, the proportion of total Met in lipid 1 (F-GlySyn) was expressed in WT cells. This chimeric rafts vs. non–lipid rafts was augmented in WT myoblasts, form localized in the non-lipid-raft region of the plasma but not in glypican-1-deficient myoblasts. Importantly, in membrane as we previously reported [38]. Figure 5 shows WT myoblasts, most of the phospho-Met was associated that mock-transfected WT myoblasts induced the activa- with lipid raft fractions. In contrast, in the glypican-1- tion of AKT and ERK1/2 in response to HGF. In myo- deficient myoblasts, most of the phospho-Met was as- blasts expressing the chimeric F-GlySyn, however, both sociated with non-lipid-raft fractions. In both WT and phospho-AKT and phospho-ERK1/2 levels decreased com- glypican-1-deficient myoblasts, phospho-ERK1/2 and pared to WT cells. These levels are comparable to levels phospho-AKT were found in the non-lipid-raft fractions. found in the glypican-1-deficient myoblasts. The figure Next, we evaluated the presence of CD44 in lipid raft also shows that diminished sensitivity to HGF, which we domains and its dependence on glypican-1. Our results had previously observed in the glypican-1-deficient cells, show that the association of CD44 with the lipid raft do- was restored after reexpressing glypican-1 by transient main is dependent on glypican expression and that this transfection with rat glypican-1. Together, these results in- association is stabilized after pretreatment with HGF dicate that glypican-1 must be associated with lipid rafts to sustain HGF-dependent signaling. (Figure 3). The results described above suggest that glypican-1 is required for the translocation and stabilization of Met to Glypican-1 physically interacts with HGF and Met in lipid lipid rafts, where it is activated. To test this possibility, rafts to form an active signaling complex cells were treated with MβCD, an antifungal drug that se- The results described above suggest that glypican-1 may lectively extracts cholesterol from the plasma membrane interact with Met and HGF in lipid rafts to form the Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 7 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 2 Hepatocyte growth factor-dependent myoblast migration requires glypican-1 expression. (A) Wild-type (WT) C2C12 and C6 myoblasts were seeded onto the upper part of transwell chambers at the same density in serum-free media. The lower chamber contained serum-free media with or without 20 ng/ml hepatocyte growth factor (HGF). After 8 hours, the cells in the upper part of the filter were scraped. The cells that had efficiently migrated through the filter were fixed with paraformaldehyde, stained with crystal violet and photographed or as shown in (B) stained with crystal violet and solubilized in phosphate-buffered saline containing 1% Triton X-100. The absorbance of the detergent soluble fraction at 595 nm was determined. Values are expressed as mean ± standard deviation of three independent experiments. ***P < 0.001 relative to WT control. The migration of WT under control conditions corresponds to a value of 1.0. ternary complex Met-HGF-glypican-1. To test this possibil- the cells were treated with HGF, the levels of coimmuno- ity, WT myoblasts were transfected with an empty vector precipitated Met increased with both forms of glypican-1, as the control or with rat glypican-1 (F-Gly) or chimeric F- though in a more pronounced way with F-Gly. Interest- GlySyn, both of which contained a FLAG epitope. Forty- ingly, when the activated form of precipitated Met was eight hours later, the cells were incubated with or without evaluated, F-Gly interacted substantially more than the 20 ng/ml [ I]HGF for 5 minutes. The cell extracts in the non-lipid-raft form of glypican-1 (F-GlySyn) with phospho- presence of phosphatase inhibitors were immunoprecipi- Met. We also found that [ I]HGF coimmunoprecipitated tated with anti-FLAG antibodies, and the precipitate was almost four times more with F-Gly than with F-GlySyn. As evaluated for total and phospho-Met. Figure 6A shows that, an immunoprecipitation control, F-Gly and F-GlySyn were in the absence of HGF, Met coimmunoprecipitated with detected with specific anti-glypican-1 antibodies. These re- both F-Gly and F-GlySyn almost to the same extent. When sults suggested that glypican-1 physically interacted with Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 8 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 3 Met is localized and activated by hepatocyte growth factor in lipid rafts by a glypican-1-dependent mechanism. C2C12 and C6 myoblasts were serum-starved for 6 hours and then treated without (control) or with 10 ng/ml hepatocyte growth factor (HGF) for 5 minutes. The cells were lysed with 1% Triton X-100 and fractionated by sucrose density gradients (5% to 45%). Twelve fractions were collected, but only the last ten fractions were analyzed (the lipid raft–enriched fraction started at fraction 4) by immunoblotting for total HGF receptor (Met), phospho-Met (Tyr 1235/1349), phosphorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2), phospho-AKT and CD44. As fractionation + + controls, the presence of the lipid raft membrane protein marker caveolin 1 (Cav 1) and the non-lipid-raft domain marker Na /K -ATPase are shown. WT, Wild type. Met and HGF preferentially located in lipid rafts, where the myoblast migration, we subjected the C57BL/10 mice to receptor was stabilized and activated in response to HGF. intramuscular coinjection of C2C12 or C6 myoblasts to- To determine if binding of HGF on the myoblast cell sur- gether with HGF in the TA muscles. Seven days after the face was modulated by glypican-1, we performed a ligand transplantation, the muscles were extracted, frozen in li- binding assay. WT and glypican-1-deficient myoblasts were quid nitrogen and cryosectioned. Prior to grafting, the incubated with [ I]HGF at 4°C to avoid endocytosis of the myoblasts were stained with the vital dialkylcarbocyanine ligand. The radioactivity associated with low- and high- dye, DiI (red fluorescence), to trace their localization in affinity binding sites, as well as the remaining radioactivity the muscle cryosections. Figure 7 shows that HGF in- in the cell extracts, was determined. Figure 6B shows that duced an increase in the number of WT myoblasts that the binding of [ I]HGF to both low- and high-affinity migrated longer distances (more than 600 μm). However, binding sites was diminished by 50% in the absence of this effect was prevented in glypican-1-deficient myo- glypican-1, suggesting that this lipid raft–associated HSPG blasts. These results suggest that glypican-1 expression is was required to concentrate HGF on the cell surface and required for efficient in vivo myoblast migration in re- for binding to Met. These results indicate that glypican-1 sponse to HGF. facilitated the binding of HGF to the Met receptor, enhan- cing its phosphorylation at lipid raft domains. Discussion One of the main functions of membrane-associated HSPGs, Migration of transplanted myoblasts in skeletal muscles is particularly for glypicans, is to regulate signaling of several enhanced by HGF and requires glypican-1 cytokines, morphogens and growth factors [38,50-53]. It The data described above demonstrates the requirement has been reported that loss of HSPG expression prevents of glypican-1 for HGF-dependent signaling and migration. the cell mitogenic response induced by HGF [54-56], but To test the in vivo role of glypican-1 on HGF-induced the specific roles and mechanisms of the different HSPGs Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 9 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 4 Disruption of lipid rafts diminishes hepatocyte growth factor–dependent signaling. (A) C2C12 myoblasts were serum-starved for 6 hours, and during the last hour the cells were treated with or without methyl-β-cyclodextrin (MβCD) at the indicated concentrations. After two washes with serum-free media, the cells were treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. The cell extracts were analyzed by immunoblotting for total HGF receptor (Met), phospho- and total AKT, phosphorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2) and total ERK1/2, and tubulin was used as a loading control. (B) Quantification from two independent experiments is shown. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. *P < 0.05, **P <0.01, ***P < 0.001. (C) C2C12 myoblasts treated with or without 10 mM MβCD for 1 hour as described in (A) were lysed and fractionated in sucrose density gradients as described in Figure 3. The distributions of total Met and caveolin 1 (Cav-1) were determined by immunoblot analysis. In (A) and (C),the molecular weight standards are shown at left. as regulators of HGF-dependent responses have not been 1 appears as an essential cell-surface, low-affinity binding studied in depth. site for HGF, likely acting as a presenter or facilitator of In the present report, we show that, in myoblasts, HGF to its high-affinity Met binding site, where it is cofrac- glypican-1 located in lipid raft membrane domains was tionated with the known HGF coreceptor CD44 [34]. required for maximum HGF-dependent signaling and cell Glypican-1, Met and HGF formed an active signaling tern- migration in vitro and in vivo. We also show that glypican- ary complex in lipid raft membrane domains. Whether Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 10 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 5 Glypican-1 is required to sustain the hepatocyte growth factor-dependent signaling in lipid rafts. Wild-type (WT) myoblasts were transiently transfected with an empty vector as the control or with a non-lipid-raft form of glypican-1 containing the extracellular domain of rat glypican-1 and the transmembrane and cytoplasmic domains of mouse syndecan-1 (F-GlySyn) [36]. C6 myoblasts were transiently transfected with an empty vector as the control or with rat glypican-1 (C6-Gly). Forty-eight hours after transfection, the cells were serum-starved for 6 hours and then treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. (A) The cell extracts were analyzed by immunoblotting for total HGF receptor (Met), phospho- and total Akt and phosphorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2) and total ERK1/2. Glypican-1 core protein levels after heparitinase digestion of endogenous and both transfected forms of glypican-1 were detected by using an anti-glypican-1 antibody. Tubulin levels were used as loading controls. (B) Quantification from two independent experiments is shown. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. *P < 0.05, **P <0.01. phospho-Met is relocated from non-lipid-raft to lipid raft protein–coupled receptors, including β-adrenergic, neuro- domains in response to HGF or whether Met is directly ac- kinin 1 receptor and muscarinic cholinergic receptors tivated in lipid rafts, where it is stabilized, are still not [61-64]. Lipid rafts can also act as a platform where recep- known. Chimeric non-lipid-raft glypican-1 (F-GlySyn) also tor signaling is turned off, such as in the case of serine- coimmunoprecipitated with Met, but not with the active threonine kinase transforming growth factor β [65] and form of the receptor or with HGF, indicating that loca- tyrosine kinase epidermal growth factor receptors, which lization of glypican-1 in lipid raft domains was unnecessary are activated in lipid rafts, but rapidly relocalized to non– for the interaction between Met and the extracellular part lipid rafts to de-activate downstream signaling [66]. We of glypican-1, but was required for binding of HGF and previously reported that glypican-1 in lipid rafts acted as a subsequent receptor activation. negative regulator of FGF-2 signaling, sequestering the The participation of lipid rafts as signaling platforms growth factor in these domains away from their transdu- to facilitate interaction of the required elements to acti- cing receptors [38]. Our present results show that, upon vate a signaling pathway has been reported for different ligand binding, Met is recruited to lipid rafts to activate receptor tyrosine kinases, such as the platelet-derived MAPK, ERK1/2 and PI3K/AKT pathways. This process growth factor, TrkA/nerve growth factor and insulin re- required the presence of structured lipid raft membrane ceptors. After ligand activation, MAPK and phosphoino- domains as well as glypican-1 in these domains to sustain sitide 3-kinase (PI3K) signaling mediators are recruited the HGF-dependent signaling. However, these results did to lipid rafts, where they are activated [57-60]. The not eliminate the possibility of other Met-dependent func- same mechanism of action has also been reported for G tions in non–lipid rafts. Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 11 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 6 Glypican-1 in lipid rafts coimmunoprecipitates with the activated form of Met and regulates hepatocyte growth factor binding to low- and high-affinity cell surface binding sites. (A) C2C12 myoblasts were transfected with rat glypican-1 (F-Gly) or the non-lipid-raft chimeric glypican-1 (F-GlySyn), as described in the Figure 5 legend. F-Gly and F-GlySyn contained a FLAG epitope at the amino terminus. Forty-eight hours after transfection, the cells were serum-starved for 4 hours and then treated with or without 20 ng/ml [ I]HGF for 5 minutes. The cell extracts were incubated with anti-FLAG M2 Affinity Gel for 3 hours at 4°C, and, after several washes, the beads were incubated with heparitinase and chondroitinase ABC for 8 hours. The immunoprecipitated (IPP) bound material was eluted with protein loading buffer and analyzed by Western immunoblotting for total hepatocyte growth factor (HGF) receptor (Met), phospho-Met and glypican-1. The membranes were exposed to a phosphorimager to detect [ I]HGF. (B) C2C12 and C6 myoblasts were serum-starved for 4 hours and then treated with or 125 125 without 10 ng/ml [ I]HGF for 2 hours at 4°C. After several washes in ice-cold binding buffer, [ I]HGF was eluted with high salt and acid to determine low- and high-affinity binding sites, respectively. Counts per minute (cpm) were determined by γ counting and corrected for protein content in cell extracts. Statistical significance was assessed by two-way analysis of variance and a Bonferroni multiple comparisons posttest. **P <0.01. HGF is involved in many different processes in which Met expression [6-8]. In the present study, we show that both cell growth and cell migration are required, such as glypican-1 was required for the migration of myoblasts in embryonic development, tissue repair and organ re- in response to HGF, both in vitro and in vivo. In vitro generation [67]. In particular, the roles of HGF and Met glypican-1-deficient myoblasts were almost unresponsive for muscle development, differentiation and regeneration to HGF as a chemoattractant in the Boyden chamber as- have been reported [7]. During limb muscle develop- says, in contrast to WT myoblasts, which migrated exten- ment, migratory muscle precursor cells delaminate from sively through the membrane toward the HGF-containing the dermomyotome, an epithelial structure that develops media. The migration capacity toward other chemoattrac- from somites, reaching their specific destination in the tants did not appear hampered, because no significant dif- limb buds [68-70] in a process dependent on HGF and ferences were observed when both types of cells were Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 12 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 7 Hepatocyte growth factor-dependent migration of myoblast in vivo requires glypican-1 expression. (A) Wild-type (WT) or C6 myoblasts (500 × 10 ), prelabeled with the vital dialkylcarbocyanine dye, DiI (red fluorescence), and suspended in 30 μl of physiological serum with or without 10 ng of carrier-free hepatocyte growth factor (HGF) were transplanted into the left and right tibialis anterior (TA) muscles, respectively, of 3-month-old C57BL/10 mice under anesthesia. One week later, both TA muscles were processed for cryosectioning. Concentric circles with annuli 200 μm from each other were superimposed on selected muscle cross-section images. The cells were counted under an inverted microscope equipped for epifluorescence. (B) The number of the cells that migrated more than 200 μm were considered the total migrating cells, and the percentages of cells that migrated more than 600 μm were calculated. The migration of untreated WT myoblasts was corrected to 100%. Values are expressed as mean ± SD of two independent experiments. ***P <0.001. challenged to migrate toward 10% FBS (data not shown). expanded in vitro couldbeimprovedbycoinjection with We determined the role of glypican-1 in myoblast mi- HGF. In addition, this effect required the expression of gration in vivo in response to HGF by intramuscular glypican-1 in the myoblast plasma membrane. coinjection of WT or glypican-1-deficient myoblasts in This result is very promising, because one of the main the presence or absence of the growth factor. In vivo problems associated with stem cell therapies for the treat- myoblast migration was improved by coinjection with ment of patients with muscular dystrophies is the poor HGF, particularly in WT cells, compared to the slight mi- migration of the transplanted cells. As a result, therapy gratory effect observed with glypican-1-deficient myoblasts. with intramuscular injection of myoblasts or SCs in sev- These results show that in vivo migration of myoblast eral clinical trials has been mostly unsuccessful [71-73]. Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 13 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Based upon our results, the use of fluorescence-activated features of stem cells. It is explained by asymmetric cell cell sorting with higher expression of glypican-1 and/or division involving some daughter cells, which continue the coinjection with HGF to improve efficiency needs to be differentiation pathway, whereas other cells exit the cell carefully evaluated. cycle and return to quiescence [90,91]. HGF concentra- As previously mentioned, HSPGs are essential compo- tions above 20 ng/ml induced the quiescence of primary nents required for the myogenic inhibitory signaling of myogenic cells. This effect was reversible because treat- FGF-2 [28,30-33,74-76] and HGF [3,77,78]. During dif- ment with low concentrations of HGF could rescue the ferentiation, expression of all syndecans was downregu- proliferation of myogenic cells after high HGF-induced lated, which is consistent with a reduction in sensitivity quiescence [77]. to the inhibitory effect of FGF-2 [32,38,79]. In contrast, It would be interesting to determine whether glypican- the expression of glypican-1 remained constant, being 1 has a potential role in the control of SC sensitivity to the main cell surface HSPG present during myogenesis extracellular HGF and to define which SCs will continue [35,39]. In addition, during muscle regeneration, expression to form new muscle and which will exit the cell cycle in of glypican-1 increased and was temporarily and histologi- asymmetric cell division to maintain the pool of muscle cally related to the newly regenerating myofiber expression stem cells. Besides its association with lipid rafts in the of embryonic myosin [80]. However, the exact role of cell membrane, glypican-1 is also endogenously proc- glypican-1 during this process has not been addressed to essed to a soluble form that is incorporated into the date. Glypican-1-knockout mice were almost indistinguish- ECM [35,38,80], where it can act as a reservoir for HGF able from WT mice in size, fertility, internal anatomy and and other heparin-binding growth factors that can be re- lifespan, with the exception of the brain, which was notice- leased upon an injury to activate SCs. More accurate future ably smaller [81]. This suggests that glypican-1 is required studies designed to determine the control mechanisms of in mammals for brain development, but not for other glypican-1 and Met expression between daughter cells dur- tissues, such as skeletal muscle. To further elucidate the ing asymmetric cell division, as well as the role of glypican- results of the present study, it would be informative to 1 during the muscle regeneration process, are therefore evaluate the skeletal muscle regeneration process in necessary. glypican-1-null mice. Glypican-1 is required for terminal myogenesis, acting Conclusion as a repressor of FGF-2 [38]. This can be explained by Glypican-1 in lipid raft membrane domains is required for the sequestration of FGF-2 by glypican-1 in lipid rafts, maximum HGF-dependent signaling and myoblast migra- away from FGF-2 receptors and syndecans that are lo- tion in vitro and in vivo. cated in non–raft domains. As we have shown, however, glypican-1 positively regulates HGF-mediated signaling Abbreviations AKT: Effector of the phosphoinositide 3-kinase/AKT pathway; by recruiting or stabilizing Met in lipid raft domains ERK: Extracellular signal-regulated kinase; FGF-2: Fibroblast growth factor 2; where it was activated, with consequential triggering of FGFR: Transducing fibroblast growth factor receptor; F-Gly: Rat glypican-1 downstream targets. Reduction of Met expression during containing a FLAG epitope in its amino terminus; F-GlySyn: Chimeric heparan sulfate proteoglycan (extracellular domain of rat glypican-1 and the trans- the myogenic differentiation process (data not shown) [82] membrane and cytoplasmic domains of mouse syndecan-1 containing a therefore seemed to circumvent the myogenic inhibitory FLAG epitope); HGF: Hepatocyte growth factor; HS: Heparan sulfate; effect of HGF in spite of the constitutive expression of HSPG: Heparan sulfate proteoglycan; Met: Transducing hepatocyte growth + + factor receptor; MβCD: Methyl-β-cyclodextrin; Na /K -ATPase: Sodium glypican-1 [35,39]. All of these changes switched the bal- potassium ATP pump; Phospho-AKT: Phosphorylated form of AKT; ance from a proliferative, migratory and antimyogenic state Phospho-ERK: Phosphorylated form of extracellular signal-regulated kinase; in response to FGF-2 and HGF to a promyogenic response Phospho-Met: Phosphorylated form of hepatocyte growth factor receptor; TA: Tibialis anterior. whereby both muscle inhibitory signals decreased, thus allowing differentiation. Competing interests Immediately after injury, low concentrations of HGF The authors declare that they have no competing interests. (2 to 3 ng/ml) are released from ECM reservoirs [83-85] in conjunction with the local release of nitric oxide. These Authors’ contributions JG participated in the design of the study, carried out the cellular and are the first cues involved in the activation (that is, exit molecular experiments, performed the statistical analysis and drafted the from quiescence) of SCs, which then proliferate to form manuscript. DC carried out the myoblast migration experiments. EB new fibers or repair the destroyed ones [10,84,86]. To conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. maintain their regenerative potential, many proliferating SCs return to quiescence, repopulating the SC niche to Acknowledgments maintain a progenitor pool, which will be activated to re- This study was supported by grants CARE-PFB-12/2007, CONICYT-79090027, pair the muscle in response to a new injury [87-89]. 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Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells

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Copyright © 2014 by Gutiérrez et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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2044-5040
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10.1186/2044-5040-4-5
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24517345
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Abstract

Background: Via the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF. Methods: C2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed. Results: Glypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration. Conclusions: Glypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains. Keywords: Glypican-1, Heparan sulfate proteoglycans, Hepatocyte growth factor, HGF-mediated signaling, Raft membrane domains, Skeletal muscle Background muscle fibers [2]. HGF was originally identified as a scatter The process of skeletal muscle regeneration is initiated factor because of its ability to increase the motility of sev- immediately after injury by the release of growth factors eral normal and neoplastic cells [4,5]. The requirement of and cytokines from injured muscles, blood vessels, infil- HGF for migration of muscle precursor cells during mouse trating inflammatory cells and extracellular matrix (ECM) muscle development has been established by the genetic reservoirs. These factors include basic fibroblast growth ablation of HGF or the HGF receptor (Met). In both cases, factor 2 (FGF-2) and hepatocyte growth factor (HGF) the result was the absence of hindlimb muscles, which are [1-3]. The factors promote the activation, proliferation, mi- formed by muscle precursor cells that migrate from the gration and survival of satellite cells (SCs), which are the dermomyotome [6-8]. In vitro studies have shown that muscle stem cells responsible for the formation of new HGF not only induces the proliferation and migration of myogenic cells but that it also delays muscle differentiation by inhibiting the expression of MyoD and myogenin, two * Correspondence: ebrandan@bio.puc.cl master myogenic regulatory transcription factors [3,9,10]. Centro de Regulación Celular y Patología (CRCP), Centro de Regeneración y The expression of HGF and Met are downregulated during Envejecimiento (CARE), Departamento de Biología Celular y Molecular, MIFAB, Pontificia Universidad Católica de Chile, Santiago, Chile © 2014 Gutiérrez et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 2 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 myogenesis, which is consistent with attenuation of myo- was clearly decreased in the absence of glypican-1, suggest- genic inhibitory signaling of HGF [11-13]. Therefore, HGF ing that glypican-1 was a positive regulator of HGF signal- plays key role during myogenesis, regulating the prolifera- ing [38]. tion, migration and subsequent differentiation of muscle Because HGF and Met are found in lipid raft domains precursor cells. [17-20], we hypothesize that glypican-1 in these domains Upon HGF binding, Met is activated by dimerization is necessary to stabilize the HGF-Met signaling complex. with subsequent trans-phosphorylation of four tyrosine In the present study, we report that the presence of residues which act as docking motifs for signaling media- glypican-1 in lipid rafts was required for maximum HGF- tors, including mitogen-activated protein kinase (MAPK), dependent signaling, localizing and stabilizing HGF and extracellular signal-regulated kinases 1 and 2 (ERK1/2) Met in its phosphorylated or activated state (phospho- and phosphoinositide 3-kinase protein kinase B (AKT), Met). We also show that glypican-1, phospho-Met and among others [14-16]. HGF interact, indicating that they form part of a signaling It has been proposed that HGF and Met form a complex complex in lipid rafts. Finally, we show that glypican-1 is in lipid rafts, which are sphingolipid- and cholesterol-rich required for myoblast migration induced by HGF in vitro domains that form phase-separated lipid rafts in the mem- and in vivo, demonstrating the requirement of glypican-1 brane. In these domains, Met is stabilized by HGF to in- expression and HGF for processes such as muscle stem duce its activation [17-20]. cell therapy, where the migration of myoblasts must be Another important component of the HFG-Met sig- enhanced. naling is the ubiquitous transmembrane glycoprotein CD44, the major receptor for hyaluronic acid [21,22]. In Methods different cell types, the activation of the MET receptor Cell culture by HGF depends on the presence of some isoforms of The mouse skeletal muscle cell line C2C12 (American CD44 [21]. As proposed, HGF, Met and CD44 would form Type Culture Collection, Manassas, VA, USA) [40] and its a complex in lipid raft membrane domains, which cor- derived clone deficient in glypican-1 expression [38] were responds to sphingolipid- and cholesterol-rich domains grown as previously described [31,38]. Myoblasts were forming phase-separated lipid rafts in the membrane, treated with HGF (R&D Systems, Minneapolis, MN, USA) where Met would be stabilized by HGF inducing its acti- as indicated in each experiment. Methyl-β-cyclodextrin vation [17,18]. (MβCD) (Sigma-Aldrich, St Louis, MO, USA) treatment HGF also binds to heparin, heparan sulfate (HS) and at 1 or 10 mM concentrations were performed as previ- dermatan sulfate [23-27]. Heparan sulfate proteoglycans ously described [38]. For the phosphorylation experiments (HSPGs), key components of the cell surface and the of Met, ERK1/2 and AKT, the cells were serum-starved ECM, regulate many processes related to cell growth for 6 hours and then treated for the indicated times. and differentiation. Cell-surface HSPGs bind soluble li- gands, increasing their local concentration and modulating Transient transfection and generation of stable clones ligand–receptor interactions [28]. For example, HSPG is The pcDNA3.0 empty vector (Invitrogen, Carlsbad, CA, required for FGF-2-dependent signaling through its recep- USA) and pcDNA3.0 vectors containing rat glypican-1 tors (FGFRs) [29-32], forming the ternary complex HSPG- and chimeric HSPG comprising the extracellular domain FGF-2-FGFR [33]. However, the exact role of HSPG in of rat glypican-1 were fused to the transmembrane and HGF signaling is poorly understood. In vitro assays have cytoplasmic domains of mouse syndecan-1 containing a shown that heparin increases the mitogenic effect of HGF FLAG epitope in their amino-terminal F-Gly and F- and facilitates its oligomerization, inducing Met dimeri- GlySyn, respectively [38]. Transfection were carried out zation and activation [34]. Previously, we showed that using Lipofectamine and PLUS reagents (Invitrogen) ac- myoblast migration induced by HGF was strongly inhib- cording to the supplier’s protocol. ited if the cells were depleted of HS chains, indicating that at least the myoblast cell response to HGF depended on Isolation of lipid rafts HS [23]. Lipid rafts were prepared as described previously, with We have also previously shown that myoblasts express some modifications [38]. All of the buffers and instru- different membrane-bound HSPGs, the four transmem- ments used in the procedure described below were used brane syndecans and glypican-1, which corresponds to a at 4°C. Briefly, C2C12 myoblasts from a 150-mm dish were glycosylphosphatidylinositol-anchored HSPG [31,32,35-39]. lysed in 400 μl of lysis buffer (25 mM 2-(N-morpholino) Glypican-1 is the only HSPG located in lipid raft microdo- ethanesulfonic acid, pH 6.5, 150 mM NaCl, with a mixture mains, which sequester FGF-2 to avoid its interaction with of protease inhibitors and 1 mM phenylmethanesulfonyl FGFRs. Thus, glypican-1-deficient cells exhibit enhanced fluoride supplemented with 1% Triton X-100). Cells were sensitivity to FGF-2. In contrast, HGF-dependent signaling incubated for 20 minutes on ice, then homogenized with Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 3 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 ten strokes of a loose-fitting Dounce homogenizer. Ho- For coimmunoprecipitation experiments, wild-type and mogenates were mixed with 400 μl of 90% sucrose (45% glypican-1-deficient myoblasts (C6) were transiently trans- final concentration), loaded at the bottom of a Sorvall 4-ml fected as indicated in the figure legends. At 48 hours after centrifuge tube (Thermo Scientific, Asheville, NC, USA) transfection, cells were serum-starved for 4 hours, then ei- and overlaid with 1.6 ml of 35% sucrose and 1.6 ml of 5% ther treated or not treated with 20 ng/ml [ I]HGF in sucrose, both in the lysis buffer without Triton X-100. The Dulbecco’s modified Eagle’s medium (DMEM) 0.1% bo- samples were centrifuged at 45,000 rpm for 18 hours at vine serum albumin (BSA) for 5 minutes. The cell extracts 4°C in an AH-650 rotor. Twelve fractions (330 μleach) in RIPA buffer plus phosphatase inhibitors were incu- were collected from top to bottom and designated as bated with anti-FLAG M2 Affinity Gel (Sigma-Aldrich) fractions 1 to 12. Only the last ten fraction were ana- for 3 hours at 4°C. The beads were sequentially washed lyzed, because the low-density lipid raft–enriched frac- in RIPA buffer, then in heparitinase reaction buffer tions started at fraction 5 in several previous assays that (20 mM Tris, 150 mM NaCl, 1 mM MgCl ,1mM Ca we performed. Cl , pH 7.4). The beads were then treated with hepariti- nase and chondroitinase ABC for 3 hours at 37°C. The SDS-PAGE, Western blot and coimmunoprecipitation assays bound material was eluted with protein loading buffer Aliquots from the last ten fractions of the different sucrose and assessed by Western blot analysis for total Met, density fractionations were separated on 8% SDS-PAGE phospho-Met and glypican-1 or exposed to a phosphori- gels (Mini-PROTEAN II; Bio-Rad Laboratories, Hercules, mager to detect [ I]HGF. CA, USA) and electrophoretically transferred to Immobilon membranes (EMD Millipore, Bedford, MA, USA). Western Biotin labeling and precipitation of biotin-labeled proteins blots were probed using the following primary antibodies: Biotin labeling was conducted as previously described rabbit anti-mouse Met (1:200) (Santa Cruz Biotechnology, [43]. Equal amounts of protein (100 μg) obtained from Santa Cruz, CA, USA), rabbit anti-phospho-Met at Tyr previously biotinylated cell extracts were precipitated for 1234 and Tyr 1235 (1:1,000) (Cell Signaling Technology, 2 hours at 4°C using streptavidin agarose resin (Thermo Danvers, MA, USA), rabbit anti-caveolin-1 (1:500) (Santa Fisher Scientific, Rockford, IL, USA). The bound material Cruz Biotechnology), rabbit anti-glypican-1 M-95 (1:500) was eluted with protein loading buffer and assessed by + + (Santa Cruz Biotechnology), mouse anti-Na /K -ATPase Western blot analysis for total Met as described above. (1:1,000) (Upstate Biotechnology, Lake Placid, NY, USA) and rat anti-CD44 (1:500) (BD Pharmingen, San Jose, CA, Transwell migration assays USA). Migration assays were conducted using 24-well, 8-μm-pore To identify glypican-1, samples containing equivalent transwell systems (EMD Millipore). C2C12 and C6 myo- amounts of protein were treated with heparitinase and blasts were seeded onto the upper part of the chamber at a chondroitinase ABC (United States Biological, Swampscott, density of 100,000 cells per well in 300 μlofserum-free MA, USA) as previously described [39,41] prior to SDS- media. The lower chamber was loaded with 500 μlof PAGE and Western blot analysis using anti-glypican-1 M- serum-free media with or without 20 ng/ml HGF or 10% 95 antibody. fetal bovine serum (FBS) (data not shown). The cells were For analysis of phosphorylated proteins, cell extracts allowed to migrate for 8 hours. Migration was assessed by were prepared in radioimmunoprecipitation assay (RIPA) removing the cells on the upper side of the transwell with buffer in the presence of phosphatase inhibitors as previ- a cotton swab, then staining the remaining cells with crys- ously described [38,42]. Aliquots with equivalent amounts tal violet, and solubilizing the cells in 1% Triton X-100 to of protein were subjected to SDS-PAGE in 8% polyacryl- measure the absorbance of the Triton X-100 solution at amide gels, electrophoretically transferred to Immobilon 595 nm [44]. membranes (EMD Millipore) and probed with the fol- lowing antibodies: rabbit anti-phospho-ERK1/2 (1:1,000), In vivo myoblast migration assay mouse anti-FLAG (1:5,000) (Stratagene, La Jolla, CA, USA), Myoblasts were labeled with the vital dialkylcarbocyanine rabbit anti-ERK1/2 (1:1,000), rabbit anti-phospho-AKT dye DiI (red fluorescence) according to the supplier’s (1:1,000) (Calbiochem, San Diego, CA, USA), mouse anti- protocol (Sigma-Aldrich). Aliquots containing 500 × 10 α-tubulin (1:5,000) (Sigma-Aldrich), mouse anti-myosin myoblasts were resuspended in 30 μl of physiological (1:5,000) (Sigma-Aldrich) and mouse anti-glyceraldehyde serum and kept on ice. Immediately before grafting, 1 μl 3-phosphate dehydrogenase (1:2,000) (Chemicon Inter- of physiological serum containing or not containing 10 ng national, Temecula, CA, USA). All immunoreactions were of carrier-free HGF was added to myoblast. Three-month- visualized by enhanced chemiluminescence (Pierce Bio- old C57BL/10 mice were used as hosts, and cells were technology, Rockford, IL, USA) using a ChemiDoc-It 410 slowly injected longitudinally in both tibialis anterior (TA) high-resolution imaging system (UVP, Upland, CA, USA). muscles of mice under isoflurane gas anesthesia. Cells Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 4 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 treated or not treated with HGF were injected into the Statistical analyses contralateral TA muscles. After 7 days, the TA muscles The number of replicates is indicated in the figure leg- were snap-frozen in isopentane before being entirely cut ends for each experiment. Data are presented as the in transversal 7-μm cross-sections. Muscle cross-sections mean ± standard deviation. Statistical significance was as- were visualized under a Nikon Diaphot inverted micro- sessed using two-way analysis of variance and a Bonferroni scope (Nikon Instruments, Melville, NY, USA) equipped multiple-comparisons posttest. Differences were consid- for epifluorescence. Concentric rings disposed 200 μmfrom ered statistically significant at P < 0.05. each other were superimposed on the selected muscle cross-section photographs. The total number of migrating Results myoblasts was determined by counting the labeled cells Myoblasts require glypican-1 expression for proper that had migrated more than 200 μm from the injection site hepatocyte growth factor signaling (which was determined by the border of the more intense To evaluate the role of glypican-1 in the myoblast response fluorescence) [45]. The percentage of cells that reached to HGF, C2C12 myoblasts and the derived clone C6, which more than 600 μm over the total migrating myoblast was expresses low levels of HSPG [38], were treated with in- quantified. These percentages were used to compare the creasing concentrations of HGF. Phosphorylation of the migration of myoblasts between the different conditions. Met receptor (phospho-Met) and the second messengers All mice had free access to water and a chow diet until they AKT (phospho-AKT) and ERK1/2 (phospho-ERK1/2) in were studied. All protocols were conducted in strict accord- response to HGF were analyzed by Western immunoblot- ance with the formal approval of the Animal Ethics Com- ting. Figure 1A shows that the phosphorylation levels of mittee of the Pontificia Universidad Católica de Chile. Met, AKT and ERK1/2 increased in a HGF concentration– dependent manner. However, glypican-1-deficient myo- blasts required higher concentrations of HGF to induce Hepatocyte growth factor affinity labeling and binding assay phosphorylation of the same proteins. The diminished re- Carrier-free HFG was radiolabeled with Na I using the sponse to HGF in the absence of glypican-1 was specific, chloramine T method as previously described for FGF-2 because glypican-1 reexpression resulted in the rescue of [38]. The biological activity of the radiolabeled HGF was HGF sensitivity. The same figure comparing wild-type determined by its ability to induce phosphorylation of (WT), glypican-1-deficient and glypican-1-overexpressing ERK1/2 compared to unlabeled HGF as described above. myoblasts also shows that the total levels of Met, AKT and The binding of [ I]HGF to cell surfaces was performed ERK1/2 were not affected by the different conditions of as described previously with some modifications [46]. glypican-1 expression. Quantification values from three in- Briefly, subconfluent myoblasts were incubated for 2 hours dependent experiments are shown in Figure 1B. Figure 1C at 4°C in DMEM containing 0.2% BSA, 25 mM 2-[4-(2- shows that expression levels of Met present at the cell hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), surface were unaltered by the presence or absence of pH 7.4, and 10 ng/ml [ I]HGF. To determine nonspecific glypican-1, as determined by labeling of the extracellu- binding, parallel cultures were incubated under the same lar proteins with biotin followed by precipitation with conditions with the addition of a 200-fold excess of un- streptavidin-agarose and detection with a specific anti- labeled HGF. After several washes in binding buffer and Met receptor antibody using Western blots. once with phosphate-buffered saline to remove unbound Becausemuscleprecursor cellsmigrate in response to ligand, the cells were sequentially washed twice with 2 M HGF during skeletal muscle development and regeneration, NaCl in 20 mM HEPES, pH 7.4, for 5 minutes (low affinity we decided to evaluate the role of glypican-1 in HGF- binding) and twice with 2 M NaCl in 20 mM NaAc, dependent migration. Figures 2A and 2B show that HGF pH 4.0, for 5 minutes (high-affinity binding) [47-49]. The induces the migration of WT myoblasts tenfold. In con- cells were extracted, and the protein content was deter- trast, less than twofold induction was found in glypican-1- mined as indicated below. The amount of radioactivity deficient myoblasts. In the absence of HGF, WT and present in the low- and high-affinity washes and cell ex- glypican-1-deficient myoblast migration was essentially the tracts was determined using a γ scintillation counter. The same. Together, these results suggest that glypican-1 is re- counts per minute (cpm) values were corrected for the quired for a proper myoblast response to HGF, as deter- protein content in the cell extracts. mined by activation of HGF-dependent signaling and myoblast migration. Protein determination Protein content in cell extracts was determined with a Met is localized and activated in lipid rafts by a HGF- and bicinchoninic acid protein assay kit (Pierce Biotechnology) glypican-1-dependent mechanism with BSA used as the standard according to the supplier’s We have shown that glypican-1 was the only HSPG as- protocol. sociated with lipid raft microdomains in myoblasts [38]. Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 5 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 1 (See legend on next page.) Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 6 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 (See figure on previous page.) Figure 1 Myoblasts require glypican-1 expression for proper hepatocyte growth factor signaling. (A) Wild-type (WT) C2C12 myoblasts and C6 myoblasts (glypican-1-deficient clone) transiently transfected with rat glypican-1 (C6-Gly), were serum-starved for 6 hours and then treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. The cell extracts were analyzed by immunoblotting for total HGF receptor (Met) levels, phospho-Met (Tyr 1235/1349), phospho- and total AKT levels, phospho- and total levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2), glypican-1 core protein (after heparitinase treatment), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin. Total Met, AKT and ERK1/2 were used as loading control of its respective phosphorylated forms. GADPH and tubulin were used as loading control of glypican-1 expression levels. The Western blot images are representative of three independent experiments. (B) Quantitation of phospho-Met, phospho-AKT and phospho-ERK1/2 from three independent experiments is shown. Values are expressed as mean ± standard deviation. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. *P < 0.05, **P < 0.01. (C) Cell surface proteins of WT and C6 myoblasts labeled with EZ-Link Sulfo-NHS-Biotin (Pierce Biotechnology) as described in Methods. Aliquots of the cell extracts containing equal amounts of protein were precipitated with streptavidin-sepharose beads. The bound material was analyzed by Western blot immunoblotting against total Met. Aliquots of each assay obtained prior to the precipitation were analyzed by Western blot immunoassay for total Met, with tubulin used as the input control. Molecular weight standards are shown at left. The results presented in Figures 1 and 2 suggest that to disrupt lipid raft structure. WT myoblasts pretreated glypican-1 acts as a positive regulator of HGF signaling. with MβDC at two different concentrations (1 nM and 10 Therefore, we studied the association of Met with lipid raft nM) were stimulated with increasing concentrations of membrane domains and the possible role of glypican-1 HGF for 5 minutes. Figure 4A shows that total Met levels and HFG in this localization. To accomplish this objective, did not change significantly after treatment, but the HGF- WT and glypican-1-deficient myoblasts were either un- dependent activation of AKT and ERK was diminished in treated or treated with 10 ng/ml HGF, then fractionated in myoblasts with disrupted lipid raft domains. In addition, sucrose density gradients. Figure 3 shows that in untreated Figure 4B shows that both Met and caveolin 1 were relo- WT myoblasts (control), Met fractionated in lipid rafts calized from lipid rafts to non-lipid-raft fractions after (fractions 5, 6 and 7) and non-lipid-raft fractions (fractions MβCD treatment. 10, 11 and 12) to almost the same extent. In contrast, in The results of the present study indicate that Met, glypican-1-deficient myoblasts, almost all Met fractionated phospho-Met and glypican-1 colocalized in lipid raft do- in the non-lipid-raft fractions. In both WT and glypican-1- mains of the plasma membrane. Moreover, glypican-1 deficient myoblasts, the basal phosphorylation level of Met expression and lipid raft integrity were required to sus- (as shown in Figure 1) was exclusively present in non- tain the HGF-dependent signaling. Next, we evaluated lipid-raft fractions. The distributions of caveolin 1 and so- whether glypican-1 per se or its presence in lipid raft do- + + dium/potassium ATPase (Na /K -ATPase) were used as mains was required to sustain the HGF signaling mediated lipid raft and non-lipid-raft markers, respectively. These by the Met receptor. A chimeric form of HSPG containing results suggest that glypican-1 is required to distribute part the extracellular domain of rat glypican-1 and the trans- of the total HGF receptor to lipid raft domains. After the membrane and cytoplasmic domains of mouse syndecan- treatment with HGF, the proportion of total Met in lipid 1 (F-GlySyn) was expressed in WT cells. This chimeric rafts vs. non–lipid rafts was augmented in WT myoblasts, form localized in the non-lipid-raft region of the plasma but not in glypican-1-deficient myoblasts. Importantly, in membrane as we previously reported [38]. Figure 5 shows WT myoblasts, most of the phospho-Met was associated that mock-transfected WT myoblasts induced the activa- with lipid raft fractions. In contrast, in the glypican-1- tion of AKT and ERK1/2 in response to HGF. In myo- deficient myoblasts, most of the phospho-Met was as- blasts expressing the chimeric F-GlySyn, however, both sociated with non-lipid-raft fractions. In both WT and phospho-AKT and phospho-ERK1/2 levels decreased com- glypican-1-deficient myoblasts, phospho-ERK1/2 and pared to WT cells. These levels are comparable to levels phospho-AKT were found in the non-lipid-raft fractions. found in the glypican-1-deficient myoblasts. The figure Next, we evaluated the presence of CD44 in lipid raft also shows that diminished sensitivity to HGF, which we domains and its dependence on glypican-1. Our results had previously observed in the glypican-1-deficient cells, show that the association of CD44 with the lipid raft do- was restored after reexpressing glypican-1 by transient main is dependent on glypican expression and that this transfection with rat glypican-1. Together, these results in- association is stabilized after pretreatment with HGF dicate that glypican-1 must be associated with lipid rafts to sustain HGF-dependent signaling. (Figure 3). The results described above suggest that glypican-1 is required for the translocation and stabilization of Met to Glypican-1 physically interacts with HGF and Met in lipid lipid rafts, where it is activated. To test this possibility, rafts to form an active signaling complex cells were treated with MβCD, an antifungal drug that se- The results described above suggest that glypican-1 may lectively extracts cholesterol from the plasma membrane interact with Met and HGF in lipid rafts to form the Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 7 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 2 Hepatocyte growth factor-dependent myoblast migration requires glypican-1 expression. (A) Wild-type (WT) C2C12 and C6 myoblasts were seeded onto the upper part of transwell chambers at the same density in serum-free media. The lower chamber contained serum-free media with or without 20 ng/ml hepatocyte growth factor (HGF). After 8 hours, the cells in the upper part of the filter were scraped. The cells that had efficiently migrated through the filter were fixed with paraformaldehyde, stained with crystal violet and photographed or as shown in (B) stained with crystal violet and solubilized in phosphate-buffered saline containing 1% Triton X-100. The absorbance of the detergent soluble fraction at 595 nm was determined. Values are expressed as mean ± standard deviation of three independent experiments. ***P < 0.001 relative to WT control. The migration of WT under control conditions corresponds to a value of 1.0. ternary complex Met-HGF-glypican-1. To test this possibil- the cells were treated with HGF, the levels of coimmuno- ity, WT myoblasts were transfected with an empty vector precipitated Met increased with both forms of glypican-1, as the control or with rat glypican-1 (F-Gly) or chimeric F- though in a more pronounced way with F-Gly. Interest- GlySyn, both of which contained a FLAG epitope. Forty- ingly, when the activated form of precipitated Met was eight hours later, the cells were incubated with or without evaluated, F-Gly interacted substantially more than the 20 ng/ml [ I]HGF for 5 minutes. The cell extracts in the non-lipid-raft form of glypican-1 (F-GlySyn) with phospho- presence of phosphatase inhibitors were immunoprecipi- Met. We also found that [ I]HGF coimmunoprecipitated tated with anti-FLAG antibodies, and the precipitate was almost four times more with F-Gly than with F-GlySyn. As evaluated for total and phospho-Met. Figure 6A shows that, an immunoprecipitation control, F-Gly and F-GlySyn were in the absence of HGF, Met coimmunoprecipitated with detected with specific anti-glypican-1 antibodies. These re- both F-Gly and F-GlySyn almost to the same extent. When sults suggested that glypican-1 physically interacted with Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 8 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 3 Met is localized and activated by hepatocyte growth factor in lipid rafts by a glypican-1-dependent mechanism. C2C12 and C6 myoblasts were serum-starved for 6 hours and then treated without (control) or with 10 ng/ml hepatocyte growth factor (HGF) for 5 minutes. The cells were lysed with 1% Triton X-100 and fractionated by sucrose density gradients (5% to 45%). Twelve fractions were collected, but only the last ten fractions were analyzed (the lipid raft–enriched fraction started at fraction 4) by immunoblotting for total HGF receptor (Met), phospho-Met (Tyr 1235/1349), phosphorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2), phospho-AKT and CD44. As fractionation + + controls, the presence of the lipid raft membrane protein marker caveolin 1 (Cav 1) and the non-lipid-raft domain marker Na /K -ATPase are shown. WT, Wild type. Met and HGF preferentially located in lipid rafts, where the myoblast migration, we subjected the C57BL/10 mice to receptor was stabilized and activated in response to HGF. intramuscular coinjection of C2C12 or C6 myoblasts to- To determine if binding of HGF on the myoblast cell sur- gether with HGF in the TA muscles. Seven days after the face was modulated by glypican-1, we performed a ligand transplantation, the muscles were extracted, frozen in li- binding assay. WT and glypican-1-deficient myoblasts were quid nitrogen and cryosectioned. Prior to grafting, the incubated with [ I]HGF at 4°C to avoid endocytosis of the myoblasts were stained with the vital dialkylcarbocyanine ligand. The radioactivity associated with low- and high- dye, DiI (red fluorescence), to trace their localization in affinity binding sites, as well as the remaining radioactivity the muscle cryosections. Figure 7 shows that HGF in- in the cell extracts, was determined. Figure 6B shows that duced an increase in the number of WT myoblasts that the binding of [ I]HGF to both low- and high-affinity migrated longer distances (more than 600 μm). However, binding sites was diminished by 50% in the absence of this effect was prevented in glypican-1-deficient myo- glypican-1, suggesting that this lipid raft–associated HSPG blasts. These results suggest that glypican-1 expression is was required to concentrate HGF on the cell surface and required for efficient in vivo myoblast migration in re- for binding to Met. These results indicate that glypican-1 sponse to HGF. facilitated the binding of HGF to the Met receptor, enhan- cing its phosphorylation at lipid raft domains. Discussion One of the main functions of membrane-associated HSPGs, Migration of transplanted myoblasts in skeletal muscles is particularly for glypicans, is to regulate signaling of several enhanced by HGF and requires glypican-1 cytokines, morphogens and growth factors [38,50-53]. It The data described above demonstrates the requirement has been reported that loss of HSPG expression prevents of glypican-1 for HGF-dependent signaling and migration. the cell mitogenic response induced by HGF [54-56], but To test the in vivo role of glypican-1 on HGF-induced the specific roles and mechanisms of the different HSPGs Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 9 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 4 Disruption of lipid rafts diminishes hepatocyte growth factor–dependent signaling. (A) C2C12 myoblasts were serum-starved for 6 hours, and during the last hour the cells were treated with or without methyl-β-cyclodextrin (MβCD) at the indicated concentrations. After two washes with serum-free media, the cells were treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. The cell extracts were analyzed by immunoblotting for total HGF receptor (Met), phospho- and total AKT, phosphorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2) and total ERK1/2, and tubulin was used as a loading control. (B) Quantification from two independent experiments is shown. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. *P < 0.05, **P <0.01, ***P < 0.001. (C) C2C12 myoblasts treated with or without 10 mM MβCD for 1 hour as described in (A) were lysed and fractionated in sucrose density gradients as described in Figure 3. The distributions of total Met and caveolin 1 (Cav-1) were determined by immunoblot analysis. In (A) and (C),the molecular weight standards are shown at left. as regulators of HGF-dependent responses have not been 1 appears as an essential cell-surface, low-affinity binding studied in depth. site for HGF, likely acting as a presenter or facilitator of In the present report, we show that, in myoblasts, HGF to its high-affinity Met binding site, where it is cofrac- glypican-1 located in lipid raft membrane domains was tionated with the known HGF coreceptor CD44 [34]. required for maximum HGF-dependent signaling and cell Glypican-1, Met and HGF formed an active signaling tern- migration in vitro and in vivo. We also show that glypican- ary complex in lipid raft membrane domains. Whether Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 10 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 5 Glypican-1 is required to sustain the hepatocyte growth factor-dependent signaling in lipid rafts. Wild-type (WT) myoblasts were transiently transfected with an empty vector as the control or with a non-lipid-raft form of glypican-1 containing the extracellular domain of rat glypican-1 and the transmembrane and cytoplasmic domains of mouse syndecan-1 (F-GlySyn) [36]. C6 myoblasts were transiently transfected with an empty vector as the control or with rat glypican-1 (C6-Gly). Forty-eight hours after transfection, the cells were serum-starved for 6 hours and then treated with the indicated concentrations of hepatocyte growth factor (HGF) for 5 minutes. (A) The cell extracts were analyzed by immunoblotting for total HGF receptor (Met), phospho- and total Akt and phosphorylated extracellular signal-regulated kinases 1 and 2 (phospho-ERK1/2) and total ERK1/2. Glypican-1 core protein levels after heparitinase digestion of endogenous and both transfected forms of glypican-1 were detected by using an anti-glypican-1 antibody. Tubulin levels were used as loading controls. (B) Quantification from two independent experiments is shown. Statistical significance was assessed using two-way analysis of variance and a Bonferroni multiple-comparisons posttest. *P < 0.05, **P <0.01. phospho-Met is relocated from non-lipid-raft to lipid raft protein–coupled receptors, including β-adrenergic, neuro- domains in response to HGF or whether Met is directly ac- kinin 1 receptor and muscarinic cholinergic receptors tivated in lipid rafts, where it is stabilized, are still not [61-64]. Lipid rafts can also act as a platform where recep- known. Chimeric non-lipid-raft glypican-1 (F-GlySyn) also tor signaling is turned off, such as in the case of serine- coimmunoprecipitated with Met, but not with the active threonine kinase transforming growth factor β [65] and form of the receptor or with HGF, indicating that loca- tyrosine kinase epidermal growth factor receptors, which lization of glypican-1 in lipid raft domains was unnecessary are activated in lipid rafts, but rapidly relocalized to non– for the interaction between Met and the extracellular part lipid rafts to de-activate downstream signaling [66]. We of glypican-1, but was required for binding of HGF and previously reported that glypican-1 in lipid rafts acted as a subsequent receptor activation. negative regulator of FGF-2 signaling, sequestering the The participation of lipid rafts as signaling platforms growth factor in these domains away from their transdu- to facilitate interaction of the required elements to acti- cing receptors [38]. Our present results show that, upon vate a signaling pathway has been reported for different ligand binding, Met is recruited to lipid rafts to activate receptor tyrosine kinases, such as the platelet-derived MAPK, ERK1/2 and PI3K/AKT pathways. This process growth factor, TrkA/nerve growth factor and insulin re- required the presence of structured lipid raft membrane ceptors. After ligand activation, MAPK and phosphoino- domains as well as glypican-1 in these domains to sustain sitide 3-kinase (PI3K) signaling mediators are recruited the HGF-dependent signaling. However, these results did to lipid rafts, where they are activated [57-60]. The not eliminate the possibility of other Met-dependent func- same mechanism of action has also been reported for G tions in non–lipid rafts. Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 11 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 6 Glypican-1 in lipid rafts coimmunoprecipitates with the activated form of Met and regulates hepatocyte growth factor binding to low- and high-affinity cell surface binding sites. (A) C2C12 myoblasts were transfected with rat glypican-1 (F-Gly) or the non-lipid-raft chimeric glypican-1 (F-GlySyn), as described in the Figure 5 legend. F-Gly and F-GlySyn contained a FLAG epitope at the amino terminus. Forty-eight hours after transfection, the cells were serum-starved for 4 hours and then treated with or without 20 ng/ml [ I]HGF for 5 minutes. The cell extracts were incubated with anti-FLAG M2 Affinity Gel for 3 hours at 4°C, and, after several washes, the beads were incubated with heparitinase and chondroitinase ABC for 8 hours. The immunoprecipitated (IPP) bound material was eluted with protein loading buffer and analyzed by Western immunoblotting for total hepatocyte growth factor (HGF) receptor (Met), phospho-Met and glypican-1. The membranes were exposed to a phosphorimager to detect [ I]HGF. (B) C2C12 and C6 myoblasts were serum-starved for 4 hours and then treated with or 125 125 without 10 ng/ml [ I]HGF for 2 hours at 4°C. After several washes in ice-cold binding buffer, [ I]HGF was eluted with high salt and acid to determine low- and high-affinity binding sites, respectively. Counts per minute (cpm) were determined by γ counting and corrected for protein content in cell extracts. Statistical significance was assessed by two-way analysis of variance and a Bonferroni multiple comparisons posttest. **P <0.01. HGF is involved in many different processes in which Met expression [6-8]. In the present study, we show that both cell growth and cell migration are required, such as glypican-1 was required for the migration of myoblasts in embryonic development, tissue repair and organ re- in response to HGF, both in vitro and in vivo. In vitro generation [67]. In particular, the roles of HGF and Met glypican-1-deficient myoblasts were almost unresponsive for muscle development, differentiation and regeneration to HGF as a chemoattractant in the Boyden chamber as- have been reported [7]. During limb muscle develop- says, in contrast to WT myoblasts, which migrated exten- ment, migratory muscle precursor cells delaminate from sively through the membrane toward the HGF-containing the dermomyotome, an epithelial structure that develops media. The migration capacity toward other chemoattrac- from somites, reaching their specific destination in the tants did not appear hampered, because no significant dif- limb buds [68-70] in a process dependent on HGF and ferences were observed when both types of cells were Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 12 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Figure 7 Hepatocyte growth factor-dependent migration of myoblast in vivo requires glypican-1 expression. (A) Wild-type (WT) or C6 myoblasts (500 × 10 ), prelabeled with the vital dialkylcarbocyanine dye, DiI (red fluorescence), and suspended in 30 μl of physiological serum with or without 10 ng of carrier-free hepatocyte growth factor (HGF) were transplanted into the left and right tibialis anterior (TA) muscles, respectively, of 3-month-old C57BL/10 mice under anesthesia. One week later, both TA muscles were processed for cryosectioning. Concentric circles with annuli 200 μm from each other were superimposed on selected muscle cross-section images. The cells were counted under an inverted microscope equipped for epifluorescence. (B) The number of the cells that migrated more than 200 μm were considered the total migrating cells, and the percentages of cells that migrated more than 600 μm were calculated. The migration of untreated WT myoblasts was corrected to 100%. Values are expressed as mean ± SD of two independent experiments. ***P <0.001. challenged to migrate toward 10% FBS (data not shown). expanded in vitro couldbeimprovedbycoinjection with We determined the role of glypican-1 in myoblast mi- HGF. In addition, this effect required the expression of gration in vivo in response to HGF by intramuscular glypican-1 in the myoblast plasma membrane. coinjection of WT or glypican-1-deficient myoblasts in This result is very promising, because one of the main the presence or absence of the growth factor. In vivo problems associated with stem cell therapies for the treat- myoblast migration was improved by coinjection with ment of patients with muscular dystrophies is the poor HGF, particularly in WT cells, compared to the slight mi- migration of the transplanted cells. As a result, therapy gratory effect observed with glypican-1-deficient myoblasts. with intramuscular injection of myoblasts or SCs in sev- These results show that in vivo migration of myoblast eral clinical trials has been mostly unsuccessful [71-73]. Gutiérrez et al. Skeletal Muscle 2014, 4:5 Page 13 of 16 http://www.skeletalmusclejournal.com/content/4/1/5 Based upon our results, the use of fluorescence-activated features of stem cells. It is explained by asymmetric cell cell sorting with higher expression of glypican-1 and/or division involving some daughter cells, which continue the coinjection with HGF to improve efficiency needs to be differentiation pathway, whereas other cells exit the cell carefully evaluated. cycle and return to quiescence [90,91]. HGF concentra- As previously mentioned, HSPGs are essential compo- tions above 20 ng/ml induced the quiescence of primary nents required for the myogenic inhibitory signaling of myogenic cells. This effect was reversible because treat- FGF-2 [28,30-33,74-76] and HGF [3,77,78]. During dif- ment with low concentrations of HGF could rescue the ferentiation, expression of all syndecans was downregu- proliferation of myogenic cells after high HGF-induced lated, which is consistent with a reduction in sensitivity quiescence [77]. to the inhibitory effect of FGF-2 [32,38,79]. In contrast, It would be interesting to determine whether glypican- the expression of glypican-1 remained constant, being 1 has a potential role in the control of SC sensitivity to the main cell surface HSPG present during myogenesis extracellular HGF and to define which SCs will continue [35,39]. In addition, during muscle regeneration, expression to form new muscle and which will exit the cell cycle in of glypican-1 increased and was temporarily and histologi- asymmetric cell division to maintain the pool of muscle cally related to the newly regenerating myofiber expression stem cells. Besides its association with lipid rafts in the of embryonic myosin [80]. However, the exact role of cell membrane, glypican-1 is also endogenously proc- glypican-1 during this process has not been addressed to essed to a soluble form that is incorporated into the date. Glypican-1-knockout mice were almost indistinguish- ECM [35,38,80], where it can act as a reservoir for HGF able from WT mice in size, fertility, internal anatomy and and other heparin-binding growth factors that can be re- lifespan, with the exception of the brain, which was notice- leased upon an injury to activate SCs. More accurate future ably smaller [81]. This suggests that glypican-1 is required studies designed to determine the control mechanisms of in mammals for brain development, but not for other glypican-1 and Met expression between daughter cells dur- tissues, such as skeletal muscle. To further elucidate the ing asymmetric cell division, as well as the role of glypican- results of the present study, it would be informative to 1 during the muscle regeneration process, are therefore evaluate the skeletal muscle regeneration process in necessary. glypican-1-null mice. Glypican-1 is required for terminal myogenesis, acting Conclusion as a repressor of FGF-2 [38]. This can be explained by Glypican-1 in lipid raft membrane domains is required for the sequestration of FGF-2 by glypican-1 in lipid rafts, maximum HGF-dependent signaling and myoblast migra- away from FGF-2 receptors and syndecans that are lo- tion in vitro and in vivo. cated in non–raft domains. As we have shown, however, glypican-1 positively regulates HGF-mediated signaling Abbreviations AKT: Effector of the phosphoinositide 3-kinase/AKT pathway; by recruiting or stabilizing Met in lipid raft domains ERK: Extracellular signal-regulated kinase; FGF-2: Fibroblast growth factor 2; where it was activated, with consequential triggering of FGFR: Transducing fibroblast growth factor receptor; F-Gly: Rat glypican-1 downstream targets. Reduction of Met expression during containing a FLAG epitope in its amino terminus; F-GlySyn: Chimeric heparan sulfate proteoglycan (extracellular domain of rat glypican-1 and the trans- the myogenic differentiation process (data not shown) [82] membrane and cytoplasmic domains of mouse syndecan-1 containing a therefore seemed to circumvent the myogenic inhibitory FLAG epitope); HGF: Hepatocyte growth factor; HS: Heparan sulfate; effect of HGF in spite of the constitutive expression of HSPG: Heparan sulfate proteoglycan; Met: Transducing hepatocyte growth + + factor receptor; MβCD: Methyl-β-cyclodextrin; Na /K -ATPase: Sodium glypican-1 [35,39]. All of these changes switched the bal- potassium ATP pump; Phospho-AKT: Phosphorylated form of AKT; ance from a proliferative, migratory and antimyogenic state Phospho-ERK: Phosphorylated form of extracellular signal-regulated kinase; in response to FGF-2 and HGF to a promyogenic response Phospho-Met: Phosphorylated form of hepatocyte growth factor receptor; TA: Tibialis anterior. whereby both muscle inhibitory signals decreased, thus allowing differentiation. Competing interests Immediately after injury, low concentrations of HGF The authors declare that they have no competing interests. (2 to 3 ng/ml) are released from ECM reservoirs [83-85] in conjunction with the local release of nitric oxide. These Authors’ contributions JG participated in the design of the study, carried out the cellular and are the first cues involved in the activation (that is, exit molecular experiments, performed the statistical analysis and drafted the from quiescence) of SCs, which then proliferate to form manuscript. DC carried out the myoblast migration experiments. EB new fibers or repair the destroyed ones [10,84,86]. To conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. maintain their regenerative potential, many proliferating SCs return to quiescence, repopulating the SC niche to Acknowledgments maintain a progenitor pool, which will be activated to re- This study was supported by grants CARE-PFB-12/2007, CONICYT-79090027, pair the muscle in response to a new injury [87-89]. 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Skeletal MuscleSpringer Journals

Published: Feb 12, 2014

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