Expression of perlecan, a proteoglycan that binds myogenic inhibitory basic fibroblast growth factor, is down regulated during skeletal muscle differentiation.
Heparan sulfate proteoglycans (HSPG) have been shown to be involved in the activation of tyrosine kinase receptors by basic fibroblasts growth factor (bFGF), a strong inhibitor of skeletal muscle differentiation. Skeletal muscle fibers contact extracellular matrix (ECM) that surrounds individual fibers (endomysium) and bundles of several fibers (perimysium). Perlecan is a HSPG present in the majority of basement membranes. In this study we evaluated the expression and localization of perlecan during differentiation of C2C12 skeletal muscle cells. C2C12 myoblasts incubated with [35S]Na2SO4 synthesize a HSPG that can be specifically immunoprecipitated with antibodies against murine perlecan. The immunoprecipitated HSPG eluted from a Sepharose CL-4B with a Kav of 0.44. Analysis of the core protein of the HSPG immunoprecipitated from [35S]methionine-labeled C2C12 after treatment with heparitinase revealed two polypeptides of 170 and over 300 kDa. The amount of polypeptides immunoprecipitated decreased with muscle differentiation. Immunocytolocalization studies indicate that perlecan is localized on the myoblast surface and by immunogold staining we have demonstrated that it is associated with patches of incipient extracellular matrix. The expression of perlecan mRNA decreased substantially during skeletal muscle differentiation, in contrast to the increase in transcripts for specific skeletal muscle proteins such as myogenin and creatine kinase. By immunofluorescence microscopy almost no perlecan staining associated with the surface of myotubes was observed. All these results suggests that perlecan, a HSPG that binds myogenic inhibitory bFGF, normally associated with basement membranes in adult tissues is present on the surface of myoblasts and its expression is down regulated during skeletal muscle differentiation.
Larraín
J
J
Unidad de Neurobiología Molecular, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, P. Universidad Católicade Chile, Casilla, Santiago.
Alvarez
J
J
Hassell
J R
JR
Brandan
E
E
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D003402:Creatine Kinase / Q000235:genetics; D005109:Extracellular Matrix / Q000737:chemistry*; D016222:Fibroblast Growth Factor 2; D005786:Gene Expression Regulation; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology*; D018008:Myogenin / Q000235:genetics; D011509:Proteoglycans / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis
D003402:Creatine Kinase; D016222:Fibroblast Growth Factor 2; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D018008:Myogenin; D011509:Proteoglycans; D012333:RNA, Messenger; C503801:Myog protein, mouse; C071927:perlecan