Cell-type specific adhesive interactions of skeletal myoblasts with thrombospondin-1.
Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein that may play important roles in the morphogenesis and repair of skeletal muscle. To begin to explore the role of thrombospondin-1 in this tissue, we have examined the interactions of three rodent skeletal muscle cell lines, C2C12, G8, and H9c2, with platelet TSP-1. The cells secrete thrombospondin and incorporate it into the cell layer in a distribution distinct from that of fibronectin. Myoblasts attach and spread on fibronectin- or thrombospondin-coated substrates with similar time and concentration dependencies. Whereas cells adherent on fibronectin organize actin stress fibers, cells adherent on TSP-1 display prominent membrane ruffles and lamellae that contain radial actin microspikes. Attachment to thrombospondin-1 or the 140-kDa tryptic fragment is mediated by interactions with the type 1 repeats and the carboxy-terminal globular domain. Attachment is not inhibited by heparin, GRGDSP peptide, or VTCG peptide but is inhibited by chondroitin sulphate A. Integrins of the beta 1 or alpha V subgroups do not appear to be involved in myoblast attachment to TSP-1; instead, this process depends in part on cell surface chondroitin sulphate proteoglycans. Whereas the central 70-kDa chymotryptic fragment of TSP-1 does not support myoblast attachment, the carboxy-terminal domain of TSP-1 expressed as a fusion protein in the bacterial expression vector, pGEX, supported myoblast attachment to 30% the level of intact TSP-1. Thrombospondin-4 (TSP-4) is also present in skeletal muscle and a fusion protein containing the carboxy-terminal domain of TSP-4 also supported myoblast adhesion, although this protein was less active on a molar basis than the TSP-1 fusion protein. Thus, the carboxyterminal domain of TSP-1 appears to contain a primary attachment site for myoblasts, and this activity is present in a second member of the thrombospondin family.
Adams
J C
JC
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
Lawler
J
J
D000199:Actins / Q000032:analysis; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D000939:Epitopes / Q000032:analysis; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006025:Glycosaminoglycans / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology* / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D019699:Thrombospondins
D000199:Actins; D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D000939:Epitopes; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D006025:Glycosaminoglycans; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019699:Thrombospondins