Abstract
The basement membrane of skeletal muscle is produced by the muscle cells it ensheathes and by nonmuscle cells located in the surrounding extracellular matrix. In this study, we have shown that platelet-derived growth factor (PDGF) stimulates secretion of three basement membrane components of skeletal muscle: laminin (70% increase), fibronectin (30%), and type IV collagen (70%). Furthermore, we have found using the signal transduction inhibitors, genistein (tyrosine kinase inhibitor), phorbol 12-myristate 13-acetate (protein kinase C (PKC) inhibitor), thapsigargin (depletes intracellular Ca2+ stores), and H89 (protein kinase A inhibitor), that PDGF-stimulated secretion of these proteins occurs through distinct signaling pathways. Densitometry of Western blots of L6 myoblast supernatant indicates that the PDGF-induced increase in secretion of laminin and type IV collagen is tyrosine kinase-dependent. The increase in type IV collagen secretion also shows dependence on PKC, as well as the release of intracellular Ca2+. Inhibition of either of these pathways reduces the increase in type IV collagen secretion to 20%. In contrast, the PDGF-induced increase in laminin secretion is unaffected by inhibition of either PKC or intracellular Ca2+ release. The increase in fibronectin secretion by PDGF uses yet a third set of signals. PDGF-induced fibronectin secretion is not dependent on tyrosine kinase activity but is dependent on protein kinase A as well as the release of intracellular Ca2+. These divergent signaling pathways provide for independent regulation of basement membrane protein secretion, allowing a muscle cell to modify both the quantity and composition of its basement membrane in response to its environment.
Highlights
Shown that the composition of the basement membrane of muscle shows regional variability in composition and thickness
Using inhibitors of cell proliferation, we were able to determine that the increase in laminin, type IV collagen, and fibronectin secretion resulting from platelet-derived growth factor (PDGF) stimulation did not result from an increase in cell density induced by PDGF stimulation
PDGF Stimulation of Laminin, Type IV Collagen, and Fibronectin Secretion Is Mediated by Distinct Sets of Second Messengers—Unidentified, soluble factors have been shown previously to stimulate secretion of extracellular matrix molecules into the supernatant of myoblasts grown in vitro [15, 16]
Summary
Cell Culture—Rat L6 myoblasts were grown on plastic tissue culture dishes in Dulbecco’s minimal essential medium (DMEM) with 10% fetal bovine serum supplemented with 1% penicillin and streptomycin at 37 °C and 5% CO2. At the end of PDGF stimulation, supernatant was collected from experimental and control cultures and centrifuged for 10 min at 3000 ϫ g. Duplicate cultures were prepared for each experimental and control time point, and supernatants and cells were collected separately for analysis by immunoblots. Two plates of each concentration were incubated for 24 h, and the supernatant was collected and prepared for SDS-PAGE. After 24 h, supernatant was collected from PDGF/uridine/FuDR cultures, PDGF cultures, and DMEM-only cultures and prepared for SDS-PAGE and immunoblots. Signal Transduction Inhibitor Experiments—The signaling pathways involved in PDGF stimulation of basement membrane protein secretion were analyzed by stimulating L6 myoblasts in the presence of a variety of signal transduction inhibitors. Supernatant was collected 24 h after PDGF stimulation and prepared for SDS-PAGE analysis
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