Abstract
Betaglycan is a membrane-anchored proteoglycan co-receptor that binds transforming growth factor beta (TGF-beta) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains. In this study we evaluated the expression of betaglycan during the C(2)C(12) skeletal muscle differentiation. Betaglycan expression, as determined by Northern and Western blot, was up-regulated during the conversion of myoblasts to myotubes. The mouse betaglycan gene promoter was cloned, and its sequence showed putative binding sites for SP1, Smad3, Smad4, muscle regulatory factor elements such as MyoD and MEF2, and retinoic acid receptor. Transcriptional activity of the mouse betaglycan promoter reporter was also up-regulated in differentiating C(2)C(12) cells. We found that MyoD, but not myogenin, stimulated this transcriptional activity even in the presence of high serum. Betaglycan promoter activity was increased by RA and inhibited by the three isoforms of TGF-beta. On the other hand, basic fibroblast growth factor, BMP-2, and hepatocyte growth factor/scatter factor, which are inhibitors of myogenesis, had little effect. In myotubes, up-regulated betaglycan was also detectable by TGF-beta affinity labeling and immunofluorescence microscopy studies. The latter indicated that betaglycan was localized both on the cell surface and in the ECM. Forced expression of betaglycan in C(2)C(12) myoblasts increases their responsiveness to TGF-beta2, suggesting that it performs a TGF-beta presentation function in this cell lineage. These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and RA modulate its expression by a mechanism that is independent of myogenin.
Highlights
Betaglycan is a membrane-anchored proteoglycan coreceptor that binds transforming growth factor  (TGF-) via its core protein and basic fibroblast growth factor through its glycosaminoglycan chains
These results indicate that betaglycan expression is up-regulated during myogenesis and that MyoD and retinoic acid (RA) modulate its expression by a mechanism that is independent of myogenin
The level of cellular betaglycan during skeletal muscle differentiation was determined by Western blot analysis of detergent extracts treated previously with heparitinase and chondroitinase ABC, a procedure that reveals the total content of betaglycan as its core protein
Summary
Cell Culture—The mouse skeletal muscle cell line C2C12 (ATCC, Manassas, VA) [23] was grown and induced to differentiate as described [15]. Rat and L6E9 cells were routinely cultured in Dulbecco’s modified Eagle’s medium containing 10% or 20% fetal bovine serum, respectively, as described [19]. Fetal bovine serum and chicken embryo extract were added to restore their complete growth medium concentrations, and the cells were cultured for a further 14 h. The cells were trypsinized and plated in 24-well plates in normal growth medium Under these conditions, FGF-2 (3 ng/ml) (Invitrogen), BMP-2 (1 nM) (generous gift from Genetic Institute, Cambridge, MA), HGF (3 ng/ml) (R & D Systems, Minneapolis, MN), TGF- (1 ng/ml) (R & D Systems), and RA (1 M) (Sigma) were added at the indicated concentrations. DNA and Protein Determination—DNA [28] and protein [29] were determined in aliquots of cell extracts as described
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