Abstract
Syndecan-3 is a member of a family of transmembrane proteoglycans that posses highly homologous cytoplasmic and transmembrane domains and function as extracellular matrix receptors and low-affinity receptors for signaling molecules such as basic fibroblasts growth factor (FGF-2). Syndecan-3 is transiently expressed in developing limb bud and absent in adult skeletal muscle. In this study we investigated the expression of syndecan-3 and its role on FGF-2-dependent inhibition of myogenesis. Syndecan-3 expression was down-regulated during skeletal muscle differentiation of C(2)C(12) myoblasts, as determined by Northern blot analyses and immunoprecipitation. To probe the function of syndecan-3 during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid coding for antisense syndecan-3 mRNA. The resulting inhibition of syndecan-3 expression caused accelerated skeletal muscle differentiation, as determined by expression of creatine kinase and myosin and myoblast fusion. Expression of a master transcription factor for muscle differentiation, myogenin, was also accelerated in antisense syndecan-3-transfected myoblasts compared with control transfected and wild type cells. Reduced expression of syndecan-3 resulted in a 13-fold decrease in sensitivity to FGF-2-dependent inhibition of myogenin expression. Addition of heparin partially reversed this effect. These results demonstrate that syndecan-3 expression is down-regulated during differentiation and the level of expression of membrane-bound heparan sulfate on myoblast surface is critical for fine modulation of responsiveness to FGF-2. These findings strongly suggest a role for syndecan-3 in regulation of skeletal muscle terminal differentiation.
Highlights
Skeletal muscle myoblasts are the precursors to skeletal muscle fibers
The results presented in this paper demonstrate that the expression of syndecan-3, an integral membrane heparan sulfate proteoglycan [14, 17], decreases during differentiation of skeletal muscle cells
The biochemical characteristics of syndecan-3 synthesized by skeletal muscle cells are in good agreement with the ones described for this heparan sulfate proteoglycan from other sources [20, 22]; it contains mainly heparan sulfate glycosaminoglycans chains and a core protein of approximately 120 kDa
Summary
Materials—The C2C12 cell line and the plasmid pRSVlacZII phagemid vector (-galactosidase reporter) were purchased from ATCC. The day the cells were rinsed twice with Hanks’ balanced salt solution and cultured in normal growth medium. Analysis of Creatine Kinase Activity—Myoblasts and myoblasts induced to differentiate for the indicated days were washed twice with phosphate-buffered saline and lysed by incubation with phosphatebuffered saline containing 0.1% Triton X-100 for 10 min at 25 °C and harvested by scraping. For transfection the cells were incubated for 6 h in Opti-MEM I containing 5 g each of the myogenin reporter plasmid pMYOCAT [12, 18] and pRSVlacZII phagemid vector (-galactosidase reporter) and 15 g of LipofectAMINE. The cells were washed twice with Hanks’ balanced salt solution and incubated for 2 days in growth medium followed by 40 h in differentiation medium. DNA and Protein Determination—DNA [26] and protein [27] were determined in aliquots of cell extracts as described
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
