Summary
Skeletal fibroblasts in vitro can acquire myofibroblast phenotypes by the development of biochemical and morphological features, mainly the expression of alpha-smooth-muscle actin (α-SMA). Myogenic differentiation is a central event in skeletal muscle development, and has commonly been studied in vitro in the context of skeletal muscle development and regeneration. Controlling this process is a complex set of interactions between myoblasts and the extracellular matrix. Osteopontin (OPN) is an acidic, phosphorylated matrix protein that contains an Arg-Gly-Asp (RGD) cell attachment sequence and has been identified as an adhesive and migratory substrate for several cell types. The aim of this study was to investigate osteopontin expression during the differentiation of skeletal fibroblasts into myofibroblasts and during myogenesis in a coculture model. Fibroblasts and myoblasts were obtained from skeletal muscle of 18-d-old Wistar strain rat fetuses by enzymatic dissociation. At 1 and 9 d, cocultures were immunolabeled, and the cells were also separately subjected to Western blotting to analyze OPN expression. Our data using confocal microscopy showed that myoblasts displayed a strong staining for OPN and that this labeling was maintained after myotube differentiation. Conversely, during fibroblast differentiation into myofibroblasts, we observed a significant increase in OPN expression. The results obtained by immunolabeling were confirmed by Western blotting. We suggest that OPN is important mainly during early stages of myogenesis, facilitating myoblast fusion and differentiation, and that the increased expression of OPN in myofibroblasts might be related to its effects as a key cytokine regulating tissue repair and inflammation.
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Ashizawa, N., Graf, K.; Do, Y. S.; Nunohiro, T.; Giachelli, C. M.; Meehan, W. P.; Tuan, T. L.; Hsueh, W. A. Osteopontin is produced by rat cardiac fibroblasts and mediates A(II)-induced DNA synthesis and collagen gel contraction. J. Clin. Invest. 98(10):2218–2227; 1996.
Bayless, K. J.; Davis, G. A.; Meininger G. E. Isolation and biological properties of osteopontin from bovine milk. Protein Expr. Purif. 9(3):309–314; 1997.
Bayless, K. J.; Meininger, G. A.; Scholtz, J. M.; Davis, G. E. Osteopontin is a ligand for the alpha4betal integrin. J. Cell Sci. 111(Pt. 9):1165–1174; 1998.
Boettiger, D.; Enomoto-Iwamoto, M.; Yoon, H. Y.; Hofer, U.; Menko, A. S.; Chiquet-Ehrismann, R. Regulation of integrin alpha 5 beta 1 affinity during myogenic differentiation. Dev. Biol. 169(1):261–272; 1995.
Buck, C. A.; Horwitz, A. F. Cell surface receptors for extracellular matrix molecules. Annu. Rev. Cell Biol. 3:179–205; 1987.
Giachelli, C. M.; Liaw, L.; Murry, C. E.; Schwartz, S. M.; Almeida, M. Osteopontin expression in cardiovascular diseases. Ann. N.Y. Acad. Sci. 760:109–126; 1995.
Graf, K.; Do, Y. S.; Ashizawa, N.; Meehan W. P.; Giachelli, C. M.; Marboe, C. C.; Fleck, E., Hsueh, W. A. Myocardial osteopontin expression is associated with left ventricular hypertrophy. Circulation 96(9):3063–3071; 1997.
Hartner, A.; Porst, M.; Gauer, S.; Prols F.; Veelken, R.; Hilgers, K. F. Glomerular osteopontin expression and macrophage infiltration in glomerulosclerosis of DOCA-salt rats. Am. J. Kidney Dis. 38(1):153–164; 2001.
Hirata, A.; Masuda, S.; Tamura, T. et al. Expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin. Am. J. Pathol. 163(1):203–215; 2003.
Komatsubara, I.; Murakami, T. Kusachi, S. et al. Spatially and temporally different expression of osteonectin and osteopontin in the infarct zone of experimentally induced myocardial infarction in rats. Cardiovasc. Pathol. 12(4):186–194; 2003.
Lafuste P.; Sonnet C.; Chazaud B.; Dreyfus P. A.; Gherardi R. K.; Wewer U. M.; Authier F. J. ADAM12 and alpha9betal integrin are instrumental in human myogenic cell differentiation. Mol. Biol. Cell 16(2):861–70; 2005.
Langen, R. C.; Schols, A. M.; Kelders, M. C.; Wouters, E. F.; Janssen-Heininger, Y. M. Enhanced myogenic differentiation by extracellular matrix is regulated at the early stages of myogenesis. In Vitro Cell. Dev. Biol. Anim. 39(3):163–169; 2003.
Li, G.; OpariL, S.; Kelpke, S. S.; Chen, Y. F.; Thompson, J. A. Fibroblast growth factor receptor-1 signaling induces osteopontin expression and vascular smooth muscle cell-dependent adventitial fibroblast migration in vitro. Circulation 106(7):854–859; 2002.
Li, Y.; Huard, J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am. J. Pathol. 161(3):895–907 2002.
Liaw, L.; Almeida, M.; Hart, C. E.; Schwartz, S. M.; Giachelli, C. M. Osteopontin promotes vascular cell adhesion and spreading and is chemotactic for smooth muscle cells in vitro. Circ. Res. 74(2):214–224; 1994.
Melo, F.; Carey, D. J.; Brandan, E. Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression. J. Cell Biochem. 62(2):227–239; 1996.
Murry, C. E.; Giachelli, C. M.; Schwartz, S. M.; Vracko, R. Macrophages express osteopontin during repair of myocardial necrosis. Am. J. Pathol. 145(6):1450–1462; 1994.
Oldberg, A.; Franzen, A.; Heinegard, D. Cloning and sequence analysis of rat bone sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc. Natl. Acad. Sci. USA 83(23):8819–8823; 1986.
O’Regan, A.; Berman, J. S. Osteopontin: a key cytokine in cell-mediated and granulomatous inflammation. Int. J. Exp. Pathol. 8(6):373–390; 2000.
Pereira, R. O.; De Carvalho, T.; Barbosa, H. S.; Porto, L. C.; Carvalho, L. Enhancement of lipid bodies during differentiation of skeletal myofibroblasts of rat’s fetus in vitro. In Vitro Cell. Dev. Biol. Anim. 40(1–2):1–3; 2004.
Powell, D. W.; Mifflin R. C.; Valentich, J. D.; Crowe, S. E.; Saada, J. L.; West, A. B. Myofibroblasts. I. Paracrine cells important in health and disease. Am. J. Physiol. 277(46):1–19; 1999.
Singh, K.; Sirokman, G.; Communal, C. Robinson, K. G.; Conrad, C. H.; Brooks, W. W.; Bing, O. H.; Colucci, W. S. Myocardial osteopontin expression coincides with the development of heart failure Hypertension 33(2):663–670; 1999.
Sun, A. J.; Gao P. J.; Liu J. J.; Ji K. D.; Zhu D. L. Identification of genes related to cell phenotypic transition by differential display analysis. Sheng Li Xue Bao. 53(6):435–439; 2001.
Tomasek, J. T.; Gabbiani, G.; Hinz, B.; Chaponier, C.; Brown, R. A. Myofibroblasts and mechanoregulation of connective tissue remodeling. Nature 3:349–363; 2002.
Yun, K.; Wold, B. Skeletal muscle determination and differentiation: story of a core regulatory network and its contex. Curr. Opin. Cell Biol. 8(6):877–89, 1996.
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Pereira, R.O., Carvalho, S.N., Stumbo, A.C. et al. Osteopontin expression in coculture of differentiating rat fetal skeletal fibroblasts and myoblasts. In Vitro Cell.Dev.Biol.-Animal 42, 4–7 (2006). https://doi.org/10.1007/s11626-006-0003-0
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DOI: https://doi.org/10.1007/s11626-006-0003-0