TGF-β1-induced synthesis of collagen fibers in skeletal muscle-derived stem cells

Abstract

The aim of this study was to investigate the mechanism of deposition of extracellular matrix induced by TGF-β1 in skeletal muscle-derived stem cells (MDSCs). Rat skeletal MDSCs were obtained by using preplate technique, and divided into four groups: group A (control group), group B (treated with TGF-β1, 10 ng/mL), group C (treated with TGF-β1 and anti-connective tissue growth factor (CTGF), both in 10 ng/mL), and group D (treated with anti-CTGF, 10 ng/mL). The expression of CTGF, collagen type-I (COL-I) and collagen type-III (COL-III) in MDSCs was examined by using RT-PCR, Western blot and immunofluorescent stain. It was found that one day after TGF-β1 treatment, the expression of CTGF, COL-I and COL-III was increased dramatically. CTGF expression reached the peak on the day 2, and then decreased rapidly to a level of control group on the day 5. COL-I and COL-III mRNA levels were overexpresed on the day 2 and 3 respectively, while their protein expression levels were up-regulated on the day 2 and reached the peak on the day 7. In group C, anti-CTGF could partly suppress the overexpression of COL-I and COL-II induced by TGF-β1 one day after adding CTGF antibody. It was concluded that TGF-β1 could induce MDSCs to express CTGF, and promote the production of COL-I and COL-III. In contrast, CTGF antibody could partially inhibit the effect of TGF-β1 on the MDSCs by reducing the expression of COL-I and COL-III. Taken together, we demonstrated that TGF-β1-CTGF signaling played a crucial role in MDSCs synthesizing collagen proteins in vitro, which provided theoretical basis for exploring the methods postponing skeletal muscle fibrosis after nerve injury.

Keywords

TGF-β1 / CTGF / muscle-derived stem cells

References

[1] WynnTA. Common and unique mechanisms regulate fibrosis in various fibroproliferative diseases. J Clin Invest, 2007, 117(3): 524-529

[2] TomasekJJ, GabbianiG, HinzB, et al.. Myofibroblasts and mechano-regulation of connective tissue remodeling. Nat Rev Mol Cell Biol, 2002, 3(5): 349-363

[3] FriedmanSL. Mechanisms of disease: mechanisms of hepatic fibrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol, 2004, 1(2): 98-105

[4] SchuppanD, RuehlM, SomasundaramR, et al.. Matrix as a modulator of hepatic fibrogenesis. Semin Liver Dis, 2001, 21(3): 351-372

[5] PhanSH. The myofibroblast in pulmonary fibrosis. Chest, 2002, 122(6): 286S-289S

[6] HagoodJS, OlmanMA. Muscle fatigue. Am J Respir Cell Mol Biol, 2007, 37(5): 503-506

[7] LiY, HuardJ. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol, 2002, 161(3): 895-907

[8] CaoB, ZhengB, JankowskiRJ, et al.. Muscle stem cells differentiate into hematopoietic lineages but retain myogenic potential. Nat Cell Biol, 2003, 5(7): 640-646

[9] MassaguéJ, BlainSW, LoRS. TGFbeta signaling in growth control, cancer, and heritable disorders. Cell, 2000, 103(2): 295-309

[10] DerynckR, AkhurstRJ. Differentiation plasticity regulated by TGF-beta family proteins in development and disease. Nat Cell Biol, 2007, 9(9): 1000-1004

[11] DavidA. Brenner. Molecular pathogenesis of liver fibrosis. Trans Am Clin Climatol Assoc, 2009, 120(2): 361-368

[12] MSW, TAWynn. Pulmonary fibrosis: pathogenesis, etiology and regulation. Mucosal Immunol, 2009, 2(2): 103-121

[13] BrownRD, AmblerSK, MitchellMD, et al.. The cardiac fibroblast: therapeutic target in myocardial remodeling and failure. Annu Rev Pharmacol Toxicol, 2005, 45(4): 657-687

[14] DesmoulièreA, DarbyIA, GabbianiG. Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis. Lab Invest, 2003, 83(12): 1689-1707

[15] MengFB, ChenJH, LiJ, et al.. Role of transforming growth factor-β1 in the process of fibrosis of denervated skeletal muscle. J Huazhong Univ Sci Technol [Med Sci], 2011, 31(1): 77-82

[16] Shi-WenX, LeaskA, AbrahamD. Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis. Cytokine Growth Factor Rev, 2008, 19(2): 133-144

[17] RachfalAW, BrigstockDR. Structural and functional properties of CCN proteins. Vitam Horm, 2005, 70(1): 69-103

[18] FanWH, PechM, KarnovskyMJ. Connective tissue growth factor (CTGF) stimulates vascular smooth muscle cell growth and migration in vitro. Eur J Cell Biol, 2000, 79(12): 915-923

[19] ChenCC, ChenN, LauLF. The angiogenic factor induces adhesive signaling primary human skin fibroblasts. J Biol Chem, 2001, 276(13): 10 443-10 452

[20] ParadisV, DargereD, BonvoustF e a1. Effects and regulation of connective tissue growth factor on hepatic stellate cells. Lab Invest, 2002, 82(6): 767-774

[21] NieMB, ChenZB, HongGX. Influence of transforming growth factor β₁ on denervated mouse muscle derived stem cell producing connective tissue growth factor at different time points in vitro. Zhongguo Xiufu Chongjian Waike Zazhi (Chinese), 2007, 21(7): 557-560

[22] GrotendorstGR, OkochiH, HayashiN. A novel transforming growth factor beta response element controls the expression of the connective tissue growth factor gene. Cell Growth Differ, 1996, 7(4): 469-480

This project was supported by the National Natural Science Foundation of China (No. 30872627).