INFLUENCE OF INHIBITION OF ACTIN POLYMERIZATION ON ADIPOGENIC DIFFERENTIATION OF RAT Achilles-DERIVED TENDON STEM CELLS IN VITRO_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHENBo ¹ ,  TANGKanglai ¹ , ZHANGJiqiang ² , GUOYupeng ¹ , LIUXiangzhou ¹ , SHIYouxin ¹

1. Department of Orthopedics, Orthopedic Center of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing, 400038, P. R. China;
2. Department of Neurobiology, Third Military Medical University;

Corresponding author：

TANGKanglai, Email: tangkanglai@hotmail.com

Keywords：

Tendon stem cells; Adipogenic differentiation; Cytochalasin D; Actin; Cytoskeleton; Rat

DOI：

10.7507/1002-1892.20150045

Video：

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Objective To investigate the effect of cytoskeleton modification on the adipogenic differentiation of rat Achilles-derived tendon stem cells (TSCs) in vitro. Methods TSCs were isolated from the tendon tissue of male Sprague Dawley rats (aged 3 weeks) by enzymatic digestion method and cultured for 3 passages. After the 3rd passage cells were cultured with DMEM medium containing 15% fetal bovine serum and cytochalasin D (CYD) at the concentrations of 0, 50, 100, 500, and 1 000 ng/mL, the cell survival condition and morphology changes were observed by inverted phase contrast microscope, the cytoskeleton was observed through fibrous actin (F-actin) staining, and the ratio of F-actin/soluble globular actin (G-actin) was detected and calculated through Western blot. According to the above results, the effective concentration of CYD was selected and used for next experiments. After TSCs were cultured for 3 and 7 days respectively with adipogenic induction media (induction group), adipogenic induction media containing CYD (CYD+induction group), ordinary medium (ordinary group), and ordinary medium containing CYD (CYD+ordinary group), the real-time quantitative PCR (qRT-PCR) and Western blot were carried out to measure the mRNA and protein expressions of adipogenic differentiation-related markers, including peroxisome proliferator-activated receptor γ (PPARγ), 1ipoprotein lipase (LPL), and fatty acid binding protein (aP2). Results The final CYD concentration of 100 ng/mL can inhibit effectively G-actin polymerization into F-actin, but could not affect TSCs survival, which was used for next experiments. qRT-PCR and Western blot suggested that the mRNA expressions of PPARγ, LPL, and aP2 and the protein expressions of PPARγ and aP2 were increased significantly in the CYD+induction group at 3 and 7 days when compared with the induction group (P<0.05). In the CYD+ordinary group, there still was a significant increase in the mRNA expressions of PPARγ, LPL, and aP2 when compared with the ordinary group (P<0.05). Conclusion Inhibition of F-actin polymerization can increase adipogenic differentiation of rat Achilles-derived TSCs in vitro, and cytoskeleton modification is a pre-requisite for TSCs differentiation into adipocytes, which might have important implications for the mechanism research of tendinopathy.

Citation： CHENBo, TANGKanglai, ZHANGJiqiang, GUOYupeng, LIUXiangzhou, SHIYouxin. INFLUENCE OF INHIBITION OF ACTIN POLYMERIZATION ON ADIPOGENIC DIFFERENTIATION OF RAT Achilles-DERIVED TENDON STEM CELLS IN VITRO. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(2): 206-212. doi: 10.7507/1002-1892.20150045 Copy

1.	Wang JC, Guo Q, Li B. Tendon biomechanics and mechano-biology-a minireview of basic concepts and recent advancements. J Hand Ther, 2012, 25(2):133-141.
2.	Woo SL, Debski RE, Zeminski J, et al. Injury and repair of ligaments and tendons. Annu Rev Biomed Eng, 2000, 2:83-118.
3.	Salingcarnboriboon R, Yoshitake H, Tsuji K, et al. Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property. Exp Cell Res, 2003, 287(2):289-300.
4.	Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med, 2007, 13(10):1219-1227.
5.	Yin Z, Chen X, Chen JL, et al. The regulation of tendon stem cell differentiation by the alignment of nanofibers. Biomaterials, 2010, 31(8):2163-2175.
6.	Rui YF, Lui PP, Li G, et al. Isolation and characterization of muhipotent rat tendon-derived stem cells. Tissue Eng Part A, 2010, 16(5):1549-1558.
7.	Zhang J, Wang JH. Mechanobiological response of tendon stem cells:implications of tendon homeostasis and pathogenesis of tendinopathy. J Orthop Res, 2010, 28(5):639-643.
8.	Zhang J, Wang JH. Production of PGE(2) increases in tendons subjected to repetitive mechanical loading and induces differentiation of tendon stem cells into non-tenocytes. J Orthop Res, 2010, 28(2):198-203.
9.	邓银栓, 唐康来, 谢美明, 等. 不同牵伸载荷对体外培养人肌腱细胞纤维型肌动蛋白的影响. 中华医学杂志, 2011, 91(25):1780-1785.
10.	胡超, 唐康来, 陈万, 等. 大鼠跟腱来源肌腱干细胞的分离培养及鉴定. 第三军医大学学报, 2013, 35(11):1097-1101.
11.	Sonowal H, Kumar A, Bhattacharyya J, et al. Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway. J Biomed Sci, 2013, 20:71.
12.	Zeng LH, Xu L, Rensing NR, et al. Kainate seizures cause acute dendritic injury and actin depolymerization in vivo. J Neurosci, 2007, 27(43):11604-11613.
13.	Kustermans G, El Benna J, Piette J, et al. Perturbation of actin dynamics induces NF-kappaB activation in myelomonocytic cells through an NADPH oxidase-dependent pathway. Biochem J, 2005, 387(Pt2):531-540.
14.	Genescà M, Sola A, Hotter G, et al. Actin cytoskeleton derangement induces apoptosis in renal ischemia/reperfusion. Apoptosis, 2006, 11(4):563-571.
15.	Bugyi B, Carlier MF. Control of actin filament treadmilling in cell motility. Annu Rev Biophys, 2010, 39:449-470.
16.	Pollard TD, Blanchoin L, Mullins RD. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct, 2000, 29:545-576.
17.	Schwartz MA, DeSimone DW. Cell adhesion receptors in mechanotransduction. Curr Opin Cell Biol, 2008, 20(5):551-556.
18.	Schwartz MA. Integrins and extracellular matrix in mechanotransduction. Cold Spring Harb Perspect Biol, 2010, 2(12):a005066.
19.	Vogel V, Sheetz MP. Cell fate regulation by coupling mechanical cycles to biochemical signaling pathways. Curr Opin Cell Biol, 2009, 21(1):38-46.
20.	Titushkin I, Cho M. Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells. Biophys J, 2007, 93(10):3693-3702.
21.	Erickson GR, Gimble JM, Franklin DM, et al. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun, 2002, 290(2):763-769.
22.	McBeath R, Pirone DM, Nelson CM, et al. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell, 2004, 6(4):483-495.
23.	Himangshu Sonowal, Atul Kumar, Jina Bhattacharyya, et al. Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway. Journal of Biomedical Science, 2013, 20:71.
24.	马林, 唐康来, 周兵华, 等. 机械牵伸对体外大鼠肌腱干细胞RhoA/Rho相关蛋白激酶分子表达的影响. 中国修复重建外科杂志, 2014, 28(5):639-643.
25.	Teramura T, Takehara T, Onodera Y, et al. Mechanical stimulation of cyclic tensile induces reduction of pluripotent related gene expressions via activation of RhoA/ROCK and subsequent decreasing of AKT phosphorylation in human indiced pluripotent stem cells. Biochem Biophys Res Commun, 2012, 417(2):836-841.

1. Wang JC, Guo Q, Li B. Tendon biomechanics and mechano-biology-a minireview of basic concepts and recent advancements. J Hand Ther, 2012, 25(2):133-141.
2. Woo SL, Debski RE, Zeminski J, et al. Injury and repair of ligaments and tendons. Annu Rev Biomed Eng, 2000, 2:83-118.
3. Salingcarnboriboon R, Yoshitake H, Tsuji K, et al. Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property. Exp Cell Res, 2003, 287(2):289-300.
4. Bi Y, Ehirchiou D, Kilts TM, et al. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat Med, 2007, 13(10):1219-1227.
5. Yin Z, Chen X, Chen JL, et al. The regulation of tendon stem cell differentiation by the alignment of nanofibers. Biomaterials, 2010, 31(8):2163-2175.
6. Rui YF, Lui PP, Li G, et al. Isolation and characterization of muhipotent rat tendon-derived stem cells. Tissue Eng Part A, 2010, 16(5):1549-1558.
7. Zhang J, Wang JH. Mechanobiological response of tendon stem cells:implications of tendon homeostasis and pathogenesis of tendinopathy. J Orthop Res, 2010, 28(5):639-643.
8. Zhang J, Wang JH. Production of PGE(2) increases in tendons subjected to repetitive mechanical loading and induces differentiation of tendon stem cells into non-tenocytes. J Orthop Res, 2010, 28(2):198-203.
9. 邓银栓, 唐康来, 谢美明, 等. 不同牵伸载荷对体外培养人肌腱细胞纤维型肌动蛋白的影响. 中华医学杂志, 2011, 91(25):1780-1785.
10. 胡超, 唐康来, 陈万, 等. 大鼠跟腱来源肌腱干细胞的分离培养及鉴定. 第三军医大学学报, 2013, 35(11):1097-1101.
11. Sonowal H, Kumar A, Bhattacharyya J, et al. Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway. J Biomed Sci, 2013, 20:71.
12. Zeng LH, Xu L, Rensing NR, et al. Kainate seizures cause acute dendritic injury and actin depolymerization in vivo. J Neurosci, 2007, 27(43):11604-11613.
13. Kustermans G, El Benna J, Piette J, et al. Perturbation of actin dynamics induces NF-kappaB activation in myelomonocytic cells through an NADPH oxidase-dependent pathway. Biochem J, 2005, 387(Pt2):531-540.
14. Genescà M, Sola A, Hotter G, et al. Actin cytoskeleton derangement induces apoptosis in renal ischemia/reperfusion. Apoptosis, 2006, 11(4):563-571.
15. Bugyi B, Carlier MF. Control of actin filament treadmilling in cell motility. Annu Rev Biophys, 2010, 39:449-470.
16. Pollard TD, Blanchoin L, Mullins RD. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct, 2000, 29:545-576.
17. Schwartz MA, DeSimone DW. Cell adhesion receptors in mechanotransduction. Curr Opin Cell Biol, 2008, 20(5):551-556.
18. Schwartz MA. Integrins and extracellular matrix in mechanotransduction. Cold Spring Harb Perspect Biol, 2010, 2(12):a005066.
19. Vogel V, Sheetz MP. Cell fate regulation by coupling mechanical cycles to biochemical signaling pathways. Curr Opin Cell Biol, 2009, 21(1):38-46.
20. Titushkin I, Cho M. Modulation of cellular mechanics during osteogenic differentiation of human mesenchymal stem cells. Biophys J, 2007, 93(10):3693-3702.
21. Erickson GR, Gimble JM, Franklin DM, et al. Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun, 2002, 290(2):763-769.
22. McBeath R, Pirone DM, Nelson CM, et al. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell, 2004, 6(4):483-495.
23. Himangshu Sonowal, Atul Kumar, Jina Bhattacharyya, et al. Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway. Journal of Biomedical Science, 2013, 20:71.
24. 马林, 唐康来, 周兵华, 等. 机械牵伸对体外大鼠肌腱干细胞RhoA/Rho相关蛋白激酶分子表达的影响. 中国修复重建外科杂志, 2014, 28(5):639-643.
25. Teramura T, Takehara T, Onodera Y, et al. Mechanical stimulation of cyclic tensile induces reduction of pluripotent related gene expressions via activation of RhoA/ROCK and subsequent decreasing of AKT phosphorylation in human indiced pluripotent stem cells. Biochem Biophys Res Commun, 2012, 417(2):836-841.

Chinese Journal of Reparative and Reconstructive Surgery

INFLUENCE OF INHIBITION OF ACTIN POLYMERIZATION ON ADIPOGENIC DIFFERENTIATION OF RAT Achilles-DERIVED TENDON STEM CELLS IN VITRO

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