Tougu Xiaotong formula induces chondrogenic differentiation in association with transforming growth factor-β1 and promotes proliferation in bone marrow stromal cells

Chen,Jiashou; Liu,Guozhong; Weng,Xiaping; Liu,Fayuan; Lin,Pingdong; Li,Huiting; Chen,Wenlie; Huang,Yunmei; Liu,Xianxiang; Ye,Hongzhi; Li,Xihai

doi:10.3892/ijmm.2014.2049

Tougu Xiaotong formula induces chondrogenic differentiation in association with transforming growth factor-β1 and promotes proliferation in bone marrow stromal cells

Authors:
- Jiashou Chen
- Guozhong Liu
- Xiaping Weng
- Fayuan Liu
- Pingdong Lin
- Huiting Li
- Wenlie Chen
- Yunmei Huang
- Xianxiang Liu
- Hongzhi Ye
- Xihai Li
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Affiliations: College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian 350005, P.R. China, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China, Fujian Key Laboratory of Integrative Medicine on Geriatrics, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China
Published online on: December 24, 2014 https://doi.org/10.3892/ijmm.2014.2049
Pages: 747-754

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Abstract

Indian hedgehog (Ihh), one of the hedgehog gene families, is indicated in the regulation of chondrocyte differentiation. Tougu Xiaotong formula (TXF), a traditional Chinese medicinal compound, has been used for the treatment of bone and joint disease. However, the underlying molecular mechanisms of TXF on the function of bone marrow stromal cells (BMSCs) remain unclear. In the present study, the affect of TXF on proliferation and chondrogenic differentiation was investigated in primary BMSCs from four‑week‑old Sprague Dawley rats. The cell viability in BMSCs treated with TXF was higher compared to the untreated cells. Additionally, the percentage of G0/G1 phase cells was significantly decreased, whereas that of the S phase cells was significantly increased. Furthermore, following TXF treatment, cyclin D1, cyclin‑dependent kinase 4 (CDK4) and CDK6 expression in BMSCs was significantly enhanced. The results showed that TXF had no cytotoxicity to BMSCs. To explore the effect of TXF on the differentiation in BMSCs, whether TXF induced chondrogenic differentiation of BMSCs by the regulation of Ihh signaling pathway was investigated. The protein expression of Ihh, Patched and Smoothened in the induction group were significantly increased when compared to those in the control group, and the highest protein level of Ihh was in the induction group that was treated with the combination of TXF and transforming growth factor‑β1 (TGF‑β1). In addition, TXF combined with TGF‑β1 significantly induced the protein expression of cartilage oligomeric matrix protein and collagen II compared to the TGF‑β1 group. Taken together, these results indicate that TXF promotes the proliferation via accelerating the G1/S transition, and induces chondrogenic differentiation in BMSCs by activation of the Ihh signaling pathway in association with TGF‑β1.

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1	Pittenger MF, Mackay AM, Beck SC, et al: Multilineage potential of adult human mesenchymal stem cells. Science. 284:143–147. 1999. View Article : Google Scholar : PubMed/NCBI
2	Charbord P: Bone marrow mesenchymal stem cells: historical overview and concepts. Hum Gene Ther. 21:1045–1056. 2010. View Article : Google Scholar : PubMed/NCBI
3	Pittenger MF and Martin BJ: Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res. 95:9–20. 2004. View Article : Google Scholar : PubMed/NCBI
4	Dominici M, Le Blanc K, Mueller I, et al: Minimal criteria for defining multipotent mesenchymal stromal cells The International Society for Cellular Therapy position statement. Cytotherapy. 8:315–317. 2006. View Article : Google Scholar
5	Li WJ, Tuli R, Okafor C, et al: A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials. 26:599–609. 2005. View Article : Google Scholar
6	Ochi M, Uchio Y, Tobita M and Kuriwaka M: Current concepts in tissue engineering technique for repair of cartilage defect. Artif Organs. 25:172–179. 2001. View Article : Google Scholar : PubMed/NCBI
7	Li X, Wei G, Wang X, et al: Targeting of the Sonic Hedgehog pathway by atractylenolides promotes chondrogenic differentiation of mesenchymal stem cells. Biol Pharm Bull. 35:1328–1335. 2012. View Article : Google Scholar : PubMed/NCBI
8	Lin J, Xiu Z and Wu Z: Effect of pilose antler polypeptides on the apoptosis of rabbit marrow mesenchymal stem cells differentiated into chondrogenic phenotype in vitro. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 20:427–430. 2006.(In Chinese). PubMed/NCBI
9	Huh JE, Park YC, Seo BK, et al: Cartilage protective and chondrogenic capacity of WIN-34B, a new herbal agent, in the collagenase-induced osteoarthritis rabbit model and in progenitor cells from subchondral none. Evid Based Complement Alternat Med. 2013:5275612013. View Article : Google Scholar
10	Mak KK, Kronenberg HM, Chuang PT, Mackem S and Yang Y: Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy. Development. 135:1947–1956. 2008. View Article : Google Scholar : PubMed/NCBI
11	St-Jacques B, Hammerschmidt M and McMahon AP: Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev. 13:2072–2086. 1999. View Article : Google Scholar : PubMed/NCBI
12	Wu Q, Zhang Y and Chen Q: Indian hedgehog is an essential component of mechanotransduction complex to stimulate chondrocyte proliferation. J Biol Chem. 276:35290–35296. 2001. View Article : Google Scholar : PubMed/NCBI
13	Hooper JE and Scott MP: The Drosophila patched gene encodes a putative membrane protein required for segmental patterning. Cell. 59:751–765. 1989. View Article : Google Scholar : PubMed/NCBI
14	Nakano Y, Guerrero I, Hidalgo A, Taylor A, Whittle JR and Ingham PW: A protein with several possible membranespanning domains encoded by the Drosophila segment polarity gene patched. Nature. 341:508–513. 1989. View Article : Google Scholar : PubMed/NCBI
15	Taipale J, Cooper MK, Maiti T and Beachy PA: Patched acts catalytically to suppress the activity of Smoothened. Nature. 418:892–897. 2002. View Article : Google Scholar : PubMed/NCBI
16	Li X, Lang W, Ye H, et al: Tougu Xiaotong capsule inhibits the tidemark replication and cartilage degradation of papain-induced osteoarthritis by the regulation of chondrocyte autophagy. Int J Mol Med. 31:1349–1356. 2013.PubMed/NCBI
17	Li XH, Wu MX, Ye HZ, et al: Experimental study on the suppression of sodium nitroprussiate-induced chondrocyte apoptosis by Tougu Xiaotong Capsule-containing serum. Chin J Integr Med. 17:436–443. 2011. View Article : Google Scholar : PubMed/NCBI
18	Zhang M, Xie R, Hou W, et al: PTHrP prevents chondrocyte premature hypertrophy by inducing cyclin-D1-dependent Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradation. J Cell Sci. 122:1382–1389. 2009. View Article : Google Scholar : PubMed/NCBI
19	Hwang SG, Song SM, Kim JR, Park CS, Song WK and Chun JS: Regulation of type II collagen expression by cyclin-dependent kinase 6, cyclin D1, and p21 in articular chondrocytes. IUBMB Life. 59:90–98. 2007. View Article : Google Scholar : PubMed/NCBI
20	Neuhuber B, Swanger SA, Howard L, Howard L, Mackay A and Fischer I: Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol. 36:1176–1185. 2008. View Article : Google Scholar : PubMed/NCBI
21	Chen ZZ, Van Bockstaele DR, Buyssens N, et al: Stromal populations and fibrosis in human long-term bone marrow cultures. Leukemia. 5:772–781. 1991.PubMed/NCBI
22	Nurse P: Ordering S phase and M phase in the cell cycle. Cell. 79:547–550. 1994. View Article : Google Scholar : PubMed/NCBI
23	Shen R, Wang X, Drissi H, Liu F, O’Keefe RJ and Chen D: Cyclin D1-cdk4 induce runx2 ubiquitination and degradation. J Biol Chem. 281:16347–16353. 2006. View Article : Google Scholar : PubMed/NCBI
24	Pufe T, Petersen W, Fändrich F, et al: Programmable cells of monocytic origin (PCMO): a source of peripheral blood stem cells that generate collagen type II-producing chondrocytes. J Orthop Res. 26:304–313. 2008. View Article : Google Scholar
25	Oldberg A, Antonsson P, Lindblom K and Heinegård D: COMP (cartilage oligomeric matrix protein) is structurally related to the thrombospondins. J Biol Chem. 267:22346–22350. 1992.PubMed/NCBI
26	Horie M, Choi H, Lee RH, et al: Intra-articular injection of human mesenchymal stem cells (MSCs) promote rat meniscal regeneration by being activated to express Indian hedgehog that enhances expression of type II collagen. Osteoarthritis Cartilage. 20:1197–1207. 2012. View Article : Google Scholar : PubMed/NCBI
27	Hettrich CM, Crawford D and Rodeo SA: Cartilage repair: third-generation cell-based technologies - basic science, surgical techniques, clinical outcomes. Sports Med Arthrosc. 16:230–235. 2008. View Article : Google Scholar : PubMed/NCBI
28	Beier F: Cell-cycle control and the cartilage growth plate. J Cell Physiol. 202:1–8. 2005. View Article : Google Scholar
29	Ito H, Rucker E, Steplewski A, et al: Guilty by association: some collagen II mutants alter the formation of ECM as a result of atypical interaction with fibronectin. J Mol Biol. 352:382–395. 2005. View Article : Google Scholar : PubMed/NCBI
30	Agarwal P, Schulz JN, Blumbach K, et al: Enhanced deposition of cartilage oligomeric matrix protein is a common feature in fibrotic skin pathologies. Matrix Biol. 32:325–331. 2013. View Article : Google Scholar : PubMed/NCBI
31	Miura Y, Parvizi J, Fitzsimmons JS and O’Driscoll SW: Brief exposure to high-dose transforming growth factor-beta1 enhances periosteal chondrogenesis in vitro: a preliminary report. J Bone Joint Surg Am. 84-A:793–799. 2002.PubMed/NCBI
32	McMahon AP, Ingham PW and Tabin CJ: Developmental roles and clinical significance of hedgehog signaling. Curr Top Dev Biol. 53:1–114. 2003. View Article : Google Scholar : PubMed/NCBI
33	Choi SW, Jeong DU, Kim JA, et al: Indian Hedgehog signalling triggers Nkx3.2 protein degradation during chondrocyte maturation. Biochem J. 443:789–798. 2012. View Article : Google Scholar : PubMed/NCBI
34	Enomoto-Iwamoto M, Nakamura T, Aikawa T, et al: Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation. J Bone Miner Res. 15:1659–1668. 2000. View Article : Google Scholar : PubMed/NCBI
35	Bruce SJ, Butterfield NC, Metzis V, Town L, McGlinn E and Wicking C: Inactivation of Patched1 in the mouse limb has novel inhibitory effects on the chondrogenic program. J Biol Chem. 285:27967–27981. 2010. View Article : Google Scholar : PubMed/NCBI