Connective tissue growth factor induces osteogenic differentiation of vascular smooth muscle cells through ERK signaling

Huang,Juan; Huang,Han; Wu,Min; Li,Jin; Xie,Hui; Zhou,Houde; Liao,Eryuan; Peng,Yiqun

doi:10.3892/ijmm.2013.1398

Connective tissue growth factor induces osteogenic differentiation of vascular smooth muscle cells through ERK signaling

Authors:
- Juan Huang
- Han Huang
- Min Wu
- Jin Li
- Hui Xie
- Houde Zhou
- Eryuan Liao
- Yiqun Peng
View Affiliations

Affiliations: Institute of Endocrinology and Metabolism, The Second Xiangya Hospital of Central South University, Changsha, Hunan, P.R. China, Central South University School of Public Health, Changsha, Hunan, P.R. China
Published online on: May 29, 2013 https://doi.org/10.3892/ijmm.2013.1398
Pages: 423-429

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of atherosclerosis by promoting vascular smooth muscle cell (VSMC) growth, migration, apoptosis, adhesion and the secretion of matrix components. The osteogenic differentiation of VSMCs is essential in the development of vascular calcification. However, the role of CTGF in the transdifferentiation and calcification of VSMCs is unclear. In the present study, we examined whether CTGF stimulates VSMC transdifferentiation. Primary VSMCs were obtained from mouse thoracic aortas by enzymatic digestion and identified by immunostaining for smooth muscle specific α-actin antibody (α-SMA). VSMC calcification was induced by the addition of CTGF to the osteogenic mediaum containing 5-10% FBS in the presence of 0.25 mM ascorbic acid and 10 mM β-glycerophosphate for 14 days. Calcified cells were determined by Alizarin Red S staining. Our results revealed that CTGF induced the expression of several bone markers, including alkaline phosphatase (ALP), osteocalcin (OC), osteoprotegerin (OPG) and core-binding factor subunit α1 (Cbfα1)/runt-related transcription factor 2 (Runx2), as well as calcification. However, the inhibition of extracellular signal-regulated kinase (ERK) activity using the ERK-specific inhibitor, PD98059, blocked the induction of these proteins and VSMC calcification. Based on these data, we conclude that CTGF stimulates the transdifferentiation of VSMCs into osteoblasts and that the ERK signaling pathway appears to play a critical role in this process.

View Figures

View References

1	London GM, Marchais SJ, Guerin AP and Metivier F: Arteriosclerosis, vascular calcifications and cardiovascular disease in uremia. Curr Opin Nephrol Hypertens. 14:525–531. 2005. View Article : Google Scholar : PubMed/NCBI
2	Mackenzie NC, Zhu D, Longley L, Patterson CS, Kommareddy S and MacRae VE: MOVAS-1 cell line: A new in vitro model of vascular calcification. Int J Mol Med. 27:663–668. 2011.PubMed/NCBI
3	Mikhaylova L, Malmquist J and Nurminskaya M: Regulation of in vitro vascular calcification by BMP4, VEGF and Wnt3a. Calcif Tissue Int. 81:372–381. 2007. View Article : Google Scholar : PubMed/NCBI
4	Otto CM, Kuusisto J, Reichenbach DD, Gown AM and O'Brien KD: Characterization of the early lesion of ‘degenerative’ valvular aortic stenosis. Histological and immunohistochemical studies. Circulation. 90:844–853. 1994.
5	Watson KE, Bostrom K, Ravindranath R, Lam T, Norton B and Demer LL: TGF-beta 1 and 25-hydroxycholesterol stimulate osteoblast-like vascular cells to calcify. J Clin Invest. 93:2106–2113. 1994. View Article : Google Scholar : PubMed/NCBI
6	Kurabayashi M: Differentiation of smooth muscle cells and vascular calcification. Clin Calcium. 20:1637–1644. 2010.(In Japanese).
7	Nakagami H, Osako MK and Morishita R: New concept of vascular calcification and metabolism. Curr Vasc Pharmacol. 9:124–127. 2011. View Article : Google Scholar : PubMed/NCBI
8	Parhami F, Morrow AD, Balucan J, et al: Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol. 17:680–687. 1997. View Article : Google Scholar
9	Shioi A, Katagi M, Okuno Y, et al: Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages. Circ Res. 91:9–16. 2002. View Article : Google Scholar : PubMed/NCBI
10	Duan XY, Xie PL, Ma YL and Tang SY: Omentin inhibits osteoblastic differentiation of calcifying vascular smooth muscle cells through the PI3K/Akt pathway. Amino Acids. 41:1223–1231. 2011. View Article : Google Scholar : PubMed/NCBI
11	Iyemere VP, Proudfoot D, Weissberg PL and Shanahan CM: Vascular smooth muscle cell phenotypic plasticity and the regulation of vascular calcification. J Intern Med. 260:192–210. 2006. View Article : Google Scholar : PubMed/NCBI
12	Yamaguchi A: Vascular calcification and bone-related factors. Clin Calcium. 12:1078–1083. 2002.(In Japanese).
13	Oemar BS, Werner A, Garnier JM, et al: Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation. 95:831–839. 1997. View Article : Google Scholar : PubMed/NCBI
14	Moussad EE and Brigstock DR: Connective tissue growth factor: what's in a name. Mol Genet Metab. 71:276–292. 2000. View Article : Google Scholar
15	Fan WH, Pech M and Karnovsky MJ: Connective tissue growth factor (CTGF) stimulates vascular smooth muscle cell growth and migration in vitro. Eur J Cell Biol. 79:915–923. 2000. View Article : Google Scholar : PubMed/NCBI
16	Hishikawa K, Oemar BS, Tanner FC, Nakaki T, Fujii T and Luscher TF: Overexpression of connective tissue growth factor gene induces apoptosis in human aortic smooth muscle cells. Circulation. 100:2108–2112. 1999. View Article : Google Scholar : PubMed/NCBI
17	Kanazawa S, Miyake T, Kakinuma T, Tanemoto K, Tsunoda T and Kikuchi K: The expression of platelet-derived growth factor and connective tissue growth factor in different types of abdominal aortic aneurysms. J Cardiovasc Surg (Torino). 46:271–278. 2005.PubMed/NCBI
18	Ding HT, Wang CG, Zhang TL and Wang K: Fibronectin enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells via ERK pathway. J Cell Biochem. 99:1343–1352. 2006. View Article : Google Scholar : PubMed/NCBI
19	Li J, Chai S, Tang C and Du J: Homocysteine potentiates calcification of cultured rat aortic smooth muscle cells. Life Sci. 74:451–461. 2003. View Article : Google Scholar : PubMed/NCBI
20	Liao XB, Zhou XM, Li JM, et al: Taurine inhibits osteoblastic differentiation of vascular smooth muscle cells via the ERK pathway. Amino Acids. 34:525–530. 2008. View Article : Google Scholar : PubMed/NCBI
21	Yang M, Huang H, Li J, Li D and Wang H: Tyrosine phosphorylation of the LDL receptor-related protein (LRP) and activation of the ERK pathway are required for connective tissue growth factor to potentiate myofibroblast differentiation. FASEB J. 18:1920–1921. 2004.
22	Huang HC, Yang M, Li JZ and Wang HY: Connective tissue growth factor promotes the proliferation of myofibroblast through Erk-1/2 signaling pathway. Zhonghua Yi Xue Za Zhi. 85:1322–1326. 2005.(In Chinese).
23	Johnson PR, Burgess JK, Ge Q, et al: Connective tissue growth factor induces extracellular matrix in asthmatic airway smooth muscle. Am J Respir Crit Care Med. 173:32–41. 2006. View Article : Google Scholar : PubMed/NCBI
24	Ponticos M, Holmes AM, Shi-wen X, et al: Pivotal role of connective tissue growth factor in lung fibrosis: MAPK-dependent transcriptional activation of type I collagen. Arthritis Rheum. 60:2142–2155. 2009. View Article : Google Scholar : PubMed/NCBI
25	Yosimichi G, Nakanishi T, Nishida T, Hattori T, Takano-Yamamoto T and Takigawa M: CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/42MAPK/extracellular-signal regulated kinase (ERK). Eur J Biochem. 268:6058–6065. 2001. View Article : Google Scholar
26	Ross R, Glomset J, Kariya B and Harker L: A platelet-dependent serum factor that stimulates the proliferation of arterial smooth muscle cells in vitro. Proc Natl Acad Sci USA. 71:1207–1210. 1974. View Article : Google Scholar : PubMed/NCBI
27	Wada T, McKee MD, Steitz S and Giachelli CM: Calcification of vascular smooth muscle cell cultures: inhibition by osteopontin. Circ Res. 84:166–178. 1999. View Article : Google Scholar : PubMed/NCBI
28	Alexander MY, Wilkinson FL, Kirton JP, et al: Identification and characterization of vascular calcification-associated factor, a novel gene upregulated during vascular calcification in vitro and in vivo. Arterioscler Thromb Vasc Biol. 25:1851–1857. 2005. View Article : Google Scholar : PubMed/NCBI
29	Shioi A: Molecular mechanisms of vascular calcification. Clin Calcium. 20:1611–1619. 2010.(In Japanese).
30	Sinha S, Eddington H and Kalra PA: Vascular calcification: lessons from scientific models. J Ren Care. 35(Suppl 1): 51–56. 2009. View Article : Google Scholar
31	Nakano-Kurimoto R, Ikeda K, Uraoka M, et al: Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition. Am J Physiol Heart Circ Physiol. 297:H1673–H1684. 2009. View Article : Google Scholar
32	Yamaai T, Nakanishi T, Asano M, et al: Gene expression of connective tissue growth factor (CTGF/CCN2) in calcifying tissues of normal and cbfa1-null mutant mice in late stage of embryonic development. J Bone Miner Metab. 23:280–288. 2005. View Article : Google Scholar : PubMed/NCBI
33	Wang JJ, Ye F, Cheng LJ, et al: Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor. J Zhejiang Univ Sci B. 10:355–367. 2009. View Article : Google Scholar : PubMed/NCBI
34	Game BA, He L, Jarido V, et al: Pioglitazone inhibits connective tissue growth factor expression in advanced atherosclerotic plaques in low-density lipoprotein receptor-deficient mice. Atherosclerosis. 192:85–91. 2007. View Article : Google Scholar
35	Steitz SA, Speer MY, Curinga G, et al: Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers. Circ Res. 89:1147–1154. 2001. View Article : Google Scholar : PubMed/NCBI
36	Johnson RC, Leopold JA and Loscalzo J: Vascular calcification: pathobiological mechanisms and clinical implications. Circ Res. 99:1044–1059. 2006. View Article : Google Scholar : PubMed/NCBI
37	Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL and Shanahan CM: Osteo/chondrocytic transcription factors and their target genes exhibit distinct patterns of expression in human arterial calcification. Arterioscler Thromb Vasc Biol. 23:489–494. 2003. View Article : Google Scholar
38	Byon CH, Javed A, Dai Q, et al: Oxidative stress induces vascular calcification through modulation of the osteogenic transcription factor Runx2 by AKT signaling. J Biol Chem. 283:15319–15327. 2008. View Article : Google Scholar
39	Liu Y and Shanahan CM: Signalling pathways and vascular calcification. Front Biosci. 16:1302–1314. 2011. View Article : Google Scholar : PubMed/NCBI
40	Son BK and Akishita M: Mechanism of vascular calcification. Clin Calcium. 17:319–324. 2007.(In Japanese).
41	Valdivielso JM: Vascular calcification: types and mechanisms. Nefrologia. 31:142–147. 2011.(In Spanish).
42	Gallop PM, Lian JB and Hauschka PV: Carboxylated calcium-binding proteins and vitamin K. N Engl J Med. 302:1460–1466. 1980. View Article : Google Scholar : PubMed/NCBI
43	Stinson RA and Hamilton BA: Human liver plasma membranes contain an enzyme activity that removes membrane anchor from alkaline phosphatase and converts it to a plasma-like form. Clin Biochem. 27:49–55. 1994. View Article : Google Scholar
44	Zhang J, Fu M, Myles D, et al: PDGF induces osteoprotegerin expression in vascular smooth muscle cells by multiple signal pathways. FEBS Lett. 521:180–184. 2002. View Article : Google Scholar : PubMed/NCBI
45	Bear M, Butcher M and Shaughnessy SG: Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells. J Cell Biochem. 105:185–193. 2008. View Article : Google Scholar : PubMed/NCBI
46	Nakahara T, Sato H, Shimizu T, et al: Fibroblast growth factor-2 induces osteogenic differentiation through a Runx2 activation in vascular smooth muscle cells. Biochem Biophys Res Commun. 394:243–248. 2010. View Article : Google Scholar : PubMed/NCBI
47	You H, Yang H, Zhu Q, et al: Advanced oxidation protein products induce vascular calcification by promoting osteoblastic trans-differentiation of smooth muscle cells via oxidative stress and ERK pathway. Ren Fail. 31:313–319. 2009. View Article : Google Scholar
48	Tanikawa T, Okada Y, Tanikawa R and Tanaka Y: Advanced glycation end products induce calcification of vascular smooth muscle cells through RAGE/p38 MAPK. J Vasc Res. 46:572–580. 2009. View Article : Google Scholar : PubMed/NCBI
49	Osako MK, Nakagami H, Koibuchi N, et al: Estrogen inhibits vascular calcification via vascular RANKL system: common mechanism of osteoporosis and vascular calcification. Circ Res. 107:466–475. 2010. View Article : Google Scholar : PubMed/NCBI
50	Peng Y, Wu M, Huang J, Xie H, Meng J and Liao E: 17-β estradiol down-regulates the expression of CTGF in vascular smooth muscle cells of mouse. In: The 10th National Endocrinology Conference of Chinese Medical Association; Suzhou, Jiangsu Province, P.R. China. 2011, (In Chinese).