Background: Connective tissue growth factor (CTGF, CCN2) plays a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and mediating many of the pro-fibrotic effects of transforming growth factor (TGF)-β. CCN2 induction by TGF-β in renal proximal tubule epithelial cells (PTECs) is likely to play an important role in the development of tubulointerstitial fibrosis. In this study, we investigated the induction of CCN2 by TGF-β1 and the possible mechanisms of this induction in human PTECs. Methods: Experiments were performed on primary and transformed (human kidney cell (HKC)-clone 8) human PTECs. Induction of CCN2 in response to TGF-β1 was studied at the gene promoter level by reporter gene assay, mRNA by semi-quantitative RT-PCR and protein by immunoblotting. While chemical inhibitors were used to assess the role of Ras/MEK/ERK1,2 signalling, an HKC cell line over-expressing Smad7 was used to assess the role of Smad signalling in induction of CCN2 by TGF-β1. Results: TGF-β1 induced CCN2 promoter activity, mRNA and protein in human PTECs. TGF-β1-dependent CCN2 promoter activity was reduced by inhibiting Ras and MEK activation. MEK inhibition also resulted in inhibition of the TGF-β1-induced secreted CCN2 protein. There was no significant increase in CCN2 gene promoter activity or protein by TGF-β1 in Smad7 over-expressing HKCs. Conclusions: TGF-β1 induces the expression of CCN2 in human PTECs. This induction is dependent on Ras/MEK/ERK and Smad signalling. Inhibiting TGF-β induced CCN2 by targeting Smad and/or Ras/MEK/ERK1,2 signalling pathways could be of therapeutic value in renal fibrosis.

Keywords:
Connective tissue growth factor, CCN2, Transforming growth factor-β, Ras/MEK/ERK, Smads, Signalling, Proximal tubule epithelial cells, Fibrosis
1.
Nath KA: Tubulointerstitial changes as a major determinant in the progression of renal damage. Am J Kidney Dis 1992;20:1–17.
2.
Crowe MJ, Doetschman T, Greenhalgh DG: Delayed wound healing in immunodeficient TGF beta knockout mice. J Invest Dermatol 2000;115:3–11.
3.
Border WA, Noble NA: TGF beta in tissue fibrosis. N Engl J Med 1994;331:1286–1292.
4.
Mori T, Kawara S, Shinozaki, M, Hayashi N, Kakinuma T, Igarashi A, Takigawa M, Nakanishi T, Takehara K: Role and interaction of CTGF with TGF-beta in persistent fibrosis: A mouse fibrosis model. J Cell Physiol 1999;181:153–159.
5.
Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, et al: Targeted disruption of the mouse TGF-beta1 gene results in multifocal inflammatory disease. Nature 1992;359:693–699.
6.
Grotendorst GR: CTGF: A mediator of TGFβ action on fibroblasts. Cytokine Growth Factor Rev 1997;8:171–179.
7.
Igarashi A, Okochi H, Bradham DM, Grotendorst GR: Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol Biol Cell 1993;4:637–645.
8.
Leask A, Abraham DJ: The Role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology. Biochem Cell Biol 2003;81:355–363.
9.
Perbal B: CCN proteins: Multifunctional signaling regulators. Lancet 2004;363:62–64.
10.
Bradham DM, Igarishi A, Potter RL, Grotendorst GR: Connective tissue growth factor: A cysteine rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol 1991;114:1285–1294.
11.
Moussad EE, Brigstock DR: CTGF: what’s in a name? Mol Genet Metab 2000;71:276–292.
12.
Rachfal AW, Brigstock DR: Connective tissue growth factor (CTGF/CCN2) in hepatic fibrosis. Hepatol Res 2003;26:1–9.
13.
Leask A, Denton C, Abraham DJ: Insights into the molecular mechanism of chronic fibrosis: The role of CTGF in scleroderma. J Invest Dermatol 2004;122:1–6.
14.
Frazier K, Williams S, Kothapalli D, Klapper H, Grotendorst GR: Stimulation of fibroblast cell growth, matrix production, and granulation tissue formation by connective tissue growth factor. J Invest Dermatol 1996;107:404–411.
15.
Abreu JG, Ketpura NI, Reversade B, De Robertis EM: CTGF modulates cell signaling by BMP and TGF beta. Nature Cell Biol 2002;4:599–603.
16.
Weston BS, Wahab NA, Mason RM: CTGF mediates TGF-beta-induced fibronectin matrix deposition by up regulating active alpha5beta1 integrin in human mesangial cells. J Am Soc Nephrol 2003;14:601–610.
17.
Yokoi H, Mukoyama M, Sugawara A, Mori K, Nagae T, Makino H, Suganami T, Yahata K, Fujinaga Y, Tanaka I, Nakao K: Role of connective tissue growth factor in fibronectin expression and tubulointerstitial fibrosis. Am J Physiol Renal Physiol 2002;282:933–942.
18.
Wahab NA, Yevdokimova N, Weston B, Roberts T, Li XJ, Brinkman H, Mason RM: Role of CTGF in the pathogenesis of diabetic nephropathy. Biochem J 2001;359:77–87.
19.
Ito Y, Aten J, Bende RJ, Oemar BS, Rabelink TJ, Weening JJ, Goldshmeding R: Expression of connective tissue growth factor in human renal fibrosis. Kidney Int 1998;53:853–861.
20.
Wang S, Denichilo M, Brubaker C, Hirschberg R: Connective tissue growth factor in tubulointerstitial injury of diabetic nephropathy. Kidney Int 2001;60:96–105.
21.
Chen Y, Blom I, Sa S, Goldschmeding R, Abraham DJ, Leask A: CTGF expression in mesangial cells: Involvement of SMADs, MAP kinase and PKC. Kidney Int 2002;62:1149–1159.
22.
Heusinger-Ribeiro J, Eberlein M, Wahab NA, Goppelt-Struebe M: Expression of connective tissue growth factor in human renal fibroblasts: Regulatory roles of RhoA and cAMP. J Am Soc Nephrol 2001;12:1853–1861.
23.
Leask A, Holmes A, Black C, Abraham DJ: Connective tissue growth factor gene regulation. Requirements for its induction by TGF beta2 in fibroblasts. J Biol Chem 2003;278:13008–13015.
24.
Phillips AO, Steadman R, Topley N, Williams JD: Elevated D-glucose concentrations modulate TGFβ1 synthesis by human cultured renal proximal tubule cells. The permissive role of platelet-derived growth factor. Am J Pathol 1995;147:362–374.
25.
Racusen LC, Monteil C, Sgrignoli A, Lucskay M, Marouillat S, Rhim JG, Morin JP: Cell lines with extended in vitro growth potential from human renal proximal tubule: Characterisation, response to inducers and comparison with established cell lines. J Lab Clin Med 1997;129:318–329.
26.
Dai C, Yang J, Liu Y: TGF-beta1 potentiates renal tubule epithelial cell death by a mechanism independent of Smad signaling. J Biol Chem 2003;278:12537–12545.
27.
Clarke HC, Kocher HM, Khwaja A, Kloog Y, Cook HT, Hendry BM: Ras antagonist farnesylthiosalicylic acid (FTS) reduces glomerular cellular proliferation and macrophage number in rat thy-1 nephritis. J Am Soc Nephrol 2003;14:848–854.
28.
Kloog Y, Cox AD, Sinensky M: Concepts in Ras-directed therapy. Expert Opin Investig Drugs 1999;8:2121–2140.
29.
Zhang C, Meng X, Zhu Z, Liu J, Deng A: Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro. Cell Biol Int 2004;28:863–873.
30.
Roberts AB, Derynck R: Meeting report: Signaling schemes for TGF beta. Sci STKE 2001;113:PE43.
31.
Mulder KM: Role of Ras and mapks in TGF beta signaling. Cytokine Growth Factor Rev 2000;11:23–35.
32.
Bakin AV, Tomlinson AK, Bhowmick NA, Moses HL, Arteaga CL: Phosphotidylinositol 3-kinase function is required for TGF beta mediated epithelial to mesenchymal transition and cell migration. J Biol Chem 2000;275:36803–36810.
33.
Funaba M, Zimmerman CM, Mathews LS: Modulation of Smad-2 mediated signaling by extracellular signal regulated kinase. J Biol Chem 2002;277:41361–41368.
34.
Roelen BA, Cohen OS, Raychowdhury MK, Chadee DN, Zhang Y, Kyriakis JM, Alessandrini AA, Lin HY: Phosphorylation of threonine 276 in Smad4 is involved in TGF beta induced nuclear accumulation. Am J Physiol Cell Physiol 2003;285:C823–C823.
35.
Yue J, Frey RS, Mulder KM: Cross talk between the Smad1 and Ras/MEK signaling pathways for TGF beta. Oncogene 1999;18:2033–2037.
36.
Kutz SM, Hordines J, McKeown-Longo PJ, Higgins PJ: TGF beta-1 induced PAI-1 gene expression requires MEK activity and cell-to substrate adhesion. J Cell Sci 2001;114:390–414.
37.
Suzuma K, Naruse K, Suzuma I, Takahara N, Ueki K, Aiello LP, King GL: Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-Akt-dependent pathways in retinal vascular cells. J Biol Chem 2000;275:40725–40731.
38.
Reif S, Weis S, Aeed H, Gana-Weis M, Zaidel L, Avni Y, Romanelli RG, Pinzani M, Kloog Y, Bruck R: The Ras antagonist, farnesylthiosalicylic acid (FTS), inhibits experimentally induced liver cirrhosis in rats. J Hepatol 1999;31:1053–1061.
39.
Lan HY, Mu W, Tomita N, Huang XR, Li JH, Zhu HJ, Morishita R, Johnson RJ: Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J Am Soc Nephrol 2003;14:1535–1548.
© 2005 S. Karger AG, Basel
2005
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