CCN3, a key matricellular protein, distinctly inhibits TGFÎ²1-mediated Smad1/5/8 signalling in human podocyte culture

Tarunkumar Hemraj Madne; Mark Edward Carl Dockrell

doi:10.14715/cmb/2018.64.3.2

Issue

Vol. 64 No. 3: Issue 3

Issue Published : March 3, 2018

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CCN3, a key matricellular protein, distinctly inhibits TGFÎ²1-mediated Smad1/5/8 signalling in human podocyte culture

https://doi.org/10.14715/cmb/2018.64.3.2

Tarunkumar Hemraj Madne

Southwest Thames Institute for Renal Research, Renal Unit, St Helier Hospital, Wrythe Lane, Carshalton, Surrey, SM5 1AA, St Georges, University of London. London, United Kingdom

Mark Edward Carl Dockrell

Southwest Thames Institute for Renal Research, Renal Unit, St Helier Hospital, Wrythe Lane, Carshalton, Surrey, SM5 1AA, St Georges, University of London. London, United Kingdom

Corresponding Author(s) : Tarunkumar Hemraj Madne

tarun2madne@yahoo.co.in

Cellular and Molecular Biology, Vol. 64 No. 3: Issue 3
Article Published : February 28, 2018

Abstract

Growth factors like TGFÎ² and CTGF (CCN2) plays a vital role in various cellular functions. TGFÎ² and CTGF are overexpressed in renal fibrosis. CTGF act as profibrotic stimuli to TGFÎ². CCN3 is a member of CCN family which also comprises CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3). CCN3 has been shown to antagonise CTGF. In this study, we investigated the role of CCN3 in TGFÎ²1-mediated signalling in human podocytes culture. This study describes the novel function of CCN3 in regulation of TGFÎ²1 mediated non-canonical Smad signalling in human podocytes culture. Experiments were conducted on conditionally immortalised human podocytes incubated with TGFÎ²1 (1.25ng/ml and 2.5ng/ml) and CCN3 (360ng/ml). Western blot study was performed to study signalling proteins. RT-PCR was performed to study alternative splicing of Fibronectin (Fn). Real time PCR was performed to look for gene expression of Fn and collagen IV and collagen I. TGFÎ²1 induced the Smad1/5/8, Smad3 and p38 phosphorylation and CCN3 downregulated the TGFÎ²1 induced Smad1/5/8 phosphorylation and did not affect Smad3 and p38 phosphorylation. In addition to this CCN3 induced alternative splicing of Extra domain A Fibronectin (EDA+Fn). CCN3 also induced collagen IV, Collagen I and Fn gene expression. This is the first evidence of downregulation of TGFÎ²-mediated activation of a Smad1/5/8 signalling pathway by CCN3 in human podocytes and in any cell type. Targeting CCN3-mediated events could provide exciting outcomes in the understanding of molecular mechanism of fibrosis.

Keywords

Podocyte TGFÎ²1 CCN3 CTGF Fibrosis.

Madne, T. H., & Dockrell, M. E. C. (2018). CCN3, a key matricellular protein, distinctly inhibits TGFÎ²1-mediated Smad1/5/8 signalling in human podocyte culture. Cellular and Molecular Biology, 64(3), 5–10. https://doi.org/10.14715/cmb/2018.64.3.2

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References

Riser BL, Najmabadi F, Perbal B, Peterson DR, Rambow JA, Riser ML, et al. CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease. Am J Pathol. 2009 May [cited 2012 Sep 27];174(5):1725–34.
Qi W, Twigg S, Chen X, Polhill TS, Poronnik P, Gilbert RE, et al. Integrated actions of transforming growth factor-beta1 and connective tissue growth factor in renal fibrosis. Am J Physiol Renal Physiol. 2005 Apr [cited 2013 Jan 8];288(4):F800-9.
Riser BL, Denichilo M, Cortes P, Baker C, Grondin JM, Yee J, et al. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol. 2000 Jan;11(1):25–38.
Nakerakanti SS, Bujor AM, Trojanowska M. CCN2 is required for the TGF-Î² induced activation of Smad1-Erk1/2 signaling network. PLoS One. 2011 Jan [cited 2013 Jan 23];6(7):e21911.
Weston BS. CTGF Mediates TGF- -Induced Fibronectin Matrix Deposition by Upregulating Active 5 1 Integrin in Human Mesangial Cells. J Am Soc Nephrol. 2003 Mar 1 [cited 2012 Nov 14];14(3):601–10.
Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J. 2004 May [cited 2012 Jul 31];18(7):816–27.
Turk T, Leeuwis JW, Gray J, Torti S V, Lyons KM, Nguyen TQ, et al. BMP signaling and podocyte markers are decreased in human diabetic nephropathy in association with CTGF overexpression. J Histochem Cytochem. 2009 Jul [cited 2013 Jan 25];57(7):623–31.
Lin CG, Leu S, Chen N, Tebeau CM, Lin S, Yeung C, et al. CCN3 ( NOV ) Is a Novel Angiogenic Regulator of the CCN Protein Family. J Biological Chemistry. 2003;278(26):24200–8.
Bark P. The modular architecture of a new family of growth regulators tissue growth factor. FEBS 1993;327(2):125–30.
Herman-Edelstein M, Thomas MC, Thallas-Bonke V, Saleem M, Cooper ME, Kantharidis P. Dedifferentiation of immortalized human podocytes in response to transforming growth factor-Î²: a model for diabetic podocytopathy. Diabetes. 2011 Jun [cited 2012 Sep 27];60(6):1779–88.
Li Y, Kang YS, Dai C, Kiss LP, Wen X, Liu Y. Epithelial-to-mesenchymal transition is a potential pathway leading to podocyte dysfunction and proteinuria. Am J Pathol. 2008 Feb [cited 2014 Oct 28];172(2):299–308.
Bottinger EP. TGF- Signaling in Renal Disease. J Am Soc Nephrol. 2002 Oct 1 [cited 2013 Jan 3];13(10):2600–10.
Whitman M. Smads and early developmental signaling by the TGFbeta superfamily. Genes Dev. 1998 Aug 15 [cited 2012 Aug 30];12(16):2445–62.
Goumans MJ, Valdimarsdottir G, Itoh S, Lebrin F, Larsson J, Mummery C, et al. Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling. Mol Cell. 2003 Oct [cited 2014 Oct 28];12(4):817–28.
Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K. Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells. 2002 Dec [cited 2014 Dec 29];7(12):1191–204.
Lan HY. Diverse roles of TGF-Î²/Smads in renal fibrosis and inflammation. Int J Biol Sci. 2011 Jan;7(7):1056–67.
Banas MC, Parks WT, Hudkins KL, Banas B, Holdren M, Iyoda M, et al. Localization of TGF-beta signaling intermediates Smad2, 3, 4, and 7 in developing and mature human and mouse kidney. J Histochem Cytochem. 2007 Mar [cited 2012 Sep 11];55(3):275–85.
Minamizato T Sakamoto K Liu T Kokubo H Katsube K Perbal B Nakamura S Yamaguchi A. CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways. Biochem Biophys Res Commun. 2007;354(2):567–573.

References

Riser BL, Najmabadi F, Perbal B, Peterson DR, Rambow JA, Riser ML, et al. CCN3 (NOV) is a negative regulator of CCN2 (CTGF) and a novel endogenous inhibitor of the fibrotic pathway in an in vitro model of renal disease. Am J Pathol. 2009 May [cited 2012 Sep 27];174(5):1725–34.

Qi W, Twigg S, Chen X, Polhill TS, Poronnik P, Gilbert RE, et al. Integrated actions of transforming growth factor-beta1 and connective tissue growth factor in renal fibrosis. Am J Physiol Renal Physiol. 2005 Apr [cited 2013 Jan 8];288(4):F800-9.

Riser BL, Denichilo M, Cortes P, Baker C, Grondin JM, Yee J, et al. Regulation of connective tissue growth factor activity in cultured rat mesangial cells and its expression in experimental diabetic glomerulosclerosis. J Am Soc Nephrol. 2000 Jan;11(1):25–38.

Nakerakanti SS, Bujor AM, Trojanowska M. CCN2 is required for the TGF-Î² induced activation of Smad1-Erk1/2 signaling network. PLoS One. 2011 Jan [cited 2013 Jan 23];6(7):e21911.

Weston BS. CTGF Mediates TGF- -Induced Fibronectin Matrix Deposition by Upregulating Active 5 1 Integrin in Human Mesangial Cells. J Am Soc Nephrol. 2003 Mar 1 [cited 2012 Nov 14];14(3):601–10.

Leask A, Abraham DJ. TGF-beta signaling and the fibrotic response. FASEB J. 2004 May [cited 2012 Jul 31];18(7):816–27.

Turk T, Leeuwis JW, Gray J, Torti S V, Lyons KM, Nguyen TQ, et al. BMP signaling and podocyte markers are decreased in human diabetic nephropathy in association with CTGF overexpression. J Histochem Cytochem. 2009 Jul [cited 2013 Jan 25];57(7):623–31.

Lin CG, Leu S, Chen N, Tebeau CM, Lin S, Yeung C, et al. CCN3 ( NOV ) Is a Novel Angiogenic Regulator of the CCN Protein Family. J Biological Chemistry. 2003;278(26):24200–8.

Bark P. The modular architecture of a new family of growth regulators tissue growth factor. FEBS 1993;327(2):125–30.

Herman-Edelstein M, Thomas MC, Thallas-Bonke V, Saleem M, Cooper ME, Kantharidis P. Dedifferentiation of immortalized human podocytes in response to transforming growth factor-Î²: a model for diabetic podocytopathy. Diabetes. 2011 Jun [cited 2012 Sep 27];60(6):1779–88.

Li Y, Kang YS, Dai C, Kiss LP, Wen X, Liu Y. Epithelial-to-mesenchymal transition is a potential pathway leading to podocyte dysfunction and proteinuria. Am J Pathol. 2008 Feb [cited 2014 Oct 28];172(2):299–308.

Bottinger EP. TGF- Signaling in Renal Disease. J Am Soc Nephrol. 2002 Oct 1 [cited 2013 Jan 3];13(10):2600–10.

Whitman M. Smads and early developmental signaling by the TGFbeta superfamily. Genes Dev. 1998 Aug 15 [cited 2012 Aug 30];12(16):2445–62.

Goumans MJ, Valdimarsdottir G, Itoh S, Lebrin F, Larsson J, Mummery C, et al. Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling. Mol Cell. 2003 Oct [cited 2014 Oct 28];12(4):817–28.

Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K. Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells. 2002 Dec [cited 2014 Dec 29];7(12):1191–204.

Lan HY. Diverse roles of TGF-Î²/Smads in renal fibrosis and inflammation. Int J Biol Sci. 2011 Jan;7(7):1056–67.

Banas MC, Parks WT, Hudkins KL, Banas B, Holdren M, Iyoda M, et al. Localization of TGF-beta signaling intermediates Smad2, 3, 4, and 7 in developing and mature human and mouse kidney. J Histochem Cytochem. 2007 Mar [cited 2012 Sep 11];55(3):275–85.

Minamizato T Sakamoto K Liu T Kokubo H Katsube K Perbal B Nakamura S Yamaguchi A. CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways. Biochem Biophys Res Commun. 2007;354(2):567–573.

Author biographies is not available.