November 4, 2007
American Society of Nephrology (ASN) Renal Week 2007, San Francisco, CA
Abstract SU-PO191

A Targeted cDNA Microarray Identifies Cytoskeletal Regulatory Proteins as Transcriptional Targets of Connective Tissue Growth Factor (CTGF)/CCN2: Implications for Diabetic Nephropathy. M Browne¹, A Gaffney², C Godson², F Martin¹, J Crean¹.

1 Diabetes Research Centre, UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin Ireland; 2 Diabetes Research Centre, UCD School of Medicine and Medical Science, University College Dublin, Ireland.

Abstract: Hyperglycaemia-induced increases in glomerular mesangial extracellular matrix production and actin cytoskeleton rearrangement are key pathological hallmarks of diabetic nephropathy. Previously, we have described the use of a Suppressive Subtractive Hybridisation (SSH) screen to identify mRNA transcripts, which are differentially expressed in human glomerular mesangial cells (HMCs) propagated in vitro under conditions of either normal (5mM) or high (30mM) physiological glucose. In this study, we have used focused cDNA microarray technology to rapidly characterise the expression profile of genes derived from this SSH in HMCs in response to a key, profibrotic mediator of diabetic nephropathy, Connective Tissue Growth Factor (CTGF/CCN2). From the SSH screen, 171 distinct clones were amplified via PCR and arrayed onto glass slides. Analysis of the focused microarrays investigating HMCs stimulated with CTGF revealed induction of 10 distinct transcripts. CTGF was observed to induce the expression of the actin/myosin-binding protein caldesmon, the myosin regulatory chain and T-plastin alongside Arp-3 and fibronectin. In addition, CTGF caused a down-regulation of tubulin alpha-3 and the F-actin capping protein. These data suggest that CTGF activates an actin binding and regulatory protein cluster, representing a previously undescribed genetic programme which likely contributes to mesangial cell dysfunction in DN. When HMCs were stimulated with CTGF and stained for tubulin and F-actin, widespread microtubular and actin rearrangement was apparent. This was accompanied by a polarized redistribution of myosin, suggesting activation of the machinery of cell migration. Redistribution of myosin was facilitated by dephosphorylation of the myosin light and heavy chains in response to CTGF, which was inhibited by the addition of the myosin inhibitor 2,3 butanedionemonoxime. Increased expression of Arp-3 in response to CTGF was associated with cdc42 dependent PAK-1 phosphorylation. This data indicates that CCN2-mediated actin rearrangement likely contributes to the pathophysiology of the glomerular mesangium in diabetic nephropathy.

See also November 7, 2007 press release