CTGF Mediates TGF- –Induced Fibronectin Matrix Deposition by Upregulating Active 5 1 Integrin in Human Mesangial Cells

Excessive deposition of fibronectin in the glomerular mesangium in diabetic nephropathy (DN) is partly due to the induction of transforming growth factor(TGF) by high glucose. TGFinduces its downstream mediator connective tissue growth factor (CTGF), which stimulates fibronectin matrix synthesis, a process that requires the presence of 5 1 integrin. Although TGFhas been shown to upregulate 5 1 integrin expression in human mesangial cells (HMC), little is known about the effect of CTGF on levels of this receptor. This study tested whether CTGF modulates 5 1 expression by HMC in culture and whether changes induced by TGFare mediated through the induction of CTGF. FACS analysis showed that both TGFand CTGF significantly increased cell-surface 5 1 levels compared with basal conditions. RTPCR indicated that the changes were at the level of transcription. Treatment of cells with TGFand antisense CTGF oligonucleotides significantly reduced the TGF–induced increases in 5 1 levels. CTGF and TGFalso significantly increased levels of ligand-occupied cell-surface 1 integrins and cell adhesion to fibronectin, the main 5 1 substrate. Antisense CTGF significantly reduced the number of adherent cells from TGF–stimulated cultures. Finally, 5 1 blocking antibodies inhibited HMC fibronectin matrix deposition, confirming the importance of this receptor for this process. Taken together, these data provide evidence that CTGF controls 5 1 expression by HMC in vitro. Alterations in 5 1 levels induced by TGFare mediated at least in part through the induction of CTGF, and specific targeting of either 5 1 or CTGF could be useful in controlling excessive fibronectin matrix production in DN. Diabetic nephropathy (DN) is characterized by excessive deposition of extracellular matrix in the glomerular mesangium (1). Although hyperglycemia in diabetes is directly related to the severity of the disease (2), it is not entirely clear how high glucose concentration exerts its effects on mesangial cells. However many of the effects of high glucose are thought to be mediated through its induction of the growth factors, transforming growth factor(TGF) (3) and connective tissue growth factor (CTGF) (4–6). The level of both factors increase in the glomerulus in DN (3,6). Connective tissue growth factor (CTGF) is a 36to 38-kD cysteine-rich secreted protein (7). CTGF is encoded by an inducible immediate early gene and is one of six distinct members of the CCN family (8). The CTGF gene contains a novel TGFresponse element in its promoter region (9) and is thought to be a downstream mediator of the some of the effects of TGF(10). Several studies support this view (11– 14), but CTGF is also induced by a variety of other factors such as thrombin and VEGF (15,16). CTGF is thought to drive glomerulosclerosis and tubulointerstitial fibrosis in renal diseases (4). CTGF levels are elevated in different renal fibrotic disorders where excessive extracellular matrix formation is observed (11,17–19) and the growth factor induces matrix protein synthesis in mesangial cells in vitro (5,6,11,19). One of the key extracellular matrix proteins secreted by human mesangial cells (HMC) is fibronectin. Levels of fibronectin are elevated in DN (20), and fibronectin secretion by HMC is increased when cultured under high glucose conditions (21–23). Fibronectin is assembled into an insoluble matrix outside the cell, and distinct regions of the molecule are required for matrix assembly (24). One of the most important regions in the fibronectin monomer for this is the cell-binding domain that incorporates the ninth and tenth type III repeats, which contain the synergy sites and RGD integrin-binding consensus respectively (24). The predominant integrin receptor that binds fibronectin and is intimately involved in matrix assembly is the 5 1 heterodimer. The importance of this receptor for fibronectin matrix assembly has been shown previously using monoclonal antibodies to block its function (25,26). Levels of the 5 1 fibronectin receptor correlate directly with the degree of fibronectin matrix accumulation in renal disease (27,28), and it is known that the profibrotic factor Received August 9, 2002. Accepted November 13, 2002. Correspondence to Dr. Roger M. Mason, Cell and Molecular Biology Section, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, South Kensington, London SW7 2AZ, UK. Phone: 44-20-7594-43019; Fax: 44-20-7594-3015; E-mail [email protected]