Transforming growth factor-beta(1) (TGF-beta(1)) functions as an important immunomodulatory cytokine in human kidney. Evidence suggests that connective tissue growth factor (CTGF) is an important downstream mediator of the profibrotic effects of TGF-beta(1). However, the role of CTGF in TGF-beta(1)-induced chemokine production remains unknown. This study was undertaken to determine whether CTGF is involved in mediating TGF-beta(1)-induced chemokine production in renal proximal tubular (HK-2) cells. Interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were measured. TGF-beta(1) induced an increase in IL-8 and MCP-1 (both P < 0.05) compared with control levels. CTGF was effectively silenced using small interference RNA (siRNA) in HK-2 cells. RT-PCR and real-time PCR confirmed a 94% reduction in CTGF mRNA. In the CTGF-silenced cells, TGF-beta(1)-stimulated IL-8 and MCP-1 secretion was not altered compared with control cells. Similarly, basal secretion of IL-8 and MCP-1 was not changed in CTGF-silenced cells. The direct effect of CTGF (20, 200, and 400 ng/ml) on IL-8 and MCP-1 was assessed at 24-, 48-, and 72-h time points and no stimulation was observed. Our studies further demonstrate that in the CTGF gene-silenced cells, CTGF partially mediates TGF-beta(1)-induced fibronectin and collagen IV secretion. These data suggest that TGF-beta(1) induced IL-8 and MCP-1 via CTGF-independent pathway. TGF-beta mediates both fibrosis and chemokine production in the proximal tubule of the kidney. However, CTGF plays a more specific role as a downstream mediator of TGF-beta(1)-induced fibrosis.
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