Transforming growth factor-1 is not only an important fibrogenic but also immunomodulatory cytokine in the human kidney. We have recently demonstrated that TGF-1 induces interleukin-8 (IL-8), macrophage chemoattractant protein-1 (MCP-1) and fibronectin production in renal proximal tubular (HK-2) cells. However, the unique dependence of IL-8, MCP-1 and fibronectin on TGF-1 expression is unknown. The TGF-1 gene was effectively silenced using siRNA. Basal secretion of IL-8 and MCP-1 decreased (both p<0.05) but paradoxically, fibronectin increased (p<0.05) in TGF-1 silenced cells compared to cells transfected with non-specific siRNA. Significant increases were observed in mRNA for the TGF-2 (p<0.05), TGF-3 (p<0.05) isoforms and pSmad2 (p<0.05) which were reflected in protein expression. Concurrent exposure to pan-specific TGF- antibody reversed the observed increase in fibronectin expression, suggesting that. TGF-2 and TGF-3 isoforms mediate the increased fibronectin expression in TGF-1 silenced cells. An increase in the DNA binding activity of activator protein-1 (AP-1; P<0.05) was also observed in TGF-1 silenced cells. In contrast, nuclear factor-kappaB (NF-B) DNA binding activity was significantly decreased (p<0.0005). These studies demonstrate that TGF-1 is a key regulator of IL-8, MCP-1 while fibronectin expression is regulated by a complex interaction between the TGF- isoforms in the HK-2 proximal tubular cell line. Decreased expression of TGF-1 reduces chemokine production in association with reduced NF-B DNA binding activity, suggesting that immunomodulatory pathways in the kidney are specifically dependent on TGF-1. Conversely, decreased expression of TGF-1 results in increased TGF-2, TGF-3, AP-1 and pSmad2, that potentially mediate the observed increase in fibronectin.