| Abstrakt: |
Transforming growth factor-β1 (TGF-β1) is not only an important fibrogenic but also immunomodulatory cytokine in the human kidney. We have recently demonstrated that TGF-β1 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-β1 expression is unknown. The TGF-β1 gene was effectively silenced in HK-2 cells using small-interference (si) RNA. Basal secretion of IL-8 and MCP-1 decreased (both P < 0.05) but, paradoxically, fibronectin increased (P < 0.05) in TGF-β1-silenced cells compared with cells transfected with nonspecific siRNA. Significant increases were observed in mRNA for the TGF-β2 (P < 0.05), TGF-β3 (P < 0.05) isoforms and pSmad2 (P < 0.05), which were reflected in protein expression. Concurrent exposure to pan-specific TGF-β antibody reversed the observed increase in fibronectin expression, suggesting that TGF-β2 and TGF-β3 isoforms mediate the increased fibronectin expression in TGF-β1-silenced cells. An increase in the DNA binding activity of activator protein-1 (AP-1; P < 0.05) was also observed in TGF-β1-silenced cells. In contrast, nuclear factor-κB (NF-κB) DNA binding activity was significantly decreased (P < 0.0005). These studies demonstrate that TGF-β1 is a key regulator of IL-8 and MCP-1, whereas fibronectin expression is regulated by a complex interaction between the TGF-β isoforms in the HK-2 proximal tubular cell line. Decreased expression of TGF-β1 reduces chemokine production in association with reduced NF-κB DNA binding activity, suggesting that immunomodulatory pathways in the kidney are specifically dependent on TGF-β1. Conversely, decreased expression of TGF-β1 results in increased TGF-β2, TGF-β3, AP-1, and pSmad2 that potentially mediates the observed increase in fibronectin. |
