Role of NAD(P)H oxidase in transforming growth factor-[beta]1-induced monocyte chemoattractant protein-1 and interleukin-6 expression in rat renal tubular epithelial cells.
Zhang, Haiyan 1,*; Jiang, Zongpei 1,*; Chang, Jie 1; Li, Xiaoyan 1; Zhu, Hengmei 1; Lan, Hui Y 2; Zhou, Shu-Feng 3; Yu, Xueqing 1
[Miscellaneous Article]
Nephrology.
14(3):302-310, April 2009.
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Aim: This study investigated the role of NAD(P)H oxidase in transforming growth factor-[beta]1 (TGF-[beta]1)-induced reactive oxygen species (ROS) generation, monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) expression in rat renal tubular epithelial NRK-52E cells.
Methods: The cells were treated with 10 ng/mL TGF-[beta]1, either in the presence or absence of the NAD(P)H oxidase inhibitor, diphenyleneiodonium (DPI), or short hairpin RNA (shRNA) suppressing p67phox expression. Expression of NAD(P)H oxidase subunits, MCP-1, and IL-6 at the mRNA levels was detected by reverse transcription polymerase chain reaction, while expression of NAD(P)H oxidase subunit p67phox protein was analyzed by western blot and MCP-1 by enzyme-linked immunosorbent assay. The cellular ROS generation was visualized using 2',7'-dichlorodihydrofluorescein diacetate by confocal microscopy.
Results: Compared to control, TGF-[beta]1 upregulated NAD(P)H oxidase subunit p67phox mRNA by 3.59-fold (P < 0.01), but had no effect on p22phox, gp91phox and p47phox NAD(P)H subunits. TGF-[beta]1 was also able to significantly increase intracellular ROS (P < 0.05), MCP-1 (P < 0.01) and IL-6 (P < 0.05) expression in NRK-52E cells. Further studies showed that generation of ROS and upregulation of MCP-1 and IL-6 by TGF-[beta]1 were significantly blocked by addition of DPI or shRNA-p67phox (P < 0.01), suggesting that these effects were NAD(P)H oxidase-dependent.
Conclusion: TGF-[beta]1 differentially regulates the expression of NAD(P)H oxidase subunits and mediates MCP-1 and IL-6 expression in rat renal tubular cells via the NAD(P)H oxidase/p67phox-dependent mechanism.
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