Role of NAD(P)H oxidase in transforming growth factor-beta1-induced monocyte chemoattractant protein-1 and interleukin-6 expression in rat renal tubular epithelial cells.
OBJECTIVE
This study investigated the role of NAD(P)H oxidase in transforming growth factor-beta1 (TGF-beta1)-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-beta1, 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-beta1 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-beta1 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-beta1 were significantly blocked by addition of DPI or shRNA-p67phox (P < 0.01), suggesting that these effects were NAD(P)H oxidase-dependent.


CONCLUSIONS
TGF-beta1 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.
Zhang
Haiyan
H
Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.
Jiang
Zongpei
Z
Chang
Jie
J
Li
Xiaoyan
X
Zhu
Hengmei
H
Lan
Hui Y
HY
Zhou
Shu-Feng
SF
Yu
Xueqing
X
D000818:Animals; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics*; D007684:Kidney Tubules / Q000378:metabolism*; D019255:NADPH Oxidases / Q000502:physiology*; D010750:Phosphoproteins / Q000502:physiology; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
D018932:Chemokine CCL2; D015850:Interleukin-6; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; C079789:neutrophil cytosol factor 67K; D019255:NADPH Oxidases