NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis
Ramón Bataller, … , John J. Lemasters, David A. Brenner
Ramón Bataller, … , John J. Lemasters, David A. Brenner
Published November 1, 2003
Citation Information: J Clin Invest. 2003;112(9):1383-1394. https://doi.org/10.1172/JCI18212.
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Article Hepatology

NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis

Abstract

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II–induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen α1(I) mRNA expression, and secretion of TGF-β1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox–/– mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox–/– mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle α-actin and expression of TGF-β1 were reduced in p47phox–/– mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

Authors

Ramón Bataller, Robert F. Schwabe, Youkyung H. Choi, Liu Yang, Yong Han Paik, Jeffrey Lindquist, Ting Qian, Robert Schoonhoven, Curt H. Hagedorn, John J. Lemasters, David A. Brenner

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Ang II stimulates intracellular signaling pathways in human HSCs in a re...
Ang II stimulates intracellular signaling pathways in human HSCs in a redox-sensitive manner. (a) Ang II stimulates phosphorylation of MAPK proteins and AKT in a redox-sensitive manner. HSCs preincubated with buffer, losartan (10–7 M), the AT2 receptor antagonist PD123319 (10–7 M), NAC (10–4 M), or DPI (10–6 M) were stimulated for 10 minutes with Ang II (10–8 M). Twenty-five micrograms of cell extracts were subjected to Western blotting using anti–phospho–ERK-2, anti–phospho–c-Jun, and anti–phospho–p38 MAPK antibodies. Anti–α-tubulin antibody was used to demonstrate equal protein loading. ERK-2, JNK, AKT, and p38 MAPK activities were assessed by specific kinase assays (see Methods). Cells were stimulated for 10 minutes with Ang II (10–8 M) in the presence or absence of losartan, PD123319, NAC, and DPI. (b) Ang II does not induce phosphorylation of STAT1 and STAT3. Cells were stimulated for 10 minutes with Ang II (10–8 M) or IFN-γ (100 U/ml), and 25 μg of cell extracts were subjected to Western blotting using anti–phospho-STAT1 and -STAT3 antibodies. Anti–α-tubulin antibody was used to demonstrate equal protein loading. (c) Ang II activates AP-1 DNA binding in human HSCs. Stimulation with Ang II (10–8 M) for 2 hours increased AP-1 binding, as assessed by electrophoretic mobility shift assay. This effect was prevented by losartan (10–7 M), NAC (10–5 M), and DPI (10–6 M), but not by PD123319 (10–7 M). Numbers underneath the gel represent fold expression compared with cells treated with buffer.