Original Contribution
Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells

https://doi.org/10.1016/j.freeradbiomed.2008.04.024Get rights and content

Abstract

The proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have been shown to participate in signaling cascades regulating proliferation induced by platelet-derived growth factor (PDGF), a powerful smooth muscle mitogen. We sought to determine the role of Nox5 in the regulation of PDGF-stimulated human aortic smooth muscle cell (HASMC) proliferation. Cultured HASMC were found to express four isoforms of Nox5. When HASMC stimulated with PDGF were pretreated with N-acetyl cysteine (NAC), proliferation was significantly reduced. Proliferation induced by PDGF was also heavily dependent on JAK/STAT activation, as the JAK inhibitor, AG490, was able to completely abolish PDGF-stimulated HASMC growth. Specific knockdown of Nox5 with a siRNA strategy reduced PDGF-induced HASMC ROS production and proliferation. Additionally, siRNA to Nox5 inhibited PDGF-stimulated JAK2 and STAT3 phosphorylation. ROS produced by Nox5 play an important role in PDGF-induced JAK/STAT activation and HASMC proliferation.

Section snippets

Materials

PDGF-BB was purchased from R&D Systems, Inc. (Minneapolis, MN), and N-acetylcysteine (NAC) was from Sigma-Aldrich (St. Louis, MO). Non-silencing siRNA and siRNA to Nox5 (siNox5, which targets all Nox5 isoforms) were purchased from Ambion (Austin, TX). Phospho-JAK2 (Tyr1007/Tyr1008) antibody was from Santa Cruz Biotechnologies (Santa Cruz, CA), phospho-STAT3 (Tyr705) and STAT3 antibodies were from Cell Signaling Technology, Inc (Danvers, MA), and anti-JAK2 antibody was from Millipore (Billerica,

HASMCs predominantly express Nox4 and all isoforms of Nox5

Real time PCR with primers to Nox family members revealed that proliferating HASMCs express Nox4 and Nox5, with Nox4 mRNA more abundant than Nox5 mRNA (23,895 ± 7,150 vs. 395 ± 56 copies per 108 18S). Using primers to Nox1 and Nox2, we were able to measure copy numbers as low as 10 per reaction, but detected minimal Nox1 and no Nox2 mRNA in HASMCs. Five variants of Nox5 have been reported: α, β, γ, δ, and a short form that is missing exon 6 [24], [31], [32]. We used reverse transcriptase PCR using

Discussion

The NADPH oxidases have emerged as important elements of signaling pathways in VSMCs. There is ample evidence that more than one Nox exists in this cell type and that the different homologues are located in different compartments [34] and serve different functions. In rat VSMCs, Nox1 appears to be required for angiotensin II- and PDGF-induced ROS production and growth [22], and Nox4 has been shown to be important for the maintenance of a contractile, non-proliferative phenotype [23]. However,

Acknowledgments

This work was supported by NIH grant HL074604 to DBJ and HL058863 to KKG. We thank Dr. Peter Sayeski for his advice on the JAK/STAT experiments.

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