Reactive Oxygen Generated by NADPH Oxidase 1 (Nox1) Contributes to Cell Invasion by Regulating Matrix Metalloprotease-9 Production and Cell Migration

Masahiro Shinohara,Yoshifumi Adachi,Junji Mitsushita,Mitsuhiro Kuwabara,Atsushi Nagasawa,Saori Harada,Shuichi Furuta,Yugen Zhang,Kajla Seheli,Hitoshi Miyazaki,Tohru Kamata

doi:10.1074/jbc.m109.071779

Masahiro Shinohara, Yoshifumi Adachi + Show 9 more

Open Access

https://doi.org/10.1074/jbc.m109.071779

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Abstract

A mediating role of the reactive oxygen species-generating enzyme Nox1 has been suggested for Ras oncogene transformation phenotypes including anchorage-independent cell growth, augmented angiogenesis, and tumorigenesis. However, little is known about whether Nox1 signaling regulates cell invasiveness. Here, we report that the cell invasion activity was augmented in K-Ras-transformed normal rat kidney cells and attenuated by transfection of Nox1 small interference RNAs (siRNAs) into the cells. Diphenyleneiodonium (DPI) or Nox1 siRNAs blocked up-regulation of matrix metalloprotease-9 at both protein and mRNA levels in K-Ras-transformed normal rat kidney cells. Furthermore, DPI and Nox1 siRNAs inhibited the activation of IKKalpha kinase and the degradation of IkappaB alpha, suppressing the NFkappaB-dependent matrix metalloprotease-9 promoter activity. Additionally, epidermal growth factor-stimulated migration of CaCO-2 cells was abolished by DPI and Nox1 siRNAs, indicating the requirement of Nox1 activity for the motogenic effect of epidermal growth factor. This Nox1 action was mediated by down-regulation of the Rho activity through the low molecular weight protein-tyrosine phosphatase-p190RhoGAP-dependent mechanism. Taken together, our findings define a mediating role of Nox1-generated reactive oxygen species in cell invasion processes, most notably metalloprotease production and cell motile activity.

Highlights

The ability of tumors to invade the neighboring extracellular matrix is critical for the metastases, which is primarily accompanied by augmented matrix metalloprotease production and cell motility
We show here that Nox1-generated reactive oxygen species (ROS) mediates oncogenic Ras-induced matrix metalloproteases (MMPs)-9 production
Unlike the induction of vascular endothelial growth factor (VEGF), where the sole activation of the Nox1 system is insufficient [9], the Nox1 signal alone seems to recapitulate the ability of K-RasVal12 to induce the MMP-9 expression

Summary

Introduction

N-7 cells markedly reduced the level of MMP-9 and MMP-2, whereas Neg-1 cells maintained the activities, indicating the involvement of Nox1 in their expression (Fig. 2A). To determine whether Nox1 regulates the NF␬B-dependent transcriptional activity of the MMP-9 promoter in response to Ras activation, KNRK cells were treated with DPI after transfection with a pGL-MMP-9-670 reporter and subjected to luciferase activity assay. Because our previous study shows that the superoxide generation is increased due to up-regulation of Nox1 in KNRK cells [3], these data collectively indicate that ROS generated by Nox1 are required for NF␬B-mediated response of the MMP-9 expression to oncogenic activation of Ras. To understand whether up-regulation of the Nox1 activity alone can initiate transcription of MMP-9, NRK cells were co-transfected with Nox1, NOXO1, and NOXA1 and analyzed for the MMP-9 promoter activity assay.

Results

Conclusion