Nox4 involvement in TGF-beta and SMAD3-driven induction of the epithelial-to-mesenchymal transition and migration of breast epithelial cells

Abstract

The epithelial-to-mesenchymal transition (EMT) is the development of increased cell plasticity that occurs normally during wound healing and embryonic development and can be coopted for cancer invasion and metastasis. TGF-beta induces EMT but the mechanism is unclear. Our studies suggest that Nox4, a member of the NADPH oxidase (Nox) family, is a source of reactive oxygen species (ROS) affecting cell migration and fibronectin expression, an EMT marker, in normal and metastatic breast epithelial cells. We found that TGF-beta induces Nox4 expression (mRNA and protein) and ROS generation in normal (MCF10A) and metastatic (MDA-MB-231) human breast epithelial cells. Conversely, cells expressing a dominant-negative form of Nox4 or Nox4-targeted shRNA showed significantly lower ROS production on TGF-beta treatment. Expression of a constitutively active TGF-beta receptor type I significantly increased Nox4 promoter activity, mRNA and protein expression, and ROS generation. Nox4 transcriptional regulation by TGF-beta was SMAD3 dependent based on the effect of constitutively active SMAD3 increasing Nox4 promoter activity, whereas dominant-negative SMAD3 or SIS3, a SMAD3-specific inhibitor, had the opposite effect. Furthermore, Nox4 knockdown, dominant-negative Nox4 or SMAD3, or SIS3 blunted TGF-beta induced wound healing and cell migration, whereas cell proliferation was not affected. Our experiments further indicate that Nox4 plays a role in TGF-beta regulation of fibronectin mRNA expression, based on the effects of dominant-negative Nox4 in reducing fibronectin mRNA in TGF-beta-treated MDA-MB-231and MCF10A cells. Collectively, these data indicate that Nox4 contributes to NADPH oxidase-dependent ROS production that may be critical for the progression of the EMT in breast epithelial cells, and thereby has therapeutic implications.

Introduction

Recent observations suggest that reactive oxygen species (ROS) play important roles in the TGF-beta-induced epithelial-to-mesenchymal transition (EMT) and cell mobility of many cell types; however, little is known about redox-dependent TGF-beta-induced mechanisms in breast epithelial cells [1], [2]. EMT is a common process during embryonic development, wound healing, and organ fibrosis in which epithelial cells undergo morphological changes resulting in increased cell plasticity and mobility as they transition into a mesenchymal-like cell phenotype. Recently, the EMT has been a focus of tumor cell migration and invasion. Hallmarks of cells undergoing EMT include cytoskeletal reorganization, loss of polarization, disruption of cell–cell junctions, and alterations in ECM production. Tumor microenvironments secrete soluble growth factors, inflammatory cytokines (TGF-beta, HGF, VEGF, PDGF), and matrix proteins that activate epithelial cells to induce specific EMT transcriptional programs. TGF-beta is established as a contributor to EMT progression. Smooth muscle actin (SMA) and fibronectin expression are established indicators of EMT progression, both of which are modulated in a redox-dependent manner [2], [3].

Redox-mediated signaling has become a focus toward understanding EMT progression. Oxidative stress is primarily associated with cytotoxicity; however, ROS are now recognized as important regulators of signaling pathways and gene transcription [4], [5]. ROS can act through oxidative modification of protein tyrosine phosphatases, redox-dependent transcription factors, and DNA [6], [7], [8], [9]. Major sources of cellular ROS include NADPH oxidase enzymes (Nox), mitochondrial electron transport, xanthine oxidase, and nitric oxide synthase [3]. Evidence suggests that the Nox family of ROS-generating enzymes (Nox1–5 and Duox1–2) are significant players in redox-mediated signaling [10]. In particular, Nox4 was identified as a TGF-beta-responsive oxidase implicated in cytoskeletal alterations in endothelial cells, osteoblast differentiation, pulmonary fibroblast proliferation, idiopathic pulmonary fibrosis, and enhanced oxidative stress in hepatitis C virus infection [11], [12], [13], [14], [15], [16].

Nox4 is a 578 amino acid, six transmembrane domain flavocytochrome that functions in generating superoxide by transporting electrons from cytosolic NADPH across biological membranes to molecular oxygen. Nox4 has 39% homology to Nox2 (gp91^phox), the Nox family prototype. Originally described in the kidney, Nox4 mRNA is also detected in other human and murine tissues including bone, vascular tissue, heart, liver, and lung[17], [18], [19], [20], [21], [22]. Nox4 is primarily localized in perinuclear regions and the ER but is also detected at the plasma membrane, focal adhesions, and associated with mitochondria [9], [22], [23], [24], [25], [26], [27]. Although Nox4 expression is regulated by TGF-beta in many cell types, little is known about its role in the EMT of breast epithelial cells.

In a previous study Nox family genes were surveyed in several human cancer cell lines and in patient tumors and adjacent normal tissues [28]. Their results showed increased Nox4 in breast cancer cell lines as well as in patient tumor samples. In addition, Graham et al. reported high expression of Nox4 in invasive and noninvasive cell lines as well as in patient breast tumor samples [29]. Furthermore, their report indicates that overexpression of Nox4 in MCF10A or MDA-MB-435 cells resulted in cellular senescence, resistance to apoptosis, and tumorigenic transformation. Interestingly, several studies reported the presence of senescent cells in tumor microenvironments. Although resistance to mitogenic stimuli is characteristic of senescent cells, these cells remain metabolically active and capable of secreting growth factors that activate tumor cells [30].

Here we identify Nox4 as the principle source of TGF-beta-induced superoxide production in MCF10A and MDA-MB-231 epithelial cells, and we show that Nox4 functions as an important player in EMT-related events including cell migration and fibronectin gene regulation.