TGF-beta-induced oxidative stress has been implicated in regulating EMT progression, including gene expression, cytoskeletal rearrangement, and cell mobility. Here we show Nox4 contributes to TGF-beta-mediated cell migration and fibronectin expression of normal (MCF10A) and metastatic (MDA-MB-231) breast epithelial cells. We found TGF-beta induced NADPH oxidase-dependent superoxide release in both MCF10A and MDA-MB-231 breast epithelial cells (). We show Nox4 mRNA and protein expression was substantially increased upon TGF-beta stimulation (). Furthermore, expression of constitutively active TGF-beta receptor type-1 increased NADPH oxidase-dependent superoxide release, and Nox4 mRNA, protein, and promoter activity ( and ). We further show that wild type and constitutively active SMAD3 significantly increase Nox4 promoter activity, whereas dominant-negative SMAD3 and SIS3, a SMAD3-specific inhibitor, decreased promoter activity (). In contrast, TGF-beta-mediated superoxide was significantly reduced in MCF10A and MDA-MB-231 cells expressing a dominant-negative form of Nox4 or Nox4-specific shRNA (), indicating Nox4 is the primary source of superoxide generation. Moreover, wound closure and cell migration assays of epithelial cells expressing dominant-negative Nox4 or Nox4-shRNA were significantly reduced in response to TGF-beta treatment (), indicating Nox4 is involved in TGF-beta regulation of breast epithelial cell migration. Inhibition of endogenous SMAD3 by transfection of a dominant-negative construct () or SIS3 treatment () also substantially reduced cell migration further suggesting SMAD3 is a key regulator of Nox4-mediated cell migration. Interestingly, dominant-negative Nox4 had no effect on TGF-beta-mediated cell proliferation (), indicating TGF-beta modulation of Nox4 plays important roles in cell migration rather than cell proliferation. We also found TGF-beta up-regulation of fibronectin, an indicator of EMT progression, was reduced in dominant-negative Nox4 or Nox4 shRNA transfected breast epithelial cells (), suggesting Nox4 is involved in redox-dependent fibronectin gene regulation during EMT. Taken together, our results indicate Nox4 plays an important role in breast epithelial cell migration and fibronectin expression driven by a TGF-beta/SMAD3 signaling mechanism, as summarized in .
Nox4 modulates TGF-beta/SMAD3-mediated breast epithelial cell migration
Previous reports indicate TGF-beta modulates Nox4TGF-beta in many cell types including vascular endothelial cells, kidney fibroblasts, hepatocytes, and lung epithelial cells [36
], however, little is know about TGF-beta mediated ROS generation in breast epithelial cells. Breast cancer epithelial cells are known to secrete high levels of soluble TGF-beta into the tumor microenvironment allowing for paracrine and autocrine TGF-beta signaling [39
]. While TGF-beta can have pro-apoptotic effects on specific cell types, MDA-MB-231 and MCF10A breast epithelial cells are resistant to TGF-beta-mediated growth inhibition [31
]. Rather, breast epithelial cells respond to TGF-beta by increasing EMT gene expression and enhancing their migratory and metastatic potential. Tobar et al
. recently reported Nox4-dependent ROS in migration of MCF-7 cells, a mildly invasive breast epithelial line [36
]. They demonstrated that TGF-beta is a soluble factor secreted by RMF-EG mammary fibroblasts that increased Nox4 mRNA, ROS production and cell migration of co-cultured MCF-7 cells. Here we show TGF-beta-mediated cell migration is activated through a TGFBR1→ SMAD3→Nox4 signaling pathway in both normal and highly metastatic breast epithelial cells.
Nox4 has been implicated in EMT gene regulation and migration of other cell types. Bondi et al. reported TGF-beta induced Nox4-dependent modulation of EMT markers alpha-smooth muscle actin (SMA) and fibronectin during conversion of rat kidney fibroblast to a myofibroblast phenotype [42
]. Nox4 was also reported to have a promigratory effect on angiotensin-II-treated vascular adventitial myofibroblasts [43
]. Recently, Nam et al. described ROS generated from Nox1 and Nox4 involved in migration of human keratinocytes co-treated with HGF and TGF-beta [37
], and Pendyala et al. indicated Nox4 is involved in hyperoxia-induced pulmonary artery endothelial cell migration [44
]. Also, Meng et al. reported Nox4 and Rac1 mediate IGF-1-induced ROS production and cell migration in vascular smooth muscle cells [45
]. They also demonstrated IGF-1-induced matrix metalloproteinase (MMP)-2 and 9 activities, indicative of EMT progression, were inhibited by knockdown of Nox4 or Rac1 inactivation through independent mechanisms.
Several ECM proteins are modulated under the control of TGF-beta including increased fibronectin expression. Fibronectin is important for directing cell attachment and migration during the cell reorganization and migratory process. Previous reports implicated Nox4 in cytoskeletal alterations and regulation of fibronectin expression. Under high glucose conditions, the diabetic rat kidney showed Nox4-derived ROS can mediate renal hypertrophy and fibronectin expression [33
]. Also, Hu et al. reported expression of dominant-negative Nox4 in HUVEC cells blocked the effect of TGF-beta on filipodia formation and F-actin assembly [13
]. In vascular smooth muscle cells, p22phox association with poldip2 appears to increase Nox4 activity, which was correlated with cytoskeletal remodeling and focal adhesion turnover [46
]. Our study not only demonstrates Nox4 is involved in TGF-beta-induced breast epithelial cell migration by wound healing and matrigel assays, but also provides evidence that Nox4 plays a role in EMT and cell mobility by modulating fibronectin expression.
The role of SMAD proteins, particularly SMAD3, during TGF-beta-induced EMT has been well established. Previous observations showed in SMAD3 knockout mice that EMT of lens epithelium after injury is blocked in the absence of SMAD3, along with decreased expression of EMT markers including, snail, SMA, and collagen [47
]. Another study using selective knockdown of SMAD2 and SMAD3 demonstrated that TGF-β–induced EMT is SMAD3 dependent in lung cancer cells [48
]. Moreover, Sturrock et al. showed TGF-beta-induced Nox4 mRNA and ROS were decreased in pulmonary smooth muscle cells overexpressing either dominant-negative SMAD2 or SMAD3 [49
]. By comparing constitutively active SMAD3 (C-terminal pseudo-phosphorylated) to dominant-negative SMAD3 (deletion of the MH2 domain), we are the first to demonstrate that phosphorylation of the C-terminus of SMAD3 positively regulates the human Nox4 promoter in breast epithelial cells. While we cannot rule out involvement of other transcriptions factors in response to TGF-beta, our findings provide an important foundation for identifying SMAD binding elements (SBE) and other key components involved in TGF-beta regulation of the Nox4 promoter.
The precise mechanisms of Nox4-derived ROS in TGF-beta-mediated cell migration and EMT progression of breast epithelial cells warrants further investigation. It is possible that Nox4-dependent ROS alter the activation states of signaling components or affect ECM proteins involved in EMT processes by direct protein oxidation. ROS have been shown to modulate cell signaling by altering oxidation states of proteins such as tyrosine phosphatases (PTP1B, SHP2, PTEN, and LMW-PTP), redox sensitive transcription factors (NFκ-B, p53, and AP-1), receptor and non-receptor tyrosine kinases (EGFR, PDGFR, and Src), and cytoskeletal proteins (beta-actin or associated proteins) [50
]. Moreover, a previous study reporting a proteomic screen for cysteine glutathionylated proteins revealed several cytoskeletal proteins including actin, cofilin, vimentin, and profilin are redox sensitive[54
]. Nox4 subcellular localization has been described in many places including the plasma membrane, ER, perinuclear, and nuclear compartments. Nox4 was recently reported to co-localization with F-actin at sites of invadopodia; and Nox4 knockdown resulted in reduced invadopodia formation in Src transformed 3T3 cells [55
]. Determination of TGF-beta-induced Nox4 localization will provide further insight into Nox4-dependent redox mechanisms involved in EMT progression and cell motility.