Howard E. Boudreau

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

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.

Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor β-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress

ABSTRACT Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor β (TGF-β) and hepatic fibrosis are hallmarks of hepatitis C virus (HCV) infection. The mechanisms of redox regulation in the pathogenesis of HCV-induced liver disease are not clearly understood. The results of our current studies suggest that reactive oxygen species (ROS) derived from Nox4, a member of the NADPH oxidase (Nox) family, could play a role in HCV-induced liver disease. We found that the expression of HCV (genotype 1a) cDNA constructs (full-length and subgenomic), core protein alone, viral RNA, or replicating HCV (JFH-AM2) induced Nox4 mRNA expression and ROS generation in human hepatocyte cell lines (Huh-7, Huh-7.5, HepG2, and CHL). Conversely, hepatocytes expressing Nox4 short hairpin RNA (shRNA) or an inactive dominant negative form of Nox4 showed decreased ROS production when cells were transfected with HCV. The promoters of both human and murine Nox4 were used to demonstrate transcriptional regulation of Nox4 mRNA by HCV, and a luciferase reporter tied to an ∼2-kb promoter region of Nox4 identified HCV-responsive regulatory regions modulating the expression of Nox4. Furthermore, the human Nox4 promoter was responsive to TGF-β1, and the HCV core-dependent induction of Nox4 was blocked by antibody against TGF-β or the expression of dominant negative TGF-β receptor type II. These findings identified HCV as a regulator of Nox4 gene expression and subsequent ROS production through an autocrine TGF-β-dependent mechanism. Collectively, these data provide evidence that HCV-induced Nox4 contributes to ROS production and may be related to HCV-induced liver disease.

Histamine stimulates hydrogen peroxide production by bronchial epithelial cells via histamine H1 receptor and dual oxidase.

Oxidative stress has been implicated in the pathogenesis of bronchial asthma. Besides granulocytes, the airway epithelium can produce large amounts of reactive oxygen species and can contribute to asthma-related oxidative stress. Histamine is a major inflammatory mediator present in large quantities in asthmatic airways. Whether histamine triggers epithelium-derived oxidative stress is unknown. We therefore aimed at characterizing human airway epithelial H2O2 production stimulated by histamine. We found that air-liquid interface cultures of primary human bronchial epithelial cells (BECs) and an immortalized BEC model (Cdk4/hTERT HBEC) produce H2O2 in response to histamine. The main source of airway epithelial H2O2 is an NADPH dual oxidase, Duox1. Out of the four histamine receptors (H1R-H4R), H1R has the highest expression in BECs and mediates the H2O2-producing effects of histamine. IL-4 induces Duox1 gene and protein expression levels and enhances histamine-induced H2O2 production by epithelial cells. Using HEK-293 cells expressing Duox1 or Duox2 and endogenous H1R, histamine triggers an immediate intracellular calcium signal and H2O2 release. Overexpression of H1R further increases the oxidative output of Duox-expressing HEK-293 cells. Our observations show that BECs respond to histamine with Duox-mediated H2O2 production. These findings reveal a mechanism that could be an important contributor to oxidative stress characteristic of asthmatic airways, suggesting novel therapeutic targets for treating asthmatic airway disease.

Peroxiredoxin 6 (Prdx6) supports NADPH oxidase1 (Nox1)-based superoxide generation and cell migration.

Nox1 is an abundant source of reactive oxygen species (ROS) in colon epithelium recently shown to function in wound healing and epithelial homeostasis. We identified Peroxiredoxin 6 (Prdx6) as a novel binding partner of Nox activator 1 (Noxa1) in yeast two-hybrid screening experiments using the Noxa1 SH3 domain as bait. Prdx6 is a unique member of the Prdx antioxidant enzyme family exhibiting both glutathione peroxidase and phospholipase A2 activities. We confirmed this interaction in cells overexpressing both proteins, showing Prdx6 binds to and stabilizes wild type Noxa1, but not the SH3 domain mutant form, Noxa1 W436R. We demonstrated in several cell models that Prdx6 knockdown suppresses Nox1 activity, whereas enhanced Prdx6 expression supports higher Nox1-derived superoxide production. Both peroxidase- and lipase-deficient mutant forms of Prdx6 (Prdx6 C47S and S32A, respectively) failed to bind to or stabilize Nox1 components or support Nox1-mediated superoxide generation. Furthermore, the transition-state substrate analogue inhibitor of Prdx6 phospholipase A2 activity (MJ-33) was shown to suppress Nox1 activity, suggesting Nox1 activity is regulated by the phospholipase activity of Prdx6. Finally, wild type Prdx6, but not lipase or peroxidase mutant forms, supports Nox1-mediated cell migration in the HCT-116 colon epithelial cell model of wound closure. These findings highlight a novel pathway in which this antioxidant enzyme positively regulates an oxidant-generating system to support cell migration and wound healing.

Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2.

In the thyroid gland Duox2-derived H2O2 is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2(thyd)) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H2O2, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.

Histone modifications affect differential regulation of TGFβ- induced NADPH oxidase 4 (NOX4) by wild-type and mutant p53

Previously, we showed wild-type (WT) and mutant (mut) p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (NOX4): p53-WT suppresses TGFβ-induced NOX4, ROS and cell migration, whereas tumor-associated mut-p53 proteins enhance NOX4 expression and cell migration. Here, we extended our findings on the effects of p53 on NOX4 in several tumors and examined the basis of NOX4 transcriptional regulation by p53 and SMAD3. Statistical analysis of expression data from primary tumors available from The Cancer Genome Atlas (TCGA) detected correlations between mut-p53 and increased NOX4 expression. Furthermore, by altering p53 levels in cell culture models we showed several common tumor-associated mutant forms support TGFβ/SMAD3-dependent NOX4 expression. Deletion analysis revealed two critical SMAD3 binding elements (SBE) required for mut-p53-dependent NOX4 induction, whereas p53-WT caused dose-dependent suppression of NOX4 transcription. ChIP analysis revealed SMAD3 and p53-WT or mut-p53 associate with SBEs and p53 response elements in a TGFβ-dependent manner. Interestingly, the repressive effects of p53-WT on NOX4 were relieved by mutation of its transactivation domain or histone deacetylase (HDAC) inhibitor treatment. Overexpression of p300, a transcriptional co-regulator and histone acetyltransferase (HAT), enhanced p53-mediated NOX4 induction, whereas HAT-inactive p300 reduced NOX4 expression. Mut-p53 augmented TGFβ-stimulated histone acetylation within the NOX4 promoter. Finally, wound assays demonstrated NOX4 and p300 promote TGFβ/mut-p53-mediated cell migration. Our studies provide new insight into TGFβ/SMAD3 and mut-p53-mediated NOX4 induction involving epigenetic control of NOX4 in tumor cell migration, suggesting NOX4 is a potential therapeutic target to combat tumor progression and metastasis.

PAC1 regulates receptor tyrosine kinase transactivation in a reactive oxygen species-dependent manner

Pituitary adenylate cyclase activating polypeptide (PACAP) is a growth factor for lung cancer cells. PACAP-27 or PACAP-38 binds with high affinity to non-small cell lung cancer (NSCLC) cells, causing elevated cytosolic Ca2+, increased proliferation and increased phosphorylation of extracellular regulated kinase (ERK) and the epidermal growth factor receptor (EGFR). The role of reactive oxygen species (ROS) was investigated in these processes. Addition of PACAP-38 to NCI-H838 or A549 cells increased the tyrosine phosphorylation of the EGFR, HER2 and ERK significantly by 4-, 3-, and 2-fold, respectively. The transactivation of the EGFR and HER2 was inhibited by gefitinib or lapatinib (tyrosine kinase inhibitors), PACAP (6-38) (PAC1 antagonist), N-acetylcysteine (NAC is an anti-oxidant) or dipheyleneiodonium (DPI is an inhibitor of Nox and Duox enzymes). PACAP-38 addition to NSCLC cells increased ROS which was inhibited by PACAP (6-38), NAC or DPI. Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2 mRNA was present in many NSCLC cell lines. PACAP-38 stimulated the growth of NSCLC cells whereas PACAP (6-38), gefitinib or DPI inhibited proliferation. The results show that ROS are essential for PAC1 to regulate EGFR and HER2 transactivation as well as proliferation of NSCLC cells.

Abstract 2215: Transcriptional co-regulation of Nox4 by p53 and SMAD3

Recent studies suggest p53 plays an important role in TGF-beta-mediated cell signaling and migration. Previously, we showed that wild-type (WT) and mutant forms of p53 differentially regulate reactive oxygen species (ROS) generation by NADPH oxidase-4 (Nox4). We found that WT-p53 is a potent suppressor of TGF-beta-induced Nox4, ROS production, and cell migration, whereas tumor-associated mutant p53 proteins enhanced Nox4 mRNA and protein expression and cell migration by both TGF-beta-dependent and independent mechanisms (Boudreau et al. Br J Cancer. 2014 May 13;110(10):2569-82). In this study, we examined the basis of human Nox4 promoter regulation by p53 and SMAD3. By constructing a deletion series of Nox4 promoter-reporter constructs, we discovered two regions (-4.76kb to -3.9kb and -1.8kb to - 0.2kb) upstream of the transcription start site that induce promoter activity upon activation of the TGF-beta/SMAD3 pathway. Interestingly, TGF-beta/SMAD3-mediated Nox4 promoter activity was abolished by heterologous expression of p53-WT in p53-null cell lines Hep3B (hepatocytes) and H1299 (lung epithelial). This activity was repressed by wild-type p53 in a dose-dependent manner. In contrast, expression of common tumor-associated mutant-p53 proteins (R175H, R248H, R249Q, R273H) did not repress but enhanced Nox4 promoter activity. We observed this enhancement of Nox4 promoter activity by mutant p53 (R273H) in H1299 cells expressing T204D, a constitutively active TGF-beta receptor. When individual p53 response elements were mutated, however, both untreated and R273H-expressing cells showed 50% reduction in promoter activity. These results indicate that the Nox4 promoter region between -3.97kb and -4.76kb, containing candidate p53 response elements, plays a role in modulating Nox4 expression. Preliminary chromatin immunoprecipitation results suggest that endogenous mutant p53 R249S in PLC/PRF/5 hepatoma cells complexes with the Nox4 promoter region between -3.97kb and -4.76kb. Furthermore, constructs lacking this region show significantly reduced Nox4 promoter activity in response to TGF-beta. Our results provide a basis for Nox4 co-regulation by SMAD3 and mutant p53 that may be involved in tumor progression and metastasis. Citation Format: Howard E. Boudreau, Jonathan J. Park, Thomas L. Leto. Transcriptional co-regulation of Nox4 by p53 and SMAD3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2215. doi:10.1158/1538-7445.AM2015-2215

Abstract 1475: Mutant p53 is a regulator of NADPH-oxidase 4 (Nox4) in TGF-beta-mediated migration of human lung and breast epithelial cells.

The epithelial-to-mesenchymal transition (EMT) leads to 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. We have previously shown 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. Recent studies have shown that p53 plays an important role in TGF-beta-mediated signaling and epithelial cell migration. Our current study investigates whether p53 is mechanistically involved in TGF-beta-regulated Nox4 expression and cell migration. Our results indicate that wild-type p53 is a potent suppressor of TGF-beta-induced Nox4 expression, ROS production, and cell migration. In contrast, expression of tumor-associated mutant p53 proteins (R175H or R280K) correlated with enhanced Nox4 expression and cell migration in a TGF-beta dependent manner. Furthermore, knockdown of the endogenous p53 mutant (R280K) in MDA-MB-231 cells by p53-targeted siRNA resulted in decreased Nox4 protein as well as phosphorylated Src and FAK proteins. TGF-beta treatment of MDA-MB-231 cells overexpressing a dominant-negative form of Nox4 also showed a decrease in phosphorylated Src and FAK proteins, thereby indicating mutant p53 (R280K) and Nox4 are regulators of Src and FAK activity. Collectively, our findings suggest important physiological functions for both wild type and mutant p53 proteins in regulating Nox4-dependent signaling in TGF-beta-mediated cell migration. Citation Format: Howard E. Boudreau, Benjamin W. Casterline, Devin J. Burke, Thomas L. Leto. Mutant p53 is a regulator of NADPH-oxidase 4 (Nox4) in TGF-beta-mediated migration of human lung and breast epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1475. doi:10.1158/1538-7445.AM2013-1475