Peroxidasin promotes diabetic vascular endothelial dysfunction induced by advanced glycation end products via NOX2/HOCl/Akt/eNOS pathway.

Reactive oxygen species (ROS) derived from NADPH oxidases (NOX) plays an essential role in advanced glycation end products (AGEs)-induced diabetic vascular endothelial dysfunction. Peroxidasin (PXDN, VPO1) is one member of peroxidases family that catalyzes hydrogen peroxide (H2O2) to hypochlorous acid (HOCl). This present study aimed to elucidate the role of PXDN in promoting vascular endothelial dysfunction induced by AGEs in diabetes mellitus. We found that, compared to non-diabetic (db/m) mice, PXDN expression was notably increased in db/db mice with impaired endothelium-dependent relaxation. Knockdown of PXDN in vivo through tail vein injection of siRNA restored the impaired endothelium-dependent relaxation function of db/db mice which is accompanied with up-regulation of eNOS Ser1177 phosphorylation and NO production. AGEs significantly elevated expression of PXDN and 3-Cl-Tyr, but decreased phosphorylation of Akt and eNOS and NO release in HUVECs. All these effects induced by AGEs were remarkable alleviated by silencing PXDN with small interfering RNAs. In addition, HOCl treatment alone as well as HOCl added with Akt inhibitor MK2206 inhibited phosphorylation of Akt and eNOS, reducing NO production. More importantly,AGEs-induced up-regulation of PXDN and 3-Cl-Tyr with endothelial dysfunction were transformed by NOX2 silencing and H2O2 scavengers. Thus, these results support the conclusion that PXDN promotes AGEs-induced diabetic vascular endothelial dysfunction by attenuating eNOS phosphorylation at Ser1177 via NOX2/HOCl/Akt pathway.

PXDN reduces autophagic flux in insulin-resistant cardiomyocytes via modulating FoxO1.

Autophagy, a well-observed intracellular lysosomal degradation process, is particularly important to the cell viability in diabetic cardiomyopathy (DCM). Peroxidasin (PXDN) is a heme-containing peroxidase that augments oxidative stress and plays an essential role in cardiovascular diseases, while whether PXDN contributes to the pathogenesis of DCM remains unknown. Here we reported the suppression of cell viability and autophagic flux, as shown by autophagosomes accumulation and increased expression level of LC3-II and p62 in cultured H9C2 and human AC16 cells that treated with 400 µM palmitate acid (PA) for 24 h. Simultaneously, PXDN protein level increased. Moreover, cell death, autophagosomes accumulation as well as increased p62 expression were suppressed by PXDN silence. In addition, knockdown of PXDN reversed PA-induced downregulated forkhead box-1 (FoxO1) and reduced FoxO1 phosphorylation, whereas did not affect AKT phosphorylation. Not consistent with the effects of si-PXDN, double-silence of PXDN and FoxO1 significantly increased cell death, suppressed autophagic flux and declined the level of FoxO1 and PXDN, while the expression of LC3-II was unchanged under PA stimulation. Furthermore, inhibition of FoxO1 in PA-untreated cells induced cell death, inhibited autophagic flux, and inhibited FoxO1 and PXDN expression. Thus, we come to conclusion that PXDN plays a key role in PA-induced cell death by impairing autophagic flux through inhibiting FoxO1, and FoxO1 may also affect the expression of PXDN. These findings may develop better understanding of potential mechanisms regarding autophagy in insulin-resistant cardiomyocytes.

Involvement of vascular peroxidase 1 in angiotensin II-induced hypertrophy of H9c2 cells.

Oxidative stress has been implicated in cardiac hypertrophy and heart failure. Vascular peroxidase 1 (VPO1), a peroxidase in the cardiovascular system, uses the hydrogen peroxide (H2O2) derived from co-expressed NADPH oxidases (NOX) to produce hypochlorous acid (HOCl) and catalyze peroxidative reactions. Our previous studies showed that VPO1 contributes to the vascular smooth muscle cell proliferation and endothelial dysfunction in spontaneous hypertensive rats (SHRs); however, the role of VPO1 in cardiomyocytes hypertrophy is still uninvestigated. The present study was therefore undertaken to examine the role of VPO1 in the angiotensin II-induced cardiac hypertrophy, and the underlying mechanism by which VPO1 regulates the redox signaling. As compared to WKY rats, the SHRs exhibited increased myocyte cross sectional area, enhanced Nox2 and VPO1 expression level in cardiac tissue, and an increased Ang II level in plasma. In cultured H9c2 cell line, Ang II increased the hypertrophy-related gene (BNP/ANF) expression and the cellular surface area, which was attenuated by knocking down of VPO1 via VPO1 siRNA or pharmacological inhibition of NOX/VPO1 pathway. Moreover, the enhanced hypochlorous acid (HOCl) production and phosphorylation of ERK1/2 was suppressed by VPO1 knockdown. Furthermore, the protective role of VPO1 siRNA transfection on H9c2 cardiomyocytes hypertrophy was abrogated on the HOCl stimulation, and the phosphorylated ERK1/2 expression level was found also upregulated after HOCl stimulation. In conclusion, these results suggest that the Nox2/VPO1/HOCl/ERK1/2 redox signaling pathway was implicated in the pathogenesis of Ang II-induced cardiac hypertrophy.

VPO1 mediates oxidation of LDL and formation of foam cells.

Deposition of oxidized-LDL in vascular walls is essential in the initiation of atherosclerosis. Oxidation of LDL has been attributed to myeloperoxidase as its generation of potent oxidants. However, the exact mechanism of LDL oxidation and foam cell formation in atherosclerosis remains to be elucidated. Vascular peroxidase-1 (VPO1), a newly-identified heme-containing peroxidase, is primarily expressed in cardiovascular systems, and secreted into the circulation. The present study evaluates VPO1-mediated LDL oxidation and its role in atherosclerosis. VPO1 was first demonstrated binding to LDL. VPO1-mediated oxidation of proteins and lipids in LDL was verified by a variety of methods including immunoblot analysis, free tryptophan assay, UV absorbance, and thiobarbituric acid assay. VPO1-oxidized LDL caused accumulation of LDL in monocyte-like cells and promoted formation of foam cells. Administration of inflammation factors, LPS or TNF-α, induced increasing expression of VPO1 in aorta and secretion to plasma. TNF-α also promoted formation and retention of VPO1-oxidized LDL in aortic walls. Our data suggest that VPO1 contributes to oxidation and retention of LDL in vessel walls, and formation foam cells, indicating VPO1 as a novel potential mediator of atherosclerosis.

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice.

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.

The role of vascular peroxidase 1 in ox-LDL-induced vascular smooth muscle cell calcification.

Reactive oxygen species (ROS)-induced osteogenic differentiation of vascular smooth muscle cells (VSMCs) is associated with the pathogenesis of vascular calcification. Vascular peroxidase 1 (VPO1), a peroxidase in the cardiovascular system, utilizes the hydrogen peroxide (H2O2) produced by co-expressed NADPH oxidases to produce hypochlorous acid (HOCl) and catalyze peroxidative reactions. The aim of this study was to determine whether VPO1 plays a role in the osteogenic differentiation of VSMCs in the setting of the vascular calcification induced by oxidized low-density lipoprotein (ox-LDL). In cultured primary rat VSMCs, we observed that the expression of VPO1 was significantly increased in combination with increases in calcification, as demonstrated via increased mineralization, as well as increased alkaline phosphatase (ALP) activity and up-regulated runt-related transcription factor 2 (Runx2) expression in ox-LDL-treated cells. Ox-LDL-induced VSMC calcification and Runx2 expression were both inhibited by knockdown of VPO1 using a small interfering RNA or by an NADPH oxidase inhibitor. Moreover, the knockdown of VPO1 in VSMCs suppressed the production of HOCl and the phosphorylation of AKT, ERK and P38 MAPK. Furthermore, HOCl treatment facilitated the phosphorylation of AKT, ERK1/2 and P38 MAPK and the expression of Runx2, whereas LY294002 (a specific inhibitor of PI3K), U0126 (a specific inhibitor of ERK1/2) and SB203580 (a specific inhibitor of P38 MAPK) significantly attenuated the HOCl-induced up-regulation of Runx2. Collectively, these results demonstrated that VPO1 promotes ox-LDL-induced VSMC calcification via the VPO1/HOCl/PI3K/AKT, ERK1/2, and P38 MAPK/Runx2 signaling pathways.

Myeloperoxidase is increased in human cerebral aneurysms and increases formation and rupture of cerebral aneurysms in mice.

BACKGROUND AND PURPOSE: Cerebral aneurysm (CA) affects 3% of the population and is associated with hemodynamic stress and inflammation. Myeloperoxidase, a major oxidative enzyme associated with inflammation, is increased in patients with CA, but whether myeloperoxidase contributes to CA is not known. We tested the hypotheses that myeloperoxidase is increased within human CA and is critical for formation and rupture of CA in mice. METHODS: Blood was drawn from the lumen of CAs and femoral arteries of 25 patients who underwent endovascular coiling of CA, and plasma myeloperoxidase concentrations were measured with ELISA. Effects of endogenous myeloperoxidase on CA formation and rupture were studied in myeloperoxidase knockout mice and wild-type (WT) mice using an angiotensin II-elastase induction model of CA. In addition, effects of myeloperoxidase on inflammatory gene expression in endothelial cells were analyzed. RESULTS: Plasma concentrations of myeloperoxidase were 2.7-fold higher within CA than in femoral arterial blood in patients with CA. myeloperoxidase-positive cells were increased in aneurysm tissue compared with superficial temporal artery of patients with CA. Incidence of aneurysms and subarachnoid hemorrhage was significantly lower in myeloperoxidase knockout than in WT mice. In cerebral arteries, proinflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2 (COX2), chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C motif) ligand (XCL1), matrix metalloproteinase (MMP) 8, cluster of differentiation 68 (CD68), and matrix metalloproteinase 13, and leukocytes were increased, and α-smooth muscle actin was decreased, in WT but not in myeloperoxidase knockout mice after induction of CA. Myeloperoxidase per se increased expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in endothelial cells. CONCLUSIONS: These findings suggest that myeloperoxidase may contribute importantly to formation and rupture of CA.

Fluorofenidone inhibits nicotinamide adeninedinucleotide phosphate oxidase via PI3K/Akt pathway in the pathogenesis of renal interstitial fibrosis.

AIM: Oxidative stress plays an important role in the progression of renal interstitial fibrosis. The nicotinamide adeninedinucleotide phosphate (NADPH) oxidase (Nox) family is considered one of the major sources of reactive oxygen species (ROS). In the present study, we investigated the inhibitory effects of a novel anti-fibrotic agent, Fluorofenidone (AKF-PD), upon Nox-mediated oxidative stress and deposition of extracellular matrix (ECM) in the development of renalinterstitial fibrosis. METHODS: AKF-PD was used to treat renal fibrosis in unilateral ureteral obstruction (UUO) obstructive nephropathy in rats. The expression of Nox homologues, p-Akt, collagen I and III were detected by immunoblotting or immunohistochemistry. Levels of 8-iso prostaglandin F2alpha (8-Iso PGF2a) was measured by enzyme linked immunosorbent assay. In addition, ROS and the expression of collagen I (1a), Nox subunits and p-Akt was measured in angiotensin (Ang) II-stimulated rat proximal tubular epithelial (NRK-52E) cells in culture. RESULTS: AKF-PD treatment significantly attenuated tubulo-interstitial injury, ECM deposition and oxidative stress in fibrotic rat kidneys. In addition, AKF-PD inhibited the expression of ROS, Collagen I (1a), Nox2, p-Akt in Ang II-stimulated NRK-52E cells. CONCLUSION: AKF-PD attenuates the progression of renal interstitial fibrosis partly by suppressing NADPH oxidase and ECM deposition via the PI3K/Akt signalling pathway, suggesting AKF-PD is a potential novel therapeutic agent against renal fibrosis.

Annexin A1, formyl peptide receptor, and NOX1 orchestrate epithelial repair.

N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1(-/-IEC) and AnxA1(-/-) mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.

Role of VPO1, a newly identified heme-containing peroxidase, in ox-LDL induced endothelial cell apoptosis.

Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91(phox) subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91(phox) subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91(phox) subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.

Vascular peroxidase-1 is rapidly secreted, circulates in plasma, and supports dityrosine cross-linking reactions.

Members of the peroxidase-cyclooxygenase superfamily catalyze biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. We recently identified a novel member of this family, vascular peroxidase-1 (VPO1), that is highly expressed in the human cardiovascular system. Its biosynthesis and enzymatic properties are largely unknown. Here, we report that VPO1 was rapidly and efficiently secreted into the extracellular space when the gene was stably expressed in human embryonic kidney (HEK) cells. Secreted VPO1 is a monomer with complex N-linked oligosaccharides and exhibits peroxidase activity. Biosynthesis of endogenous VPO1 by cultured human umbilical vein endothelial cells (HUVECs) shares features exhibited by heterologous expression of recombinant VPO1 (rVPO1) in HEK cells. The proinflammatory agents lipopolysaccharide and tumor necrosis factor-α induce expression of VPO1 mRNA and protein in HUVECs. Furthermore, murine and bovine sera and human plasma contain enzymatically active VPO1. rVPO1 exhibits spectral and enzymatic properties characteristic of the peroxidase-cyclooxygenase family, except with regard to its heat stability. rVPO1 catalyzes tyrosyl radical formation and promotes dityrosine cross-linking. Taken together, these data demonstrate that VPO1 is a glycosylated heme peroxidase that is actively secreted into circulating plasma by vascular endothelial cells and shares several features with other members of the peroxidase-cyclooxygenase family, including the catalysis of dityrosine formation.

Nox1 is over-expressed in human colon cancers and correlates with activating mutations in K-Ras.

The NADPH-oxidase 1 (Nox1) is a homolog of gp91phox, the catalytic subunit of the phagocyte superoxide-generating NADPH-oxidase. Nox1 is expressed in normal colon epithelial cells and in colon tumor cell lines, and overexpression in model cells has been implicated in stimulation of mitogenesis and angiogenesis and inhibition of apoptosis. This suggests that aberrant expression of Nox1 could contribute to the development of colorectal cancer. Herein, we examine the expression of Nox1 mRNA in 24 colon tumors of various stages compared with paired adjacent normal tissue from the same patient, and correlate expression with some common mutations associated with colon cancer. Nox1 was overexpressed compared with paired normal tissue in 57% of tumors as early as the adenoma stage, with no correlation of expression level with tumor stage. Overexpression of Nox1 mRNA correlated with Nox1 protein levels assessed by immunofluorescence and immunohistochemistry with an antibody specific for Nox1. There was a strong correlation between Nox1 mRNA level and activating mutations in codons 12 and 13 of K-Ras. Eighty percent (8/10) of tumors with codons 12 and 13 mutations had a 2-fold or more increase in Nox1 mRNA, and 70% (7/10) had a 5-fold or greater increase. Transgenic mice expressing K-Ras(G12V) in the intestinal epithelium also expressed markedly elevated Nox1 in both small and large intestine. There was no correlation between inactivating mutations in the tumor suppressor p53 and Nox1 expression. We conclude that Nox1 mRNA and protein are overexpressed in colon cancer and are strongly correlated with activating mutations in K-Ras.

Nox1-dependent reactive oxygen generation is regulated by Rac1.

Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91(phox), is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild-type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1(G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91(phox) (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation.

Evaluation of two anti-gp91phox antibodies as immunoprobes for Nox family proteins: mAb 54.1 recognizes recombinant full-length Nox2, Nox3 and the C-terminal domains of Nox1-4 and cross-reacts with GRP 58.

Progress in the study of Nox protein expression has been impeded because of the paucity of immunological probes. The large subunit of human phagocyte flavocytochrome b558 (Cytb), gp91phox, is also the prototype member of the recently discovered family of NADPH oxidase (Nox) proteins. In this study, we have evaluated the use of two anti-gp91phox monoclonal antibodies, 54.1 and CL5, as immunoprobes for Nox family proteins. Sequence alignment of gp91phox with Nox1, Nox3 and Nox4 identified regions of the Nox proteins that correspond to the gp91phox epitopes recognized by mAb 54.1 and CL5. Antibody 54.1 produced positive immunoblots of recombinant C-terminal fragments of these homologous proteins expressed in E. coli. Furthermore, only mAb 54.1 recognized full-length murine and human Nox3 expressed in HEK-293 cells, in immunoblots of alkali-stripped or detergent-solubilized membranes. 54.1 recognized Nox3 expression-specific proteins with Mr 30,000, 50,000, 65,000 and 88,000 for the murine protein and Mr of 38,000-58,000, 90,000, 100,000-130,000 and a broad species of higher than 160,000 for the human protein. We conclude that mAb 54.1 can serve as a probe of Nox3 and possibly other Nox proteins, if precautions are taken to remove GRP 58 and other crossreactive membrane-associated or detergent-insoluble proteins from the sample to be probed.

Point mutations in the proline-rich region of p22phox are dominant inhibitors of Nox1- and Nox2-dependent reactive oxygen generation.

The integral membrane protein p22phox is an indispensable component of the superoxide-generating phagocyte NADPH oxidase, whose catalytic core is the membrane-associated gp91phox (also known as Nox2). p22phox associates with gp91phox and, through its proline-rich C terminus, provides a binding site for the tandem Src homology 3 domains of the activating subunit p47phox. Whereas p22phox is expressed ubiquitously, its participation in regulating the activity of other Nox enzymes is less clear. This study investigates the requirement of p22phox for Nox enzyme activity and explores the role of its proline-rich region (PRR) for regulating activity. Coexpression of specific Nox catalytic subunits (Nox1, Nox2, Nox3, Nox4, or Nox5) along with their corresponding regulatory subunits (NOXO1/NOXA1 for Nox1; p47phox/p67phox/Rac for Nox2; NOXO1 for Nox3; no subunits for Nox4 or Nox5) resulted in marked production of reactive oxygen. Small interfering RNAs decreased endogenous p22phox expression and inhibited reactive oxygen generation from Nox1, Nox2, Nox3, and Nox4 but not Nox5. Truncated forms of p22phox that disrupted the PRR-inhibited reactive oxygen generation from Nox1, Nox2, and Nox3 but not from Nox4 and Nox5. Similarly, p22phox (P156Q), a mutation that disrupts Src homology 3 binding by the PRR, potently inhibited reactive oxygen production from Nox1 and Nox2 but not from Nox4 and Nox5. Expression of p22phox (P156Q) inhibited NOXO1-stimulated Nox3 activity, but co-expression of NOXA1 overcame the inhibitory effect. The P157Q and P160Q mutations of p22phox showed selective inhibition of Nox2/p47phox/p67phox, and selectivity was specific for the organizing subunit (p47phox or NOXO1) rather than the Nox catalytic subunit. These studies stress the importance of p22phox for the function of Nox1, Nox2, Nox3, and Nox4, and emphasize the key role of the PRR for regulating Nox proteins whose activity is dependent upon p47phox or NOXO1.

The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction.

Insulin stimulation of target cells elicits a burst of H(2)O(2) that enhances tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins as well as distal signaling events in the insulin action cascade. The molecular mechanism coupling the insulin receptor with the cellular oxidant-generating apparatus has not been elucidated. Using reverse transcription-PCR and Northern blot analyses, we found that Nox4, a homolog of gp91phox, the phagocytic NAD(P)H oxidase catalytic subunit, is prominently expressed in insulin-sensitive adipose cells. Adenovirus-mediated expression of Nox4 deletion constructs lacking NAD(P)H or FAD/NAD(P)H cofactor binding domains acted in a dominant-negative fashion in differentiated 3T3-L1 adipocytes and attenuated insulin-stimulated H(2)O(2) generation, insulin receptor (IR) and IRS-1 tyrosine phosphorylation, activation of downstream serine kinases, and glucose uptake. Transfection of specific small interfering RNA oligonucleotides reduced Nox4 protein abundance and also inhibited the insulin signaling cascade. Overexpression of Nox4 also significantly reversed the inhibition of insulin-stimulated IR tyrosine phosphorylation induced by coexpression of PTP1B by inhibiting PTP1B catalytic activity. These data suggest that Nox4 provides a novel link between the IR and the generation of cellular reactive oxygen species that enhance insulin signal transduction, at least in part via the oxidative inhibition of cellular protein-tyrosine phosphatases (PTPases), including PTP1B, a PTPase that has been previously implicated in the regulation of insulin action.

Identification of Novel Regulatory Elements of Human Stem Cell Factor Gene in MCF Cell.

Human stem cell factor(hSCF)is a pluripotent growth factor that regulates proliferation, differentiation and migration of certain mammalian stem cells, such as primordial germ cells etc. It is shown that hSCF and its receptor are commonly co-expressed in human breast cancer cells. Up to now, the definite regulatory mechanism of hSCF gene in breast cancer cells is unclear, except that its 5'flanking sequence contains essential elements for regulating transcription. To localize the regulatory elements responsible for the regulation of the hSCF gene, we performed transient transfection study in MCF cells, with a series of luciferase reporter gene constructs, containing different 5x end deletions of hSCF gene. This study indicates that the region of -1190 -853 significantly enhanced the luc gene expression, while the region of -339 -162 inhibited the expression. Eletrophoretic mobility shift assay confirmed that MCF nuclear extract proteins bound to both -1190 -853 and -339 -273 regions, forming specific DNA-protein complexes, indicating that there were nuclear protein binding sites in these regions. The results suggest that both -1190 -853 and -339 -273 DNA fragments of the hSCF 5'flanking sequence may be novel regulatory elements, and may play a role in the regulation of hSCF gene expression in MCF cells.