A comparative study of bronchopulmonary slowly adapting receptors between rabbits and rats.

Pulmonary mechanosensory receptors provide important inputs to the respiratory center for control of breathing. However, what is known about their structure-function relationship is still limited. In these studies, we explored this relationship comparing bronchopulmonary slowly adapting receptor (SAR) units in rabbits and rats. In morphological studies, sensory units in tracheobronchial smooth muscle labeled with anti-Na+ /K+ -ATPase (α3 subunit) were found to be larger in the rabbit. Since larger structures may result from increased receptor size or more numerous receptors, further examination showed receptor size was the same in both species, but more receptors in a structure in rabbits than rats, accounting for their larger structure. In functional studies, SAR units were recorded electrically in anesthetized, open-chest, and artificially ventilated animals and responses to lung inflation were compared at three different constant airway pressures (10, 20, and 30 cmH2 O). At each level of the inflation, SAR discharge frequencies were found to be higher in rabbits than rats. We conclude that a relatively larger number of receptors in a sensory unit may be responsible for higher SAR activities in rabbit SAR units.

The profibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression.

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and ß-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

Effect of vinyl chloride exposure on cardiometabolic toxicity.

Vinyl chloride (VC) is an organochlorine mainly used to manufacture its polymer polyvinyl chloride, which is extensively used in the manufacturing of consumer products. Recent studies suggest that chronic low dose VC exposure affects glucose homeostasis in high fat diet-fed mice. Our data suggest that even in the absence of high fat diet, exposure to VC (0.8 ppm, 6 h/day, 5 day/week, for 12 weeks) induces glucose intolerance (1.0 g/kg, i.p.) in male C57BL/6 mice. This was accompanied with the depletion of hepatic glutathione and a modest increase in lung interstitial macrophages. VC exposure did not affect the levels of circulating immune cells, endothelial progenitor cells, platelet-immune cell aggregates, and cytokines and chemokines. The acute challenge of VC-exposed mice with LPS did not affect lung immune cell composition or plasma IL-6. To examine the effect of VC exposure on vascular inflammation and atherosclerosis, LDL receptor-KO mice on C57BL/6 background maintained on western diet were exposed to VC for 12 weeks (0.8 ppm, 6 h/day, 5 day/week). Unlike the WT C57BL/6 mice, VC exposure did not affect glucose tolerance in the LDL receptor-KO mice. Plasma cytokines, lesion area in the aortic valve, and markers of lesional inflammation in VC-exposed LDL receptor-KO mice were comparable with the air-exposed controls. Collectively, despite impaired glucose tolerance and modest pulmonary inflammation, chronic low dose VC exposure does not affect surrogate markers of cardiovascular injury, LPS-induced acute inflammation in C57BL/6 mice, and chronic inflammation and atherosclerosis in the LDL receptor-KO mice.

Chronic Benzene Exposure Aggravates Pressure Overload-Induced Cardiac Dysfunction.

Benzene is a ubiquitous environmental pollutant abundant in household products, petrochemicals, and cigarette smoke. Benzene is a well-known carcinogen in humans and experimental animals; however, little is known about the cardiovascular toxicity of benzene. Recent population-based studies indicate that benzene exposure is associated with an increased risk for heart failure. Nonetheless, it is unclear whether benzene exposure is sufficient to induce and/or exacerbate heart failure. We examined the effects of benzene (50 ppm, 6 h/day, 5 days/week, and 6 weeks) or high-efficiency particulate absorbing-filtered air exposure on transverse aortic constriction (TAC)-induced pressure overload in male C57BL/6J mice. Our data show that benzene exposure had no effect on cardiac function in the Sham group; however, it significantly compromised cardiac function as depicted by a significant decrease in fractional shortening and ejection fraction, as compared with TAC/Air-exposed mice. RNA-seq analysis of the cardiac tissue from the TAC/benzene-exposed mice showed a significant increase in several genes associated with adhesion molecules, cell-cell adhesion, inflammation, and stress response. In particular, neutrophils were implicated in our unbiased analyses. Indeed, immunofluorescence studies showed that TAC/benzene exposure promotes infiltration of CD11b+/S100A8+/myeloperoxidase+-positive neutrophils in the hearts by 3-fold. In vitro, the benzene metabolites, hydroquinone, and catechol, induced the expression of P-selectin in cardiac microvascular endothelial cells by 5-fold and increased the adhesion of neutrophils to these endothelial cells by 1.5- to 2.0-fold. Benzene metabolite-induced adhesion of neutrophils to the endothelial cells was attenuated by anti-P-selectin antibody. Together, these data suggest that benzene exacerbates heart failure by promoting endothelial activation and neutrophil recruitment.

Subclinical markers of cardiovascular toxicity of benzene inhalation in mice.

Benzene is a ubiquitous environmental pollutant. Recent population-based studies suggest that benzene exposure is associated with an increased risk for cardiovascular disease. However, it is unclear whether benzene exposure by itself is sufficient to induce cardiovascular toxicity. We examined the effects of benzene inhalation (50 ppm, 6 h/day, 5 days/week, 6 weeks) or HEPA-filtered air exposure on the biomarkers of cardiovascular toxicity in male C57BL/6J mice. Benzene inhalation significantly increased the biomarkers of endothelial activation and injury including endothelial microparticles, activated endothelial microparticles, endothelial progenitor cell microparticles, lung endothelial microparticles, and activated lung and endothelial microparticles while having no effect on circulating levels of endothelial adhesion molecules, endothelial selectins, and biomarkers of angiogenesis. To understand how benzene may induce endothelial injury, we exposed human aortic endothelial cells to benzene metabolites. Of the metabolites tested, trans,trans-mucondialdehyde (10 µM, 18h) was the most toxic. It induced caspases-3, -7 and -9 (intrinsic pathway) activation and enhanced microparticle formation by 2.4-fold. Levels of platelet-leukocyte aggregates, platelet macroparticles, and a proportion of CD4+ and CD8+ T-cells were also significantly elevated in the blood of the benzene-exposed mice. We also found that benzene exposure increased the transcription of genes associated with endothelial cell and platelet activation in the liver; and induced inflammatory genes and suppressed cytochrome P450s in the lungs and the liver. Together, these data suggest that benzene exposure induces endothelial injury, enhances platelet activation and inflammatory processes; and circulatory levels of endothelial cell and platelet-derived microparticles and platelet-leukocyte aggregates are excellent biomarkers of cardiovascular toxicity of benzene.

Maternal undernutrition during pregnancy alters the epigenetic landscape and the expression of endothelial function genes in male progeny.

Recent studies point to the important role of in utero malnutrition in gene programming and in the development of vascular diseases. We hypothesize that maternal undernutrition affects vascular function in the offspring by promoting epigenetic changes that drive the differential expression of genes involved in endothelial function. To test this, we exposed mice to nutrient deprivation in utero and analyzed its effect on global DNA methylation and expression of endothelium-specific genes in the pulmonary endothelium of the adult progeny. Mice were kept either on ad libitum (AL) or energy-restricted (ER) diet during the second and third trimesters of gestation. Mice in the ER group received 65% of energy compared to mice in the AL diet group. Pulmonary endothelial cells were isolated from 6-week-old male offspring mice (AL-F1 and ER-F1). The expression of genes in the pulmonary endothelium was analyzed using quantitative reverse-transcription polymerase chain reaction array and confirmed by qRT-PCR. Several genes including fibronectin 1 and plasminogen activator inhibitor 1 were upregulated in the endothelium of male ER-F1 mice, whereas the expression of genes involved in regulation of histone acetylation was significantly attenuated. At the same time, the global DNA methylation did not change in pulmonary endothelial cells of ER-F1 mice compared to AL-F1 mice. Overall, we found that maternal undernutrition during pregnancy affects the expression of genes involved in regulation of endothelial cell function in the pulmonary vasculature of male progeny, which could potentially promote pulmonary vascular remodeling.

Role of SOD3 in silica-related lung fibrosis and pulmonary vascular remodeling.

BACKGROUND: Work-place exposure to silica dust may lead to progressive lung inflammation culminating in the development of silicosis, an irreversible condition that can be complicated by onset of pulmonary hypertension (PH). The molecular mechanisms leading to the development of PH and lung fibrosis in response to silica are not well understood. Oxidant/antioxidant imbalance in the lung may promote fibroproliferation and vascular smooth muscle proliferation, ultimately leading to the development of PH. Herein, we analyze the development of PH and lung fibrosis in mice deficient in extracellular superoxide dismutase (SOD3), an enzyme with anti-oxidant activity. METHODS: PH and silicosis were induced in wild-type and Sod3-/- mice through intratracheal injection of crystalline silica at dose 0.4 g/kg. Pulmonary hypertension and lung fibrosis were characterized by changes in right ventricular systolic pressure (RVSP) and collagen deposition 28 days following silica injections. Vascular remodeling was analyzed using immunohistochemistry and morphometric analysis. The expression of genes were analyzed using qRT-PCR and Western blot. RESULTS: C57BL6 mice exposed to silica showed attenuated expression of Sod3 in the lung suggesting a protective role for Sod3. Consistent with this, Sod3-/- mice developed more severe fibrotic inflammatory nodules with increased collagen deposition. Furthermore, the expression of genes involved in tissue remodeling (Timp1), fibrotic lesion formation (Fsp1) and inflammatory response (Mcp1) were significantly elevated in Sod3-/- mice compared to Sod3+/+ mice treated with silica. Infiltration of neutrophils and activated macrophages into affected lung was significantly higher in Sod3 deficient mice. In addition, silica produced more profound effects on elevation of RVSP in Sod3-/- compared to wild-type littermate. Increase in RVSP was concomitant with hypertrophy of pulmonary arteries located in silicotic nodules of both mouse strains, however, vascular remodeling in unaffected areas of lung was detected only in Sod3-/- mice. CONCLUSIONS: Our data suggest that Sod3 and extracellular oxidative stress may play an important role in the development of pneumoconiosis and pulmonary vascular remodeling following exposure to environmental and occupational silica.

Epigenetic regulation of EC-SOD expression in aging lung fibroblasts: Role of histone acetylation.

Lung disorders characterized by fibroproliferation and excessive deposition of extracellular matrices occur in late adulthood, and their pathological manifestations become more prominent with aging. The exact mechanisms linking aging and fibroproliferative disorders are unknown, but increased oxidative stress resulting in the accumulation of damaged proteins, DNA, and lipids is considered a major factor. In the lung, and especially in the pulmonary fibroblasts, the extracellular superoxide dismutase (EC-SOD) is a major antioxidant enzyme that has been implicated in pulmonary fibrosing disorders, among others. Here, we investigate the regulation of EC-SOD in pulmonary lung fibroblasts derived from young (up to 3 month) and old (24 month) C57BL6 mice. We found that old fibroblasts have marginally elevated levels of reactive oxidant species (ROS), which coincides with attenuated expression a number of antioxidant enzymes including EC-SOD. Exposure of old fibroblasts to the DNA methyltransferase inhibitor 5-aza-dC did not restore expression of EC-SOD. On the other hand, repression of EC-SOD expression was associated with deacetylation of lysine 9 on histone H3 and lysines 5, 8, 12 and 16 on histone H4 located at the gene promoter. Interestingly, the repressive tri-methylation of lysine 27 on histone H3 was elevated in old compared to young fibroblasts. In addition, exposure of old lung fibroblasts to HDAC class 1 and class 2 inhibitors restored EC-SOD expression to the level observed in young fibroblasts. While the exact mechanism of age-dependent downregulation of EC-SOD is yet to be defined, our studies indicate a potential role of epigenetic mechanisms including histone deacetylation in this process.

Pulmonary hypertension and vascular remodeling in mice exposed to crystalline silica.

BACKGROUND: Occupational and environmental exposure to crystalline silica may lead to the development of silicosis, which is characterized by inflammation and progressive fibrosis. A substantial number of patients diagnosed with silicosis develop pulmonary hypertension. Pulmonary hypertension associated with silicosis and with related restrictive lung diseases significantly reduces survival in affected subjects. An animal model of silicosis has been described previously however, the magnitude of vascular remodeling and hemodynamic effects of inhaled silica are largely unknown. Considering the importance of such information, this study investigated whether mice exposed to silica develop pulmonary hypertension and vascular remodeling. METHODS: C57BL6 mice were intratracheally injected with either saline or crystalline silica at doses 0.2 g/kg, 0.3 g/kg and 0.4 g/kg and then studied at day 28 post-exposure. Pulmonary hypertension was characterized by changes in right ventricular systolic pressure and lung histopathology. RESULTS: Mice exposed to saline showed normal lung histology and hemodynamic parameters while mice exposed to silica showed increased right ventricular systolic pressure and marked lung pathology characterized by a granulomatous inflammatory reaction and increased collagen deposition. Silica-exposed mice also showed signs of vascular remodeling with pulmonary artery muscularization, vascular occlusion, and medial thickening. The expression of pro-inflammatory genes such as TNF-α and MCP-1 was significantly upregulated as well as the expression of the pro-remodeling genes collagen type I, fibronectin and the metalloproteinases MMP-2 and TIMP-1. On the other hand, the expression of several vasculature specific genes involved in the regulation of endothelial function was significantly attenuated. CONCLUSIONS: We characterized a new animal model of pulmonary hypertension secondary to pulmonary fibrosis induced by crystalline silica. Our data suggest that silica promotes the damage of the pulmonary vasculature through mechanisms that might involve endothelial dysfunction, inflammation, and vascular remodeling.

Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice.

Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed as EhCySS. Cultured cells condition their media to reproduce physiological EhCySS, but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the media EhCySS before and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellular EhCySS than young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment.

Regulation of Oxidative Stress in Pulmonary Artery Endothelium. Modulation of Extracellular Superoxide Dismutase and NOX4 Expression Using Histone Deacetylase Class I Inhibitors.

An imbalance between oxidants and antioxidants is considered a major factor in the development of pulmonary vascular diseases. Oxidative stress seen in pulmonary vascular cells is regulated by increased expression of prooxidant enzymes (e.g., nicotinamide adenine dinucleotide phosphate reduced oxidases) and/or decreased production of antioxidants and antioxidant enzymes (e.g., superoxide dismutases). We and others have shown that expression of antioxidant genes in pulmonary artery cells is regulated by epigenetic mechanisms. In this study, we investigate the regulation of oxidative stress in pulmonary artery cells using inhibitors of histone deacetylases (HDACs). Human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells were exposed to an array of HDAC inhibitors followed by analysis of anti- and prooxidant gene expression using quantitative RT-PCR and quantitative RT-PCR array. We found that exposure of HPAECs to scriptaid, N-[4-[(hydroxyamino)carbonyl]phenyl]-α-(1-methylethyl)-benzeneacetamide, and trichostatin A for 24 hours induced expression of extracellular superoxide dismutase (EC-SOD) up to 10-fold, whereas expression of the prooxidant gene NADPH oxidase 4 was decreased by more than 95%. We also found that this differential regulation of anti- and prooxidant gene expression resulted in significant attenuation in the cellular levels of reactive oxygen species. Induction of EC-SOD expression was attenuated by the Janus kinase 2 protein kinase inhibitor AG490 and by silencing Janus kinase 2 expression. Augmentation of EC-SOD expression using scriptaid was associated with increased histone H3 (Lys27) acetylation and H3 (Lys4) trimethylation at the gene promoter. We have determined that oxidative stress in pulmonary endothelial cells is regulated by epigenetic mechanisms and can be modulated using HDAC inhibitors.

The c10orf10 gene product is a new link between oxidative stress and autophagy.

The human c10orf10 gene product, also known as decidual protein induced by progesterone (DEPP), is known to be differentially regulated in mouse tissues in response to hypoxia and oxidative stress, however its biological function remains unknown. We found that mice lacking extracellular superoxide dismutase (EC-SOD) show attenuated expression of DEPP in response to acute hypoxia. DEPP mRNA levels, as well as the activity of a reporter gene expressed under the control of the DEPP 5'-flanking region, were significantly upregulated in Hep3B and Vero cells overexpressing EC-SOD. Subcellular fractionation and immunofluorescent microscopy indicated that overexpressed DEPP is co-localized with both protein aggregates and aggresomes. Further biochemical characterization indicates that DEPP protein is unstable and undergoes rapid degradation. Inhibition of proteasome activities significantly increases DEPP protein levels in soluble and insoluble cytosolic fractions. Attenuation of autophagosomal activity by 3-methyladenine increases DEPP protein levels while activation of autophagy by rapamycin reduced DEPP protein levels. In addition, ectopic overexpression of DEPP leads to autophagy activation, while silencing of DEPP attenuates autophagy. Collectively, these results indicate that DEPP is a major hypoxia-inducible gene involved in the activation of autophagy and whose expression is regulated by oxidative stress.

A bioluminescent transgenic mouse model: real-time in vivo imaging of antioxidant EC-SOD gene expression and regulation by interferon gamma.

Extracellular superoxide dismutase (EC-SOD) is the main antioxidant enzyme in the extracellular matrix. We developed transgenic mice to analyze the EC-SOD promoter activity in vivo in real time and to identify the important cis-elements flanking the 5' region of the murine EC-SOD gene. Using this model, we demonstrated that luciferase reporter activity correlates closely with endogenous EC-SOD expression, although several interesting differences were also observed. Specifically, luciferase activity was detected at the highest levels in testes, aorta and perirenal fat. Reporter expression was regulated by interferon gamma, a finding that is in agreement with published endogenous EC-SOD gene expression studies. Thus, the 5'-flanking region of mouse EC-SOD gene is responsible, at least in part, for cell specific and inducible expression.

Histone acetylation regulates the cell-specific and interferon-γ-inducible expression of extracellular superoxide dismutase in human pulmonary arteries.

Extracellular superoxide dismutase (EC-SOD) is the major antioxidant enzyme present in the vascular wall, and is responsible for both the protection of vessels from oxidative stress and for the modulation of vascular tone. Concentrations of EC-SOD in human pulmonary arteries are very high relative to other tissues, and the expression of EC-SOD appears highly restricted to smooth muscle. The molecular basis for this smooth muscle-specific expression of EC-SOD is not known. Here we assessed the role of epigenetic factors in regulating the cell-specific and IFN-γ-inducible expression of EC-SOD in human pulmonary artery cells. The analysis of CpG site methylation within the promoter and coding regions of the EC-SOD gene demonstrated higher levels of DNA methylation within the distal promoter region in endothelial cells compared with smooth muscle cells. Exposure of both cell types to DNA demethylation agents reactivated the transcription of EC-SOD in endothelial cells alone. However, exposure to the histone deacetylase inhibitor trichostatin A (TSA) significantly induced EC-SOD gene expression in both endothelial cells and smooth muscle cells. Concentrations of EC-SOD mRNA were also induced up to 45-fold by IFN-γ in smooth muscle cells, but not in endothelial cells. The IFN-γ-dependent expression of EC-SOD was regulated by the Janus tyrosine kinase/signal transducers and activators of transcription proteins signaling pathway. Simultaneous exposure to TSA and IFN-γ produced a synergistic effect on the induction of EC-SOD gene expression, but only in endothelial cells. These findings provide strong evidence that EC-SOD cell-specific and IFN-γ-inducible expression in pulmonary artery cells is regulated, to a major degree, by epigenetic mechanisms that include histone acetylation and DNA methylation.

Extracellular superoxide dismutase attenuates release of pulmonary hyaluronan from the extracellular matrix following bleomycin exposure.

The major pulmonary antioxidant enzyme involved in the protection of the lung interstitium from oxidative stress is extracellular superoxide dismutase (EC-SOD). It has been previously shown that EC-SOD knock-out mice are more susceptible to bleomycin-induced lung injury, however, the molecular mechanism(s) remains unclear. We report here that bleomycin-induced lung damage, in EC-SOD KO mice, is associated with increased hyaluronan release into alveolar fluid. Analysis of hyaluronan synthase gene expression and hyaluronan molecular weight distribution suggested that elevated levels of hyaluronan in the alveolar fluid are mostly due to its release from the interstitium. Our results indicate that EC-SOD attenuates bleomycin-induced pulmonary injury, at least in part, by preventing superoxide-mediated release of hyaluronan into alveolar space.

CpG methylation attenuates Sp1 and Sp3 binding to the human extracellular superoxide dismutase promoter and regulates its cell-specific expression.

Extracellular superoxide dismutase (EC-SOD) plays an important role in maintaining normal redox homeostasis in the lung. It is expressed at very high levels in pulmonary fibroblasts, alveolar type II epithelial cells, and smooth muscle cells. The molecular mechanisms governing this cell-specific expression of EC-SOD are mostly unknown. In our previous studies we showed that EC-SOD cell-specific expression was not attributable to differential transcriptional regulation, suggesting that other, possibly epigenetic, mechanisms are involved in regulation of its expression. In this paper, we show high levels of promoter methylation in A549 cells and correspondingly low levels of methylation in MRC5 cells. Inhibition of DNA methyltransferase activity by 5-azacytidine in A549 cells reactivated EC-SOD transcription (2.75+/-0.16-fold, P<0.001), demonstrating the importance of methylation in the repression of EC-SOD expression. Furthermore, methylation of cytosines in the promoter markedly decreased Sp1/Sp3-driven promoter activity to 30.09+/-2.85% (P<0.001) compared to unmethylated promoter. This attenuation of transcription of the promoter/reporter construct was, at least in part, attributable to the binding of the methyl-binding protein MeCP2 in the insect cells. However, no binding of MeCP2 or MBD2 protein to the EC-SOD promoter was detected in mammalian cells in vivo. We also found marked differences in the chromatin organization of the EC-SOD promoter between these two cell lines, further supporting the important role epigenetic modifications play in the regulation of EC-SOD expression.

Novel mechanism for regulation of extracellular SOD transcription and activity by copper: role of antioxidant-1.

Extracellular superoxide dismutase (SOD3), a secretory copper-containing antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Although cofactor copper is required for SOD3 activity, it remains unknown whether it can regulate SOD3 transcription. We previously demonstrated that SOD3 activity requires the copper chaperone antioxidant-1 (Atox1), involved in copper delivery to SOD3 at the trans-Golgi network (TGN). Here we show that copper treatment in mouse fibroblasts significantly increases mRNA and protein levels of SOD3, but not SOD1, which is abolished in Atox1-deficient cells. Copper promotes Atox1 translocation to the nucleus. Promoter deletion analysis identifies copper- and Atox1-response elements (REs) at the SOD3 promoter. Gel-shift and ChIP assays reveal that Atox1 directly binds to the Atox1 RE in a copper-dependent manner in vitro and in vivo. Adenovirus-mediated reexpression in Atox1(-/-) cells of nucleus-targeted Atox1 (Atox1-NLS), but not TGN-targeted Atox1 (Atox1-TGN), increases SOD3 transcription without affecting SOD3 activity. Importantly, reexpression of both Atox1-NLS and Atox1-TGN together, but not either alone, in Atox1(-/-) cells increases SOD3 activity. SOD3 transcription is positively regulated by copper through the transcription factor function of Atox1, whereas the full activity of SOD3 requires both the copper chaperone and the transcription factor functions of Atox1. Thus, Atox1 is a potential therapeutic target for oxidant stress-dependent cardiovascular disease.

Transcription factors sp1 and sp3 regulate expression of human extracellular superoxide dismutase in lung fibroblasts.

The molecular mechanisms that govern the transcription of human extracellular superoxide dismutase (EC-SOD), the major extracellular antioxidant enzyme, are largely unknown. To elucidate the mechanisms involved in human EC-SOD gene regulation and expression, we localized multiple transcription start sites to a finite region located 3.9 kb upstream of the ATG initiation codon. Within this segment, we subcloned a 2.7-kb fragment upstream of a luciferase reporter gene; the resulting construct exhibited strong in vivo promoter activity in two lung-derived cell lines. Deletion analysis of the EC-SOD 5'-flanking sequences identified a minimal 0.3-kb region that had strong basal promoter activity. Computer sequence analysis revealed a putative Sp1-like binding site within the EC-SOD proximal promoter region that lacked a TATA-box and showed a high frequency of GC nucleotides. Binding of Sp1 and Sp3 transcription factors to the EC-SOD promoter was confirmed by DNase I footprint analysis, electophoretic mobility shift assay, and competition and supershift assays. Cotransfection of the EC-SOD promoter-luciferase reporter constructs with plasmids encoding Sp1 and Sp3 into Sp-deficient insect SL2 cells showed strong activation of luciferase gene expression. The occupancy of the EC-SOD promoter by Sp1/Sp3 and RNA polymerase II in vivo was determined by chromatin immunoprecipitation assay and correlated well with levels of EC-SOD expression in lung epithelial cells (A549) and pulmonary fibroblasts (MRC5). Collectively, our results demonstrate the important role Sp1 and Sp3 plays in regulating the expression of human EC-SOD in the lung.

Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox).

Chronic exposure to low-O2 tension induces pulmonary arterial hypertension (PAH), which is characterized by vascular remodeling and enhanced vasoreactivity. Recent evidence suggests that reactive oxygen species (ROS) may be involved in both processes. In this study, we critically examine the role superoxide and NADPH oxidase plays in the development of chronic hypoxic PAH. Chronic hypoxia (CH; 10% O2 for 3 wk) caused a significant increase in superoxide production in intrapulmonary arteries (IPA) of wild-type (WT) mice as measured by lucigenin-enhanced chemiluminescence. The CH-induced increase in the generation of ROS was obliterated in NADPH oxidase (gp91phox) knockout (KO) mice, suggesting that NADPH oxidase was the major source of ROS. Importantly, pathological changes associated with CH-induced PAH (mean right ventricular pressure, medial wall thickening of small pulmonary arteries, and right heart hypertrophy) were completely abolished in NADPH oxidase (gp91phox) KO mice. CH potentiated vasoconstrictor responses of isolated IPAs to both 5-hydroxytryptamine (5-HT) and the thromboxane mimetic U-46619. Administration of CuZn superoxide dismutase to isolated IPA significantly reduced CH-enhanced superoxide levels and reduced the CH-enhanced vasoconstriction to 5-HT and U-46619. Additionally, CH-enhanced superoxide production and vasoconstrictor activity seen in WT IPAs were markedly reduced in IPAs isolated from NADPH oxidase (gp91phox) KO mice. These results demonstrate a pivotal role for gp91phox-dependent superoxide production in the pathogenesis of CH-induced PAH.

Extracellular superoxide dismutase functions as a major repressor of hypoxia-induced erythropoietin gene expression.

Hypoxia and biological responses to hypoxia are commonly encountered in both normal and pathologic cellular processes. Here we report that extracellular superoxide dismutase (EC-SOD) plays a major role in regulating the magnitude of hypoxia-induced erythropoietin (Epo) gene expression, thus implicating superoxide as an intermediary signal transduction molecule critical to this process. We found that mice which have the EC-SOD gene inactivated show a marked more than 100-fold elevation in hypoxia-induced Epo gene expression, compared with wild-type controls, which was both dose and time dependent. These mice also showed a significant increase in serum Epo levels after 1 d hypoxia. Interestingly, despite elevated Epo levels, reciprocal changes in hematocrit and reticulocyte counts were not found, suggesting that this newly synthesized Epo lacks functional hematopoietic effects. When EC-SOD was overexpressed in Hep3B cells, we found a significant reduction in Epo gene induction by both CoCl2 (50 microM) and hypoxia (1% O2). Similar findings were noted with another hypoxia-inducible gene, carbonic anhydrase IX. We conclude that EC-SOD functions as a major repressor of hypoxia-induced Epo gene expression, which implicates superoxide as a signaling intermediate whose downstream effects, at least in part, may be mediated by HIF-1alpha.