PPAR{gamma} regulates hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells through NF-{kappa}B - PubMed (original) (raw)

PPAR{gamma} regulates hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells through NF-{kappa}B

Xianghuai Lu et al. Am J Physiol Lung Cell Mol Physiol. 2010 Oct.

Abstract

NADPH oxidases are a major source of superoxide production in the vasculature. The constitutively active Nox4 subunit, which is selectively upregulated in the lungs of human subjects and experimental animals with pulmonary hypertension, is highly expressed in vascular wall cells. We demonstrated that rosiglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor-γ (PPARγ), attenuated hypoxia-induced pulmonary hypertension, vascular remodeling, Nox4 induction, and reactive oxygen species generation in the mouse lung. The current study examined the molecular mechanisms involved in PPARγ-regulated, hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells (HPASMC). Exposing HPASMC to 1% oxygen for 72 h increased Nox4 gene expression and H(2)O(2) production, both of which were reduced by treatment with rosiglitazone during the last 24 h of hypoxia exposure or by treatment with small interfering RNA (siRNA) to Nox4. Hypoxia also increased HPASMC proliferation as well as the activity of a Nox4 promoter luciferase reporter, and these increases were attenuated by rosiglitazone. Chromatin immunoprecipitation assays demonstrated that hypoxia increased binding of the NF-κB subunit, p65, to the Nox4 promoter and that binding was attenuated by rosiglitazone treatment. The role of NF-κB in Nox4 regulation was further supported by demonstrating that overexpression of p65 stimulated Nox4 promoter activity, whereas siRNA to p50 or p65 attenuated hypoxic stimulation of Nox4 promoter activity. These results provide novel evidence for NF-κB-mediated stimulation of Nox4 expression in HPASMC that can be negatively regulated by PPARγ. These data provide new insights into potential mechanisms by which PPARγ activation inhibits Nox4 upregulation and the proliferation of cells in the pulmonary vascular wall to ameliorate pulmonary hypertension and vascular remodeling in response to hypoxia.

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Figures

Fig. 1.

Rosiglitazone (Rosi) attenuates hypoxia-induced human pulmonary artery smooth muscle cell (HPASMC) Nox4 expression, H2O2 generation, and proliferation. HPASMC were exposed for 72 h to control (21% O2; C) or hypoxic (1% O2; H) conditions, and during the last 24 h of exposure, selected cells were treated with rosiglitazone (10 μM) or an equivalent volume of vehicle. In A, HPASMC were collected, and RNA was isolated and reverse-transcribed to analyze mRNA levels using Nox4 and GAPDH primers. Cells treated without and with methylcellulose vehicle (M) were examined. Each bar represents the mean ± SE copies of Nox4 mRNA normalized to copies of GAPDH expressed as fold change relative to control from 2 experiments performed in triplicate. *P < 0.05 vs. C; **P < 0.05 vs. H. In B, HPASMC media were collected and subjected to assays of H2O2 concentration. Each bar represents the mean ± SE H2O2 concentration from 2 experiments, each performed in triplicate. In C, data from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assays were recorded in absorbance units. Each bar represents HPASMC proliferation as the mean ± SE fold change relative to control. n = 3. *P < 0.05 for H vs. C; **P < 0.05 for H+Rosi vs. H.

Fig. 2.

Nox4 RNA interference reduced hypoxia-induced H2O2 generation and HPASMC proliferation. HPASMC were transfected with control scrambled (siC) or Nox4 siRNA (siNox4) and subsequently exposed for 72 h to control (21% O2) or hypoxic (1% O2) conditions, and during the last 24 h of exposure, selected cells were treated with rosiglitazone (10 μM) or an equivalent volume of vehicle. HPASMC media were thereafter collected and subjected to assays of H2O2 concentration. In A, each bar represents the mean ± SE H2O2 concentration from 2 experiments, each performed in triplicate. *P < 0.05 for C+siNox4 vs. C+siC; **P < 0.05 for H+siC vs. C+siC; ***P < 0.05 for H+siNox4 vs. H+siC. Representative results from real-time PCR of Nox4 mRNA are presented (inset), demonstrating effective small interfering RNA (siRNA)-mediated knockdown of Nox4. Knockdown of Nox4 in protein levels by siNox4 is also shown in the representative Western blot under the graph. In B, data from MTT cell proliferation assays were recorded in absorbance units. Each bar represents HPASMC proliferation as the mean ± SE fold change relative to control from 2 experiments performed in quadruplicate. *P < 0.05 for H+siC vs. C+siC; **P < 0.05 for H+siNox4 vs. H+siC. CDK4, cyclin-dependent kinase 4.

Fig. 3.

Map of the human Nox4 promoter sequence −718 to +241 bp showing predicted binding sites for transcription factors based on homology with known consensus sequences. Homologous binding sequences were determined at >90% probability using MatInspector (Genomatix, Munich, Germany). The base pairs are numbered relative to the Nox4 start site, shown as +1. The translation start site methionine is coded 238 bp downstream (data not shown). Response elements for selected transcription factors that may be activated in hypoxic conditions are labeled in the figure, including peroxisome proliferator-activated receptor (PPAR) response element (PPRE), forkhead domain factor (FKHD), hypoxia response element (HRE), nuclear respiratory factor-1 (NRF-1), and NF-κB p65 and c-rel sites. RXR, retinoid X receptor; HIF, hypoxia-inducible factor.

Fig. 4.

Hypoxia, rosiglitazone, and NF-κB regulate Nox4 promoter activity. C3H/10T1/2 cells were transfected with the human Nox4 promoter luciferase reporter construct along with Renilla as a control for transfection efficiency. In A, cells were exposed for 72 h to control (21% O2) or hypoxic (1% O2) conditions, and during the last 24 h of exposure, selected cells were treated with rosiglitazone (10 μM) or an equivalent volume of vehicle. Cells were then harvested and subjected to assays for luciferase activity. Each bar represents the mean ± SE luciferase activity in each sample relative to Renilla expressed in arbitrary units from 3 experiments performed in triplicate. *P < 0.05 vs. C; **P < 0.05 vs. H. In B, C3H/10T1/2 cells were transfected with the human Nox4 promoter luciferase reporter construct along with Renilla and with either pcDNA3 NF-κB-p50 or pcDNA3 NF-κB-p65 to overexpress NF-κB subunits or empty pcDNA3 vector (control). After 48 h, cells were harvested for luciferase activity assays. Each bar represents the mean ± SE luciferase activity relative to Renilla in each sample expressed in arbitrary units from 3 separate experiments. *P < 0.05 vs. C; **P < 0.05 vs. C. In C, C3H/10T1/2 cells were transfected with the human Nox4 promoter luciferase reporter construct along with a Renilla luciferase construct. The cells were also cotransfected with the NF-κB inhibitor, pcDNA3 cylindromatosis (CYLD), or empty pcDNA3 vector (vector control; VC) and exposed to hypoxic (1% O2) conditions. After 48 h, the cells were harvested for luciferase activity assays. Each bar represents the mean ± SE luciferase activity relative to Renilla in each sample expressed in arbitrary units from 3 separate experiments. *P < 0.05 for H+CYLD vs. H+VC. In D, C3H/10T1/2 cells were transfected with the human Nox4 promoter luciferase reporter construct along with Renilla luciferase construct. The cells were also cotransfected with siRNA to p50 (si-p50) or p65 (si-p65) or with siC and exposed to hypoxic (1% O2) conditions for 48 h. The cells were then harvested for luciferase activity assays. Each bar represents the mean ± SE luciferase activity relative to Renilla in each sample expressed in arbitrary units from 3 separate experiments. *P < 0.05 vs. H+siC.

Fig. 5.

NF-κB binding to the Nox4 promoter is stimulated by hypoxia and attenuated by rosiglitazone treatment. HPASMC were exposed for 72 h to control (21% O2) or hypoxic (1% O2) conditions, and during the last 24 h of exposure, selected cells were treated with rosiglitazone (10 μM) or an equivalent volume of vehicle. Chromatin immunoprecipitation (ChIP) assays were performed with antibodies against human NF-κB p65 or control IgG. Immunoprecipitated DNA was analyzed by real-time PCR using primers specific for the Nox4 promoter surrounding the putative binding sites. Each bar represents the mean relative NF-κB binding to the Nox4 promoter ± SE from 2 experiments performed in triplicate. *P < 0.05 vs. C; **P < 0.05 vs. H.

References

1. Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem 279: 45935–45941, 2004 - PubMed
1. Ameshima S, Golpon H, Cool CD, Chan D, Vandivier RW, Gardai SJ, Wick M, Nemenoff RA, Geraci MW, Voelkel NF. Peroxisome proliferator-activated receptor gamma (PPARgamma) expression is decreased in pulmonary hypertension and affects endothelial cell growth. Circ Res 92: 1162–1169, 2003 - PubMed
1. Anrather J, Racchumi G, Iadecola C. NF-kappaB regulates phagocytic NADPH oxidase by inducing the expression of gp91phox. J Biol Chem 281: 5657–5667, 2006 - PubMed
1. Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87: 245–313, 2007 - PubMed
1. Bonello S, Zahringer C, BelAiba RS, Djordjevic T, Hess J, Michiels C, Kietzmann T, Gorlach A. Reactive oxygen species activate the HIF-1alpha promoter via a functional NFkappaB site. Arterioscler Thromb Vasc Biol 27: 755–761, 2007 - PubMed

PPAR{gamma} regulates hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells through NF-{kappa}B - PubMed (original) (raw)