Hypoxia up-regulates hypoxia-inducible factor-1alpha transcription by involving phosphatidylinositol 3-kinase and nuclear factor kappaB in pulmonary artery smooth muscle cells

Abstract

The oxygen sensitive alpha-subunit of the hypoxia-inducible factor-1 (HIF-1) is a major trigger of the cellular response to hypoxia. Although the posttranslational regulation of HIF-1alpha by hypoxia is well known, its transcriptional regulation by hypoxia is still under debate. We, therefore, investigated the regulation of HIF-1alpha mRNA in response to hypoxia in pulmonary artery smooth muscle cells. Hypoxia rapidly enhanced HIF-1alpha mRNA levels and HIF-1alpha promoter activity. Furthermore, inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT but not extracellular signal-regulated kinase 1/2 pathway blocked the hypoxia-dependent induction of HIF-1alpha mRNA and HIF-1alpha promoter activity, suggesting involvement of a PI3K/AKT-regulated transcription factor. Interestingly, hypoxia also induced nuclear factor-kappaB (NFkappaB) nuclear translocation and activity. In line, expression of the NFkappaB subunits p50 and p65 enhanced HIF-1alpha mRNA levels, whereas blocking of NFkappaB by an inhibitor of nuclear factor-kappaB attenuated HIF-1alpha mRNA induction by hypoxia. Reporter gene assays revealed the presence of an NFkappaB site within the HIF-1alpha promoter, and mutation of this site abolished induction by hypoxia. In line, gel shift analysis and chromatin immunoprecipitation confirmed binding of p50 and p65 NFkappaB subunits to the HIF-1alpha promoter under hypoxia. Together, these findings provide a novel mechanism in which hypoxia induces HIF-1alpha mRNA expression via the PI3K/AKT pathway and activation of NFkappaB.

Figure 1.

Hypoxia increases HIF-1α mRNA and protein levels. (A) Left, Mice were exposed for 2 h to 10% O₂ (H) or room air (N). Lungs were dissected and HIF-1α mRNA levels were determined by RT-PCR by using specific HIF-1α primers. 18S RNA levels were determined by RT-PCR and used as control. Accuracy of RT-PCR was tested by omitting cDNA (−Ctr). Right, lungs were dissected and exposed ex vivo to 21% O₂ (N) or 1% O₂ (H) for 2 h. HIF-1α mRNA levels were determined by Northern blot by using an HIF-1α RNA probe. 18S RNA levels used as loading control. Representative experiments are shown. (B) PASMC were incubated for different times under hypoxia (1% oxygen). HIF-1α mRNA or protein levels were determined by Northern or Western blots by using a HIF-1α RNA probe or a HIF-1α antibody. 18S or actin were used as loading controls. Representative blots of three experiments are shown. (C) PASMC were pretreated with actinomycin D (Act; 5 μM) for 1 h and exposed to hypoxia for 1 h. HIF-1α mRNA and protein levels were determined by Northern blot and Western blot, respectively. Blots are representative of three experiments. (D) rSMC were transfected with luciferase constructs where part of the HIF-1α promoter was cloned in front of the luciferase gene (pHIF1α-538 and pHIF1α-106) and exposed to hypoxia for 2 h. Luciferase activity was measured (n = 3; *p < 0.05 vs. Ctr).

Figure 2.

Hypoxia activates HIF-1α transcription via a NFκB site located at −197/−188 base pairs within the HIF-1α promoter. (A) PASMC were incubated for 30 min under hypoxia (1% oxygen). The subcellular localization of the NFκB subunits p50 and p65 was investigated by immunofluorescence by using specific antibodies directed against each subunit. As negative control, the first antibody was omitted (2AB). Pictures are representative of three experiments. (B) rSMC were transfected with luciferase constructs containing the wild-type HIF-1α promoter (pHIF1α-538) or the HIF-1α promoter mutated at the NFκB site at −197 [pHIF1α-538m(−197)] or the putative NFκB site at +149 [pHIF1α-538m(+149)]. In addition, cells were transfected with luciferase constructs driven by five NFκB sites. Transfected cells were exposed to hypoxia for 2 h before luciferase activity was measured (n = 3; *p < 0.05 vs. Ctr). (C) Cells were incubated for 1 h under hypoxia (1% oxygen) and chromatin immunoprecipitation was performed using antibodies against p50 and p65. PCR was performed using specific oligonucleotides for the HIF-1α promoter or for the PAI-1 promoter as negative control. (D) Cells were exposed for 1 h to hypoxia, and EMSAs were performed using labeled oligonucleotides containing the putative NFκB binding site located at −197/−188 base pairs of the HIF-1α promoter. Competition assays were performed by adding a 25-fold molar excess of unlabeled wild-type (WT) or mutated oligonucleotides (MT). Gels are representative of three experiments.

Figure 3.

NFκB increases HIF-1α mRNA levels and promoter activity. (A) PASMC were cotransfected with vectors encoding for p50 and p65 (NFκB), IκBdn, or control (Ctr) vector. Cells were incubated for 1 h under hypoxia (1% oxygen). HIF-1α mRNA and protein levels were investigated by Northern and Western blot, respectively. 18S and actin were used as loading controls. Blots are representative of three experiments. (B) rSMC were cotransfected with luciferase constructs driven by the HIF-1α promoter (pHIF1α-538) and with vectors encoding for p50 and p65, IκBdn, or Ctr. Cells were stimulated with hypoxia for 2 h and luciferase activity was measured (n = 3; *p < 0.05 vs. Ctr; #p <0.05 vs. hypoxia-stimulated Ctr).

Figure 4.

Hypoxia activates HIF-1α transcription via PI3K/AKT and NFκB. (A) PASMC were pretreated for 1 h with LY294002 (LY; 10 μM), wortmannin (20 nM), or PD98059 (PD; 25 μM) and exposed to hypoxia for 15 min. Phosphorylation levels of AKT and ERK1/2 were determined by Western blot. Actin was used as loading control. Blots are representative of three experiments. (B) PASMC were pretreated for 1 h with 10 μM LY, 20 nM wortmannin (Wo) or 25 μM PD98059 (PD) and exposed to hypoxia for 1 h. HIF-1α mRNA levels were measured by Northern blot analysis. Blots are representative of three experiments. (C) rSMC were transfected with luciferase constructs driven by the HIF-1α promoter (pHIF1α-538) or by five NFκB sites. Cells were pretreated for 1 h with 10 μM LY, 20 nM wortmannin, or 25 μM PD and exposed to hypoxia for 2 h before luciferase activity was measured (n = 3; *p < 0.05 vs. Ctr; #p < 0.05 vs. hypoxia-stimulated Ctr).