The regulation of hypoxic genes by calcium involves c-Jun/AP-1, which cooperates with hypoxia-inducible factor 1 in response to hypoxia

Abstract

Hypoxia causes the accumulation of the transcription factor hypoxia-inducible factor 1 (HIF-1), culminating in the expression of hypoxia-inducible genes such as those for vascular endothelial growth factor (VEGF) and NDRG-1/Cap43. Previously, we have demonstrated that intracellular calcium (Ca(2+)) is required for the expression of hypoxia-inducible genes. Here we found that, unlike with hypoxia or hypoxia-mimicking conditions, the elevation of intracellular Ca(2+) neither induced the HIF-1alpha protein nor stimulated HIF-1-dependent transcription. Furthermore, the elevation of intracellular Ca(2+) induced NDRG-1/Cap43 mRNA in HIF-1alpha-deficient cells. It also increased levels of c-Jun protein, causing its phosphorylation. The protein kinase inhibitor K252a abolished c-Jun induction and activator protein 1 (AP-1)-dependent reporter expression caused by Ca(2+) ionophore or hypoxia. K252a also significantly decreased hypoxia-induced VEGF and NDRG-1/Cap43 gene expression in both human and mouse cells. Using a set of deletion VEGF-Luc promoter constructs, we found that both HIF-1 and two AP-1 sites contribute to hypoxia-mediated induction of transcription. In contrast, only AP-1 sites contributed to Ca(2+)-mediated VEGF-Luc induction. A dominant-negative AP-1 prevented Ca(2+)-dependent transcription and partially impaired hypoxia-mediated transcription. In addition, dominant-negative AP-1 diminished the expression of the NDRG-1/Cap43 gene following hypoxia. We conclude that during hypoxia, an increase in intracellular Ca(2+) activates a HIF-1-independent signaling pathway that involves AP-1-dependent transcription. Cooperation between the HIF-1 and AP-1 pathways allows fine regulation of gene expression during hypoxia.

FIG. 1.

Calcium ionophore A23187 does not induce HIF-1-dependent transcription in MEF or in A549 cells. (A) HIF-1-dependent transcription was not affected by the calcium ionophore in A549 cells. Cells were transfected with 1 μg of HRE-Luc or 1 μg of plasmid CMV-Luc by using TransFast transfection reagent. On the day after transfection, cells were treated in some instances with 0.16 or 0.8 mM nickel chloride, hypoxia (1% O₂), or 5 μM A23187 for 20 h. Cells were lysed and luciferase activity was determined as described previously (28). The data are the ratios of luciferase expression on HRE-Luc to that on CMV-Luc (HRE/CMV Ratio). (B) HIF-1-dependent transcription was not affected by calcium ionophore in MEF. The exposure conditions were similar to those described for panel A. (C) The HIF-1α protein level was not affected by calcium ionophore. Sixty micrograms of nuclear extracts from A549 cells untreated or treated with 5 μM A23187, 10 μM BAPTA-AM, or 260 μM DFX for 20 h was resolved by SDS-8% PAGE and subjected to Western blot analysis for detection of HIF-1α and p53 as described in Materials and Methods. The detection of another nuclear protein, p53, in the same samples was conducted to confirm protein loading in the cells.

FIG. 2.

Calcium ionophore A23187 stimulated expression of NDRG-1/Cap43 in a HIF-1-independent manner. (A) Northern blot analysis of NDRG-1/Cap43 expression in HIF-proficient and HIF-deficient fibroblasts. Cells were exposed to 0.5 or 1 mM nickel chloride or 1 or 5 μM A23187 for 20 h. Total RNA was isolated and subjected to Northern blot analysis. NDRG-1/Cap43 cDNA was used as a probe. The bottom panel shows ethidium bromide staining of the same gel. (B) Quantitation of NDRG-1/Cap43 expression with a PhosphorImager. The labeling is identical to that described for panel A. The multiple of induction over the control level is shown on top of each bar. (C) Western blot analysis of NDRG-1/Cap43 protein levels in HIF-proficient and HIF-deficient fibroblasts. Cells were exposed to 1 mM nickel chloride, 300 μM cobalt chloride, or 4 μM A23187 for 20 h. Cells were lysed and extracts were resolved by SDS-10% PAGE.

FIG. 3.

Cyclosporine did not suppress NDRG-1/Cap43 gene induction. (A) Northern blot analysis of NDRG-1/Cap43 expression in A549 cells. Cells were exposed to 0.5 or 1 mM nickel chloride or 5 μM A23187 in the absence or presence of cyclosporine for 20 h. Total RNA was isolated and subjected to Northern blot analysis. The bottom panel shows ethidium bromide staining of the same gel. (B) Northern blot analysis of NDRG-1/Cap43 expression in MEF. The exposure was similar to that described for panel A.

FIG. 4.

The AP-1 transcription factor was involved in calcium-dependent expression of hypoxic genes. (A) c-Jun protein was induced by calcium ionophore A23187 and was sensitive to the K252a inhibitor. A549 cells were exposed to 1 mM nickel chloride, hypoxia, or 5 μM A23187 in the presence or absence of the KN93 or K252a inhibitor. Forty micrograms of protein extracts from untreated or treated cells was resolved by SDS-10% PAGE and subjected to Western blot analysis for c-Jun protein as described in Materials and Methods. (B) AP-1-dependent transcription was induced by the calcium ionophore and was sensitive to the K252a inhibitor. A549 cells were transfected with 1 μg of the HRE-Luc or 1 μg of the AP-1-Luc reporter plasmid as described in Materials and Methods. The efficiency of transfection has been corrected by using plasmid CMV-Luc.

FIG. 5.

NDRG-1/Cap43 and VEGF expression was sensitive to the K252a inhibitor. Shown are the results of a Northern blot analysis of NDRG-1/Cap43 (A) and VEGF (B) expression in A549 cells. Cells were exposed to 1 mM nickel chloride, hypoxia, or 5 μM A23187 in the presence or absence of the KN93 or K252a inhibitor. Total RNA was isolated and subjected to Northern blot analysis by using the NDRG-1/Cap43 (A) and VEGF (B) probes. The darker panels show ethidium bromide staining of the same gels.

FIG. 6.

Role of AP-1 in the induction of VEGF-Luc by hypoxia or calcium. A549 cells were transfected with 1 μg of the VEGF-Luc (A) or 1 μg of the HRE-Luc (B) reporter plasmid as described in Materials and Methods. + DN, cells cotransfected with 1 μg of DN AP-1. In all other cases, cells were cotransfected with an empty vector. After transfection, cells were either incubated at 1% O₂ for 16 h (hypoxia) or treated with a 5 μM concentration of calcium ionophore A23187. Cells were lysed and luciferase activity was determined as described previously (28). Results were calculated as percentages of the control level and are means ± standard deviations.

FIG. 7.

Role of AP-1 in the induction of VEGF-Luc by hypoxia or calcium. A549 cells were transfected with 1 μg of pGL3-V2274, containing the full-length VEGF promoter (bars labeled 1); pGL3-V1012, containing the full-length promoter minus two AP-1 sites (bars labeled 2); pGL3-V789, minus the HIF-1 site (bars labeled 3); and pGL3-V267, minus a third AP-1 site (bars labeled 4). Cells were either incubated under 1% O₂ for 16 h (hypoxia; shaded bars), treated with 260 μM DFX (striped bars), treated with a 5 μM concentration of calcium ionophore (solid bars), or left untreated (control; open bars). Cells were lysed and luciferase activity was determined as described previously (28). Results are expressed as percentages of the control level (the activity of full-length VEGF-Luc left untreated) and are means ± standard deviations.

FIG. 8.

Role of AP-1 sites in the induction of pGL3-V2274 by hypoxia. A549 cells were transfected with 1 μg of VEGF-Luc (solid bars) reporter plasmid or 1 μg of pGL3-V1012 reporter plasmid lacking two AP-1 sites (open bars) as described in Materials and Methods. + DN, cells cotransfected with 1 μg of DN AP-1. Otherwise, cells were cotransfected with an empty vector. Cells were either incubated with 1% O₂ for 16 h (hypoxia and hypoxia + DN) or left at normal levels of oxygen (control and DN). Cells were lysed and luciferase activity was determined as described previously (28). Results were calculated as percentages of the control level (the activity of full-length VEGF-Luc under normal oxygen conditions) and are means ± standard deviations.

FIG. 9.

Role of AP-1 in hypoxia-mediated induction of NDRG-1/Cap43. The results of a Northern blot analysis of NDRG-1/Cap43 expression in A549 cells are shown. + DN, cells cotransfected with 10 μg of DN AP-1. Otherwise, cells were cotransfected with an empty vector. Cells were either incubated at 1% O₂ for 16 h (hypoxia and hypoxia + DN) or left at normal levels of oxygen (control and DN). Total RNA was isolated and subjected to Northern blot analysis by using the NDRG-1/Cap43 probe. The bottom panel shows ethidium bromide staining of the same gel.

FIG. 10.

Illustration of the regulation of hypoxia-inducible genes. Under hypoxic conditions, the accumulation of HIF-1a was accompanied by an elevation of intercellular calcium. HIF-1 directly transactivates hypoxia-inducible genes. Ca activates the JNK/AP-1 pathway, which contributes to transactivation, and potentially may participate in the stabilization of the mRNA of hypoxia-inducible genes.