Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells

Abstract

Background: Pathogenesis of pulmonary hypertension is complex and involves activation of the transcription factor, hypoxia-inducible factor-1 (HIF-1) that shifts cellular metabolism from aerobic respiration to glycolysis, in part, by increasing the expression of its downstream target pyruvate dehydrogenase kinase-1 (PDK-1), thereby promoting a proliferative, apoptosis-resistant phenotype in pulmonary vascular cells. Activation of the nuclear hormone transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), attenuates pulmonary hypertension and pulmonary artery smooth muscle cell (PASMC) proliferation. In the current study, we determined whether PPARγ inhibits HIF-1α and PDK-1 expression in human PASMCs.

Methods: HPASMCs were exposed to normoxia (21% O2) or hypoxia (1% O2) for 2-72 hours ± treatment with the PPARγ-ligand, rosiglitazone (RSG, 10μM).

Results: Compared to normoxia, HIF-1α mRNA levels were elevated in HPASMC at 2 hours hypoxia and reduced to baseline levels by 24-72 hours. HIF-1α protein levels increased following 4 and 8 hours of hypoxia and returned to baseline levels by 24 and 72 hours. PDK-1 protein levels increased following 24 hours hypoxia and remained elevated by 72 hours. RSG treatment at the onset of hypoxia attenuated HIF-1α protein and PDK-1 mRNA and protein levels at 4, 8 and 24 hours of hypoxia, respectively. However, RSG treatment during final 24 hours of 72-hour hypoxia, an intervention that inhibits HPASMC proliferation, failed to prevent hypoxia-induced PDK-1 expression.

Conclusion: Hypoxia causes transient activation of HPASMC HIF-1α that is attenuated by RSG treatment initiated at hypoxia onset. These findings provide novel evidence that PPARγ modulates fundamental and acute cellular responses to hypoxia through both HIF-1-dependent and HIF-1-independent mechanisms.

Keywords: HIF-1α; PDK-1; PPARγ; Pulmonary hypertension; Vascular smooth muscle cell.

FIGURE 1

Time course of hypoxia-induced HIF-1α activation in HPASMCs. (A) HPASMC HIF-1α mRNA levels were measured with qRT-PCR following exposure to hypoxia (1% O₂) for 2, 8, 24 or 72 hours. Data are expressed relative to ribosomal 9S and displayed as fold change versus (vs.) normoxic (21% O₂) control ± SE at the same time point (n = 4−16). ^*P < 0.05 vs. normoxia and ^**P < 0.01 vs. normoxia. (B) Quantitative densitometric analysis of Western blots for HIF-1α in nuclear protein extracts of HPASMCs exposed to normoxia (N, 21% O₂) or hypoxia (1% O₂) for 4 and 8 hours. In separate experiments, cells were exposed to normoxia or hypoxia for 72 hours. Each bar represents mean ± SE HIF-1α nuclear protein relative to fibrillarin levels in the same sample expressed as fold change over normoxia (n = 3−7). ^*P < 0.05 vs. normoxia and ^**P < 0.01 vs. normoxia. SE, standard error.

FIGURE 2

Rosiglitazone attenuates early HIF-1α expression in HPASMCs exposed to hypoxia. HPASMCs were exposed to normoxia (21% O₂) or hypoxia (1% O₂) and simultaneously treated with DMSO (Veh) or rosiglitazone (RSG) (10 μM) for 2–4 hours. (A) HPASMC HIF-1α mRNA levels following treatment for 2 hours. Each bar graph represents mean ± SE HIF-1α mRNA normalized to GAPDH in the same sample expressed as fold change over normoxia (n = 3). SE, standard error. (B) Representative immunoblots and averaged desitometric analysis of Western blotting for HIF-1α levels in HPASMC nuclear protein extracts treated for 4 hours. Each bar represents mean ± SE nuclear HPASMC HIF-1α protein levels relative to fibrillarin in the same sample expressed as fold change over normoxia (n = 3). **P < 0.05 versus (vs.) Normoxia-Veh and ^##P < 0.001 vs. Hypoxia-Veh.

FIGURE 3

Hypoxia-induced PDK-1 expression in HPASMCs is attenuated by rosiglitazone. (A) HPASMCs PDK-1 mRNA levels were measured with qRT-PCR following exposure to hypoxia (1% O₂) for 0, 2, 8 or 24 hours. Each bar represents the mean ± SE PDK-1 mRNA relative to ribosomal 9S expressed as fold change versus (vs.) normoxic control (n = 4). HPASMCs were then exposed to normoxia (21% O₂) or hypoxia (1% O₂) for 8 or 24 hours and simultaneously treated with DMSO (Veh) or rosiglitazone (RSG, 10 μM). (B) HPASMC PDK-1 mRNA levels were determined with qRT-PCR following exposure to hypoxia for 8 hours. Each bar represents mean ± SE PDK-1 mRNA relative to ribosomal 9S expressed as fold change vs. normoxic control (n = 4). ^***P < 0.001 vs. Normoxia-Veh and ^#P < 0.05 vs. Hypoxia-Veh. (C) PDK-1 protein levels were examined in HPASMC lysates by Western blot analysis following exposure to hypoxia for 24 hours. Each bar represents mean ± SE PDK-1 relative to CDK4 levels in the same sample expressed as fold change over normoxia (n = 7). ^*P < 0.05 vs. normoxia. ^##P < 0.01 vs. Hypoxia-Veh. SE, standard error.

FIGURE 4

Rosiglitazone fails to attenuate chronic hypoxia-induced PDK-1 and GLUT1 protein expression in HPASMC. HPASMCs were exposed to normoxia (21% O₂) or hypoxia (1% O₂) for 72 hours. During the final 24 hours of exposure, HPASMCs were treated with DMSO (Veh) or rosiglitazone (RSG, 10 μM). Western blotting was performed to determine PDK-1 (A), GLUT1 (B) or HIF-2α (C) levels in HPASMC. Each bar represents the mean ± SE PDK-1, GLUT1, or HIF-2α levels relative to β-actin in the same sample expressed as fold change over normoxia (n = 6). ^*P < 0.05 versus Normoxia. SE, standard error.

FIGURE 5

Depletion of HIF-1α attenuates chronic hypoxia-induced PDK-1 expression in HPASMC. HPASMCs were transfected with control siRNA or HIF-1α siRNA (to deplete HIF-1α) and then exposed to normoxia or hypoxia for 72 hours. Western blotting was performed to determine PDK-1 levels. Depletion of HIF-1α was verified by Western blotting using an antibody against HIF-1α. Each bar represents mean ± SE PDK-1 level relative to β-actin in the same sample expressed as fold change over normoxia (n = 3). ^**P < 0.01 versus (vs.) Normoxia; ^##P < 0.01 vs. hypoxia. SE, standard error.