Hydrogen sulfide inhibits hypoxia- but not anoxia-induced hypoxia-inducible factor 1 activation in a von hippel-lindau- and mitochondria-dependent manner

Abstract

Aims: In addition to nitric oxide and carbon monoxide, hydrogen sulfide (H(2)S) is an endogenously synthesized gaseous molecule that acts as an important signaling molecule in the living body. Transcription factor hypoxia-inducible factor 1 (HIF-1) is known to respond to intracellular reduced oxygen (O(2)) availability, which is regulated by an elaborate balance between O(2) supply and demand. However, the effect of H(2)S on HIF-1 activity under hypoxic conditions is largely unknown in mammalian cells. In this study, we tried to elucidate the effect of H(2)S on hypoxia-induced HIF-1 activation adopting cultured cells and mice.

Results: The H(2)S donors sodium hydrosulfide and sodium sulfide in pharmacological concentrations reversibly reduced cellular O(2) consumption and inhibited hypoxia- but not anoxia-induced HIF-1α protein accumulation and expression of genes downstream of HIF-1 in established cell lines. H(2)S did not affect HIF-1 activation induced by the HIF-α hydroxylases inhibitors desferrioxamine or CoCl(2). Experimental evidence adopting von Hippel-Lindau (VHL)- or mitochondria-deficient cells indicated that H(2)S did not affect neosynthesis of HIF-1α protein but destabilized HIF-1α in a VHL- and mitochondria-dependent manner. We also demonstrate that exogenously administered H(2)S inhibited HIF-1-dependent gene expression in mice.

Innovation: For the first time, we show that H(2)S modulates intracellular O(2) homeostasis and regulates activation of HIF-1 and the subsequent gene expression induced by hypoxia by using an in vitro system with established cell lines and an in vivo system in mice.

Conclusions: We demonstrate that H(2)S inhibits hypoxia-induced HIF-1 activation in a VHL- and mitochondria-dependent manner.

FIG. 1.

Hydrogen sulfide donors inhibited 1% O₂-induced HIF-1 protein expression and HIF-1–dependent gene expression. Hep3B cells were exposed to the indicated concentrations of NaHS (A) or Na₂S (B) for 4 h under 20% or 1% O₂. After treatment, cells were harvested, and whole-cell lysates were subjected to an immunoblot assay for HIF-1α, HIF-1β, or β-actin protein expression. (C) Hep3B cells were exposed to the indicated concentrations of NaHS for 2 or 8 h under 20% or 1% O₂. Experiments were repeated thrice. Representative immunoblots are shown (A, B, and C; left panels). Band intensities were densitometrically analyzed. Fold induction relative to lane 1 was plotted as mean±SD (A and B; right panels). *p<0.05 compared with control; ^#p<0.05 for comparisons between the indicated groups. (D) Hep3B cells were exposed to the indicated concentrations of NaHS for 8 h under 20% or 1% O₂ and harvested for semi-quantitative RT-PCR for vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), Lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase-1 (PDK-1). Experiments were repeated at least thrice in triplicate. *p<0.05 compared with the control (20% and no treatment), ^#p<0.05 for comparisons between the indicated groups. N.S., not statistically significant; HIF-1, hypoxia-inducible factor 1; NaHS, sodium hydrosulfide; Na₂S, sodium sulfide.

FIG. 2.

Inhibition of HIF-1 by NaHS was observed in cells from various kinds of tissues. SH-SY5Y cells (A) or HeLa cells (B) were exposed to the indicated concentrations of NaHS for 4 h under 20% or 1% O₂. After treatment, cells were harvested for Western blot analysis, and whole-cell lysates were subjected to an immunoblot assay for HIF-1α and HIF-1β (A and B). Representative immunoblots are shown (A and B; left panels). SH-SY5Y cells (A; right panel), HeLa cells (B; right panel), primary cultured mouse hepatocytes (C), or human aortic smooth muscle cells (HASMCs) (D) were exposed to the indicated concentrations of NaHS for 8 h under 20% or 1% O₂ and harvested for semi-quantitative RT-PCR by using primer pairs for indicated genes. Experiments were repeated at least thrice in triplicate. *p<0.05 compared with the control (20% and no treatment), ^#p<0.05 for comparisons between the indicated groups.

FIG. 3.

NaHS inhibited hypoxia-induced HIF-1α stabilization. (A and B) Hep3B cells were exposed to the indicated concentrations of NaHS for 4 h under 20% O₂ conditions and subjected to a cytotoxicity assay by using MTS/PES as described in Materials and Methods section (A), and cells were subjected to an immunoblot assay for PARP and cleaved Caspase 3 expression (B). c-caspase-3; cleaved Caspase 3 (C) Hep3B cells or HeLa cells were exposed to the indicated concentrations of NaHS for 4 h under 20% or 1% O₂. After treatment, cells were harvested for Western blot analysis, and whole-cell lysates were subjected to an immunoblot assay for HIF-1α or HIF-2α. (D) Hep3B cells were exposed to the indicated concentrations of NaHS for 8 h under 20% or 1% O₂ and harvested for semi-quantitative RT-PCR by using primer sets for HIF-1α and HIF-1β. (E) Hep3B cells were exposed to 1% O₂ for 4 h, and then cycloheximide (CHX) was added to a final concentration of 100 μM with or without 100 μM NaHS in a hypoxic workstation. Cells were incubated for 0–90 min, and whole-cell lysates were subjected to an immunoblot assay by using anti-HIF-1α or anti-HIF-1β antibodies (left panel). Relative intensities of HIF-1α signals were quantified by densitometric analysis of the immunoblots. Normalized values to time 0 as 1 were plotted against time (right panel). A representative blot is shown in the left panel. Results are mean±SD of three independent experiments. (F) Hep3B cells were transfected with the HIF-1–dependent reporter gene p2.1, encoding firefly luciferase, downstream of an HRE and SV40 promoter, and pRL-SV40, encoding Renilla luciferase. After 6-h incubation, cells were treated with the indicated concentrations of NaHS for 4 h. The ratio of firefly to Renilla luciferase activity was normalized to the value obtained for untreated cells to obtain a relative light unit (RLU). Results are mean±SD of three independent transfections. *p<0.05 compared with respective control; ^#p<0.05 for comparisons between the indicated groups (G) HeLa/ODD-Luc cells were exposed to 20% or 1% O₂ conditions under treatment with 1000 μM NaHS or 2000 μM sodium azide for 4 h. Cells were harvested, and luciferase activity in each well was measured by using the same amount of cell lysate in the dual-luciferase reporter assay system (Promega). The luciferase activity was normalized to the value obtained for untreated cells to obtain a relative light unit (RLU). Results are mean±SD of three independent wells. *p<0.05 compared with respective control under 20% O₂ conditions; ^#p<0.05 for comparisons between the indicated groups (H) Hep3B cells were treated with 1000 μM NaHS, 10 μM MG132, and/or 100 μM CHX for 4 h under 1% O₂ and harvested for immunoblot assays for HIF-1α and HIF-1β. (I) Hep3B cells were exposed to 1000 μM NaHS under 20% and 1% O₂ for 1 h. Cells were harvested, and lysates were subjected to an immunoblot assay. The phosphorylation status of Erk1/2, Akt, mTOR, and p70 S6K was examined in an immunoblot assay with anti-phosphospecific antibodies raised against the respective kinases. HRE, hypoxia-response element.

FIG. 4.

NaHS inhibited hypoxia-induced but not anoxia-induced HIF-1α protein expression. (A) Hep3B cells were exposed to 1000 μM NaHS for 4 h under 20%, 3%, 1%, or 0.1% O₂. Cells were harvested for Western blot analysis after treatment. Whole-cell lysates were subjected to an immunoblot assay for HIF-1α and HIF-1β. A representative immunoblot is shown (A; left panel). Band intensities were densitometrically analyzed from two independent immunoblots, and mean relative expression ratios of band density of NaHS-treated cells to those of cells without NaHS treatment are shown (A; right panel). (B) Hep3B cells were exposed to 1000 μM NaHS for 8 h under 20%, 1%, or 0.1% O₂ and harvested for semi-quantitative RT-PCR with a primer set for VEGF. Results are mean±SD of three independent experiments (B; left panel). HeLa/5HRE-Luc cells were treated with the 1000 μM NaHS for 8 h under 20%, 1%, and 0.1% O₂. The firefly luciferase activity was determined and normalized to the value required for untreated cells to obtain relative light units (RLU) (B; right panel). Results are mean±SD of three independent transfections. *p<0.05 compared with the control, ^#p<0.05 for comparisons between the indicated groups. (C) Hep3B cells were exposed to 100 μM CoCl₂ or 100 μM DFX with or without the indicated concentrations of NaHS for 4 h. (D) RCC4 and RCC4-von Hippel-Lindau cells were exposed to 20% and 1% O₂ with or without 1000 μM NaHS for 4 h. After treatment, cells were harvested, and whole-cell lysates were subjected to immunoblot assay for HIF-1α and HIF-1β.

FIG. 5.

Involvement of mitochondria in inhibition of HIF-1 activation by NaHS. (A) Hep3B cells were exposed to the indicated concentrations of NaHS with or without 10 mM NAC treatment for 4 h and harvested for immunoblot assays with anti-HIF-1α. (B) Hep3B cells were exposed to 1% O₂ with (+) or without (−) treatment with the indicated reagents (0.5 μM rotenone, 1 μg/ml antimycin A, and 5 mM sodium azide). After treatment, cells were harvested, and whole-cell lysates were subjected to an immunoblot assay for HIF-1α. (C) EB8 and HeEB1 cells were exposed to 20% and 1% O₂ and treated with the indicated concentrations of NaHS for 4 h. After treatment, the cells were harvested, and whole-cell lysates were subjected to an immunoblot assay for HIF-1α and HIF-1β. (D) Oxygen concentration curves were generated by using a Clark electrode for HeLa, EB8, and HeEB1 cell suspensions. Arrows indicate addition of 2 mM sodium azide or 1 mM NaHS. The slope of the curve is a measure of the rate of O₂ consumption.

FIG. 6.

NaHS inhibited HIF-1–dependent gene expression in vivo. NaHS (2.0 mg/kg) was intraperitoneally administered to mice under 20% and 10% O₂ atmosphere for 4 h. VEGF, GLUT-1, and HIF-1α mRNA levels in brain (A), kidney (B), and liver (C) were analyzed by quantitative real-time RT-PCR. Results are mean±SD of three independent mice. *p<0.05 compared with the control (20% and no treatment), ^#p<0.05 for comparisons between the indicated groups.