Specific inhibition of hypoxia inducible factor 1 exaggerates cell injury induced by in vitro ischemia through deteriorating cellular redox environment

Abstract

Hypoxia inducible factor 1 (HIF-1) has been suggested to play a critical role in the fate of cells exposed to hypoxic stress. However, the mechanism of HIF-1-regulated cell survival is still not fully understood in ischemic conditions. Redox status is critical for decisions of cell survival, death and differentiation. We investigated the effects of inhibiting HIF-1 on cellular redox status in SH-SY5Y cells exposed to hypoxia or oxygen and glucose deprivation (OGD), coupled with cell death analyses. Our results demonstrated that inhibiting HIF-1alpha expression by HIF-1alpha specific small interfering RNA (siRNA) transfection increased reactive oxygen species generation, and transformed the cells to more oxidizing environments (low GSH/GSSG ratio, low NADPH level) under either hypoxic or OGD exposure. Cell death increased dramatically in the siRNA transfected cells, compared to non-transfected cells after hypoxic/OGD exposures. In contrast, increasing HIF-1alpha expression by desferrioxamine, a metal chelator and hydroxylase inhibitor, induced a more reducing environment (high GSH/GSSG ratio, high NADPH level) and reduced cell death. Further studies showed that HIF-1 regulated not only glucose transporter-1 expression, but also the key enzymes of the pentose phosphate pathway such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. These enzymes are important in maintaining cellular redox homeostasis by generating NADPH, the primary reducing agent in cells. Moreover, catalase significantly decreased cell death in the siRNA-transfected cells induced by hypoxia and OGD. These results suggest that maintenance of cellular redox status by HIF-1 protects cells from hypoxia and ischemia mediated injuries.

Fig. 1

HIF-1α specific siRNA down-regulated HIF-1α expression in SH-SY5Y cells. A) The transfection of HIF-1α specific siRNA into SH-SY5Y cells. (a) A SH-SY5Y cell transfected with HIF-1α siRNA under light microscope. (b) SH-SY5Y cell transfected with HIF-1α siRNA under a fluorescence microscopy. The siRNA was labeled with Alexa Fluor 488 (green) on the 3′ end of the sense strand. (c) SH-SY5Y cell nuclear strained with Hoechst 33258 (blue). (d) Merged image of (b) and (c). Images were taken with a 40 objective. Images shown are representative of at least 10 fields of view. B) A representative of immunoblot of HIF-1α. C) Average of densitometric analyses of HIF-1α expression normalized to β-actin levels. D) The total HIF-1α level in SH-SY5Y cells detected by HIF-1α-Duoset-ELISA. E) The activity of HIF-1α in SH-SY5Y cells detected by HIF-1α activity-Duoset-ELISA. In B, C, D and E, cells were exposed to hypoxia (1%) for 3 hrs. N: normal cells; NC: cells transfected with negative control siRNA; siRNA: cells transfected with HIF-1α specific siRNA (5 nM). CoCl₂ and DFO: cells treated with cobalt or DFO as positive HIF-1α control. Data are mean ± SEM, n=3. *p<0.01 v.s. normal cells; ^#p<0.01 v.s. negative control cells.

Fig. 2

Effect of down-regulation of HIF-1α on cell death after hypoxia and OGD exposures. SH-SY5Y cells were incubated under normal condition (21% O₂) (Normoxia), hypoxic condition (1% O₂) (Hypoxia), and oxygen and glucose deprivation (OGD) for 3 hrs. DFO treatment was used to increase HIF-1 expression. Cell death was assessed by LDH release assay. Data are expressed as mean ± SEM, n=6. N: normal cells; NC: cells transfected with negative control siRNA; siRNA: cells transfected with HIF-1α specific siRNA (5 nM). *p<0.01 v.s. normal cells; ^#p<0.01 v.s. negative control cells.

Fig. 3

Effects of HIF-1α siRNA on intracellular ROS levels after hypoxia and OGD treatments. A) Representatives of cell images with ROS fluorescence. Images were taken with a 10 objective. Images shown are representative of at least 10 fields of view. B) ROS level. The ROS level was measured with the cell-permeable probe dichlorofluorescin diacetate (DCFH-DA). Cells were incubated with 100 μM DCFH-DA (dissolved in DMSO) for 30 min at 37 °C. N: normal control cells; NC: negative control cells; siRNA: HIF-1α siRNA transfected cells (5 nM). All cells were incubated under normal condition (21% O₂) (Normoxia), hypoxic condition (1% O₂) (Hypoxia), and oxygen and glucose deprivation (OGD) for 3 hrs. The results were normalized by protein content. Data are the mean ± SEM. n=3. *p<0.01 v.s. normal control cells. ^#p<0.01 v.s. negative control cells.

Fig. 4

Effects of HIF-1α siRNA on cellular GSH/GSSG ratio after hypoxia treatments. All cells were incubated under hypoxic condition (1% O₂) for 3 hrs. N: normal control cells; NC: negative control cells; siRNA: HIF-1α siRNA transfected cells (5 nM). DFO was used as a positive control. Data are expressed as mean ± SEM (n=6). *p<0.05 v.s. normal control cells; ^#p<0.05 v.s. negative control cells.

Fig. 5

The effect of HIF-1α knockdown on the expression of GLUT1, G6PD, PGD and the total cellular NADPH under hypoxic conditions in SH-SY5Y cells. A) Representative western blot of three independent experiments. β-actin serves as protein loading control. B) Average of densitometric analyses normalized to β-actin. C) The total cellular NADPH concentration. N: normal control cells; NC: negative control cells; siRNA: HIF-1α siRNA transfected cells; DFO was used as positive control. All cells were incubated under a hypoxic condition (1% O₂) for 3 hrs. Data are expressed as mean ± SEM (n=3). ^$p<0.05, *p<0.01 v.s. normal control cells; ^& p<0.05, ^#p<0.01 v.s. negative control cells.

Fig. 6

Effects of SOD mimic MnTMPyP and catalase on cell death and ROS levels of HIF-1α siRNA transfected cells. A) Effect of MnTMPyP and catalase on cell death in HIF-1α siRNA transfected cells exposed to hypoxia and OGD. Cells were incubated under normal condition (21% O₂) (Normoxia), hypoxic condition (1% O₂) (Hypoxia), and oxygen and glucose deprivation (OGD) for 3 hrs. B) Effect of MnTMPyP and catalase on ROS levels in HIF-1α siRNA transfected cells exposed to hypoxia and OGD. C) Effect of MnTMPyP on cell death in control siRNA and HIF-1α siRNA transfected cells exposed to OGD. D) Effect of catalase on cell death in control siRNA and HIF-1α siRNA transfected cells exposed to OGD. MnTMPyP at 5 μM and catalase at 500 units/ml were used to treat cells. Cell death/viability was assessed by LDH release assay. Data are expressed as mean ± SEM (n=6). *p<0.01 v.s. control cells; ^#p<0.01 v.s. MnTMPyP treated cells.

Fig. 7

Dose-depended inhibition of HIF-1α by HIF-1α specific siRNA in SH-SY5Y cells. A) Concentration-dependent transfection rate of HIF-1α specific siRNA. B) A representative of immunoblot of HIF-1α expression in SY-SY5Y cells transfected with HIF-1α specific siRNA at various concentrations. C) Concentration-dependent effect of HIF-1α specific siRNA on ROS levels in the cells exposed to hypoxia and OGD. D) Concentration-dependent effect of HIF-1α specific siRNA on GSH/GSSG ratio in the cells exposed to hypoxia and OGD. E) Concentration -dependent effect of HIF-1α specific siRNA on cell death in the cells exposed to hypoxia and OGD. Cells were transfected with HIF-1α specific siRNA at the concentrations of 0, 0.1, 1.0, 2.5, and 5.0 nM. They were exposed to hypoxia (1%) or OGD for 3 hrs. N: normal cells; NC: cells transfected with negative control siRNA. DFO was used as a positive HIF-1α control. Data are mean ± SEM, n=3.

Scheme 1

HIF-1 may provide cytoprotection through maintaining cellular redox status under hypoxic/ischemic exposures.