Antioxidants, HIF prolyl hydroxylase inhibitors or short interfering RNAs to BNIP3 or PUMA, can prevent prodeath effects of the transcriptional activator, HIF-1alpha, in a mouse hippocampal neuronal line

Abstract

Hypoxia-inducible factor (HIF) is a transcriptional activator that promotes death or survival in neurons. The regulators and targets of HIF-1alpha-mediated death remain unclear. We found that prodeath effects of HIF-1 are not attributable to an imbalance in HIF-1alpha and HIF-1beta expression. Rather, the synergistic death caused by oxidative stress and by overexpression of an oxygen-resistant HIF-VP16 in neuroblasts was attributable to transcriptional upregulation of BH3-only prodeath proteins, PUMA or BNIP3. By contrast, overexpression of BNIP3 was not sufficient to potentiate oxidative death. As acidosis is known to activate BNIP3-mediated death, we examined other secondary stresses, such as oxidants or prolyl hydroxylase activity are necessary for exposing the prodeath functions of HIF in neurons. Antioxidants or prolyl hydroxylase inhibition prevented potentiation of death by HIF-1alpha. Together, these studies suggest that antioxidants and PHD inhibitors abrogate the ability of HIF-mediated transactivation of BH3-only proteins to potentiate oxidative death in normoxia. The findings offer strategies for minimizing the prodeath effects of HIF-1 in neurologic conditions associated with hypoxia and oxidative stress, such as stroke and spinal cord injury.

FIG. 1.

Effect of HIF-1β expression on HT22 cell sensitivity to glutamate toxicity. HT22 cells were infected with MSCV-ARNT-IRES-GFP retrovirus at MOI of 10 and collected based on their green fluorescence by using a fluorescence-activated cell sorter (FACS). FACS-selected cells were infected with pBabe-VP16 or pBabe HIF-VP16 and further selected with puromycin. (A) HIF-1β protein expression. (1) HT22-control, (2) HT22 treated with DFO, (3) HT22 infected with MSCV-ARNT-IRES-GFP retrovirus, (4) HT22 co-infected with MSCV-ARNT-IRES-GFP and VP16 retroviruses, (5) HT22 co-infected with MSCV-ARNT-IRES-GFP and HIF-VP16 retroviruses. (B) The viability of HT22 cells carrying different viral constructs after treatment with 5 mM glutamate. HT22 cells were infected with MSCV-ARNT-IRES-GFP and sorted by FACS for green fluorescence. After cell stabilization, they were infected with pBabe-puro-VP16 or pBabe-puro-HIF-VP16 and selected with puromycin. HT22, control cells; VP16, HT22 cells infected with VP16 retrovirus; HIF-VP16, HT22 cells infected with HIF-VP16 retrovirus; ARNT-VP16, HT22 cells co-infected with MSCV-ARNT-IRES-GFP and VP16 retroviruses; ARNT-HIF, HT22 cells infected with MSCV-ARNT-IRES-GFP and HIF-1α-VP16 retroviruses. *p < 0.0001.

FIG. 2.

Forced expression of HIF-VP16 induces the expression of several prodeath BH3-only family genes. RT-PCR of BH3-only, prodeath proteins BNIP3, NIX, and PUMA. Lane 1, control HT22 cells infected with VP16; lane 2, HT22 cells infected with VP16 and treated with DFO; and lane 3, HT22 cells infected with HIF-VP16 and treated with DFO. β-Actin mRNA level (known not to change with hypoxia or hypoxia mimetics) was used as a loading control.

FIG. 3.

BNIP3 localization in mouse HTT hippocampal neuroblasts. (A) BNIP3 expression induced by HIF-VP16. (B) Cells co-transfected with plasmid expressed EGFP-BNIP3 fusion protein and pDsRed-mito. Nucleus is stained with DAPI.

FIG. 4.

Enhanced sensitivity of HIF-VP16-expressing HT22 cells but not BNIP3-expressing HT22 cell to glutamate-induced oxidative stress. HT22 cells were plated onto a 96-well plate at the density of 5 × 10⁴ cells/ml and infected with retroviruses expressing murine BNIP, VP16, or fusion protein HIF-VP16 at MOI from 1 to 50. Twenty-four hours after infection, cells were treated with 10 mM glutamate overnight. HT22, c, HT22 cells noninfected and nontreated; HT22, Glu-noninfected cells treated with glutamate; BNIP, c, cells infected with retrovirus expressing BNIP3, nontreated; BNIP, Glu, cells infected with retrovirus expressing BNIP3, treated with glutamate; HIF, c, cells infected with retrovirus expressing HIF-VP16, nontreated; HIF, Glu, cells infected with retrovirus expressing BNIP3, treated with glutamate. Data were normalized to the untreated and noninfected cells (100%). *p < 0.002. **p < 0.02.

FIG. 5.

Downregulation of BNIP3 expression by shRNA in mouse HT22 cells. (A) BNIP3 mRNA level in cells infected with the viral construct encoding shRNA against BNIP3. BNIP3 message was induced by 100 μM DFO or by infection with an HIF-VP16-encoding virus. (B) BNIP3 protein level as determined by immunoblot. (C) BNIP3 mRNA level in cells infected with viral construct encoding shRNA against GFP or HIF-1α and treated with 100 μM DFO overnight. (D) BNIP3 protein expression in cells infected with retrovirus encoding shRNA against BNIP3, HIF-1α, or GFP, treated (+) or nontreated (-) with DFO. (E) BH-3–only protein mRNA level in cells infected with VP16 alone (lane 1), treated with 100 μM DFO (lane 2), or infected with VP16 and virus-encoded shRNA against BNIP3 and treated with DFO (lane 3). β-Actin is used as a housekeeping gene.

FIG. 6.

Effect of BNIP3 downregulation on cell death induced by glutathione depletion. HT22 cells were plated at the density of 5 × 10⁴ cells/ml onto a 96-well plate, incubated overnight, and infected with retroviruses encoding VP16 or HIF-VP16 in combination with virus encoding shRNA against BNIP3 or GFP (MOI 5 for each virus). Twelve hours after infection, cells were treated with 5 mM glutamate. The viability of cells was measured 12 h after treatment. Data are normalized relative to the untreated cells (100%). *p < 0.002.

FIG. 7.

Downregulation of PUMA expression by shRNA in mouse HT22 cells and its effect on cell viability under oxidative stress. (A) PUMA protein expression in cells infected with retrovirus encoding shRNA against GFP or PUMA, nontreated control, or treated with DFO or DMOG. (B) HT22 cells were plated at the density of 5 × 10⁴ cells/ml onto a 96-well plate, incubated overnight, and infected with retroviruses encoding VP16 or HIF-VP16 in combination with virus encoding shRNA against PUMA or GFP (MOI 5 for each virus). Twelve hours after infection, cells were treated with 10 mM HCA. The viability of cells was measured 12 h after treatment. Data are normalized relative to the untreated cells (100%). *p < 0.0001.

FIG. 8.

Effects of antioxidants, low-molecular-weight HIF PHD inhibitors, or peptide HIF PHD inhibitors on cell survival in the presence of 10 m M glutamate. Cells with the stable expression of the fusion protein HIF-VP16 or of VP16 only as a control were plated onto a 96-well plate at the density of 5 × 10⁴ cells/ml for HIF-VP16 expression or at 2.5 × 10⁴ cells/ml for VP16 expression. (A) Twenty-four hours after the plating, cells were treated with either 10 mM glutamate, 10 μM DFO, 10 μM BH, or 100 μM NAC or a combination of glutamate with one of these compounds; these compounds are known to be effective in deterring the glutamate-induced toxicity. (B) Cells were treated with either HCA, DHB, DMOG, short HIF ODD wild-type peptide (HIF-wt), or mutated (HIF-mut). Data are normalized relative to the untreated cells (100%). Values between VP16 and HIF-VP16 infected and treated with glutamate or HCA, p < 0.001.

FIG. 9.

Model of HIF-1–induced activation of BH3-only Bcl2 family members. At least two models exist for HIF-1α transcriptional participation to promote cell death. One model proposes that an imbalance of HIF-1α and HIF-1β results in the dimerization of HIF-1α with p53, leading to stabilization of p53 and upregulation of its known prodeath genes, such as PUMA, NOXA, NIX, and BNIP3. The results herein do not support this model. A second model supported by the data herein is that HIF-1α and HIF-1β dimerize and regulate the expression of prodeath (and prosurvival) genes in living neurons. These neurons are triggered to commit death only when a secondary stress such as oxidative stress (or acidosis) is imposed on the cell. Such a secondary stress could induce death directly by modifying BNIP3 or indirectly by inducing the expression of an additional prodeath protein (PUMA).