Prosurvival and Prodeath Effects of Hypoxia-inducible Factor-1α Stabilization in a Murine Hippocampal Cell Line

Hoon Ryu,Andrew Kung,Joseph C. LaManna,Rajiv R. Ratan,Juan C. Chavez,Leila R. Aminova,Junghee Lee

doi:10.1074/jbc.m409223200

Abstract

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in adaptation to hypoxic stress. Previous studies from our laboratory demonstrated that pharmacological activators of HIF-1 (e.g. deferoxamine, cobalt chloride) could also protect cultured primary neurons or an immortalized hippocampal neuroblast line (HT22) from oxidative stress-induced death. However, whether HIF-1 activation is sufficient to abrogate neuronal death resulting from oxidative stress or other hypoxia-independent death inducers remains unclear. To address this question we utilized a HIF-1alpha fusion protein that partially lacks the domain required for oxygen-dependent degradation of HIF-1alpha and that has a VP16 transcriptional activation domain from herpes simplex virus. HT22 cells were infected with a retrovirus encoding either the HIF-1alpha-VP16 fusion protein or the activation domain of the VP16 protein alone as a control. Expression of HIF-1alpha-VP16, but not VP16 alone, increased luciferase activity driven by a canonical hypoxia response element, increased mRNA of established HIF-1 target genes, and increased activity of one of these HIF-1 target genes. Unexpectedly, enhanced HIF-1 activity in HT22 cells enhanced sensitivity to oxidative death induced by glutathione depletion. Accordingly, suppression of HIF-1alpha expression using RNA interference prevented oxidative death. By contrast, HIF-1alpha-VP16-expressing HT22 cells were more resistant to DNA damage (induced by camptothecin) or endoplasmic reticulum stress (induced by thapsigargin and tunicamycin) than were VP16-expressing cells, and suppression of HIF-1alpha expression using RNA interference rendered HT22 cells more sensitive to death induced by DNA damage or endoplasmic reticulum stress. Together, these data demonstrate that HIF-1 can mediate prodeath or prosurvival responses in the same cell type depending on the injury stimulus.

Highlights

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in adaptation to hypoxic stress
Both antibodies under normoxic conditions recognized a band corresponding to the HIF-1␣-VP16 protein, and it migrated to the predicted molecular mass of about 90 kDa in HT22 cells infected with the HIF1␣-VP16 retrovirus
These findings demonstrate that the HIF-1␣ fusion protein, unlike endogenous HIF-1␣, is expressed under normoxic conditions

Summary

EXPERIMENTAL PROCEDURES

Plasmid and Retroviruses—A plasmid encoding a fusion protein of amino acids 1–529 of HIF-1␣ and the herpes simplex virus VP16 transactivation domain (pBABE-puro-HIF-1␣-VP16) and a control plasmid encoding only VP16 (pBABE-puro-VP16) were described previously [31]. Nuclear Extracts and DNA Electrophoretic Mobility Shift Assay— Following infection or drug treatment, cells were scraped into cold phosphate-buffered saline, centrifuged, and washed in five packed cell volumes of buffer (10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 10 mM KCl) freshly supplemented with 0.5 mM dithiothreitol, 1 mM sodium orthovanadate, 0.4 mM phenylmethylsulfonyl fluoride, and 10 ␮l/ml of protease inhibitor mixture (Sigma). This was followed by 10 min of incubation in the same buffer on ice and homogenization in a glass Dounce homogenizer (type B pestle) [34, 35]. Scientific) were used at concentrations from 1 to 10 ␮M as noted

RESULTS

TABLE I Primer sequences

DISCUSSION