Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis

Abstract

Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). Here, we report the identification and cloning of a novel HIF-1-responsive gene, designated RTP801. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H(2)O(2)-triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.

FIG. 1.

Regulation of RTP801 expression by hypoxia. (A) RTP801; (B) VEGF; (C) TSP1 (thrombospondin 1). N, normoxia; H4, 4 h of hypoxia; H16, 16 h of hypoxia. (Top) Microarray images of hybridization signals. T, total-RNA-derived probe; Nu, nuclear-RNA-derived probe. The numbers indicate the fold increase (+) and decrease (−) in gene expression under hypoxic (H4, H16) conditions versus that under normoxic (N) conditions. (Bottom) Northern analysis of gene expression using 2 μg of poly(A) RNA extracted from C6 cells cultured under either normoxic (N) or hypoxic (H4, H16) conditions. The three probes for RTP801 and the VEGF, and TSP1 genes (used to demonstrate that the normoxic slot does not contain less RNA than the hypoxic one) were consequently hybridized to the same membrane.

FIG. 2.

Sequence analysis of RTP801 cDNA and protein. (A) Multiple alignment of rat and human RTP801 putative proteins, two Drosophila proteins encoded by scylla and charybde, and mouse RTP801L. The alignment was performed by using the Prettybox program of the Genetics Computer Group sequence analysis package. (B) Northern blot analysis of RTP801 expression in various human tissues. A human 12-tissue Northern blot membrane was purchased from Origene (HB2010).

FIG. 3.

Transcriptional regulation of RTP801. (A) Northern blot analysis of RTP801 transcription in wild-type mouse ES cells (ES+/+) and in HIF-1α null mouse ES cells (ES−/−) cultured under normoxic (N) or hypoxic conditions (H) for 16 h. Total RNA (15 μg) was loaded in each slot. (B) Nucleotide sequences of immediate upstream genomic regions of mouse and human RTP801 orthologues. The initiation ATG codon is in boldface, and the position of T is counted as +1. The TATA box is shaded gray. White letters in black background, putative HRE; dashed line, putative Egr-1 binding site. (C) EMSA and supershift analysis of mouse RTP801 promoter region. All the binding reactions except for those whose mixtures are loaded in lanes 2, 4, and 5 were performed with nuclear extracts prepared from wild-type ES cells cultured under hypoxic conditions for 16 h. The reaction mixture loaded in lane 2 contains nuclear extract prepared from wild-type ES cells cultured in normoxia, whereas reaction mixtures loaded in lanes 4 and 5 contain nuclear extracts from HIF-1α^−/− ES cells maintained in normoxic and hypoxic conditions, respectively. Lane 1, ³²P-TR-HRE oligonucleotide; lanes 2 to 5, ³²P-RTP801-HRE oligonucleotide; lane 6, ³²P-RTP801-HRE oligonucleotide and the excess of nonlabeled RTP801-HRE oligonucleotide; lane 7, ³²P-RTP801-HRE oligonucleotide and the excess of nonlabeled TR-HRE oligonucleotide; lane 8, ³²P-RTP801-HRE oligonucleotide and anti-HIF-1α antibodies; lane 9, ³²P-RTP801-HRE oligonucleotide and anti-Flag antibodies; lane 10, ³²P-RTP801-MHRE oligonucleotide. For details see Results and Materials and Methods. (D) Northern blot analysis demonstrating the p53 independence of hypoxic transactivation of RTP801. H1299 is a human lung carcinoma p53-negative cell line that was engineered to express the wild-type p53 under the control of a tetracycline-repressible promoter. The cells were cultured either in the absence (left) or presence (right) of tetracycline to induce (left) or to suppress (right) p53 expression, respectively. Both p53-positive and p53-negative H1299 cells were maintained either under normal (N) or hypoxic (H) conditions or in the presence of doxorubicin (D). Total RNA (15 μg) derived from each experiment was analyzed by Northern blotting using the probes for human RTP801 and for Waf1 (as a positive control for p53-dependent transactivation).

FIG. 4.

In situ hybridization analysis of RTP801 expression in the rat MCAO model 24 h after artery occlusion. (A and B) Microphotographs of distribution of radioactive hybridization signal specific to RTP801 (A) and VEGF (B) mRNA in coronal sections of rat brain 24 h after the MCAO (right hemisphere). Brain regions one (infarct boundary) and two (more-distant area) (arrowheads) are enlarged in panels C and D, respectively. (C and D) Microphotographs of coronal rat brain sections, bright field, with hematoxylin-eosin staining. Arrows, red neurons (C) and morphologically normal neurons (D) expressing RTP801. The RTP801-specific hybridization signal appears as black dots concentrated over expressing cells.

FIG. 5.

Analysis of MCF7-Tet-Off and PC12-Tet-Off transfectants expressing RTP801 cDNA under the control of a tetracycline-repressible promoter. (A) Northern blot analysis of tetracycline-dependent expression of RTP801 in stably transfected cell clones (15 μg of total RNA per lane). Lane 1, PC801-10; lane 2, MCF801-11; lane 3, MCF801-12; lane 4, MCF801-8. Lanes N and H contain 15 μg of total cellular RNA extracted from parental MCF7-Tet-Off cells or PC12-Tet-Off cells cultured under normoxic and hypoxic conditions, respectively. (B) Western blot analysis with anti-Flag antibodies (Sigma) of expression of RTP801-Flag in total protein extracts from MCF801-8 and control cells. The position of the 35-kDa RTP801-specific band is indicated. For induction of RTP801 expression, cells usually cultured in the presence of 1 μg of tetracycline/ml and 100 μg of hygromycin/ml were washed with phosphate-buffered saline and seeded directly in culture medium without tetracycline for 72 h.

FIG. 6.

Analysis of viability of RTP801-transfected cells. (A) Assessment of cytotoxic effect of H₂O₂ treatment, hypoxia and glucose deprivation, and serum deprivation conditions in control and RTP801-expressing MCF7-Tet-Off cells. (B) Assessment of the influence of H₂O₂, hypoxia and glucose deprivation, and RTP801 expression on generation of ROS in MCF7-Tet-Off cells. For panels A and B, 1 indicates data for MCF7-vector (a clone of MCF7-Tet-Off cells transfected with the empty pSHTet vector), 2 indicates data for MCF801-8, and 3 indicates data for MCF801-12. (C) Assessment of cytotoxic effect of H₂O₂ treatment, hypoxia and glucose deprivation, and serum deprivation conditions in control and RTP801-expressing PC12-Tet-Off cells. (D) Assessment of the influence of H₂O₂, hypoxia and glucose deprivation, and RTP801 expression on generation of ROS in PC12-Tet-Off cells. (E) Assessment of cytotoxic effect of H₂O₂ treatment and hypoxia and glucose deprivation in control and RTP801-expressing differentiated PC12-Tet-Off cells. (F) Inhibition of RTP801-induced cytotoxicity in differentiated PC12-Tet-Off cells by Boc-D (OMe)-FMK. Casp.inhib., pan-caspase inhibitor. For panels C to F, 1 indicates data for PC12-vector (a clone of PC12-Tet-Off cells transfected with the empty pSHTet vector) and 2 indicates data for PC801-10. For experimental details, see Results and Materials and Methods. Data are means of three independent experiments performed in triplicate.

FIG. 7.

Liposomal delivery of RTP801 cDNA into mouse lungs. (A) Northern blot analysis of RNA (15 μg per lane) extracted from lungs of mice injected with liposomes containing either pcDNA3 DNA (lanes 1 to 3) or pcDNA3-RTP801 (lanes 4 to 6). The position of the RTP801-specific band is indicated. (B) TUNEL staining of representative histological sections of control and RTP801-expressing mouse lungs. The experiment was repeated twice with independent liposomal preparations.