Cellular response to hypoxia plays an important role in numerous biological processes, such as hematopoiesis (1
), wound healing (36
), ischemic stroke, myocardial infarction, retinopathy, and carcinogenesis (reviewed in reference 41a
). Hypoxia-responsive genes may thus be important targets for the development of drugs modulating these major normal and pathological conditions. Therefore, we were interested in the identification of novel genes whose expression is altered in response to changing oxygen concentrations.
One such gene, designated RTP801, was identified by us as sharply up-regulated in C6 rat glioma cells in response to hypoxia. Its enhanced transcription in hypoxic tissues in vivo, for example, in permanent MCAO model of stroke, was also documented. Human and rat full-length cDNAs for RTP801 were cloned and found to represent a novel gene encoding a protein without any defined structural domains. A search of public databases revealed that RTP801 is homologous to two Drosophila homeobox target genes, scylla and charybde (Fig. ). The homology between RTP801 and the two identified Drosophila genes is maximal within regions encoding the C-terminal regions of the associated proteins, implying that the RTP801 sequence encodes a previously uncharacterized protein domain. This suggestion is further supported by the identification of a putative RTP801-like protein, designated RTP801L, in the mammalian genome. The maximal similarity between putative proteins RTP801 and RTP801L also resides within their C termini. The pattern of expression of RTP801L in embryogenesis is different from that of RTP801 (A. Faerman and E. Feinstein, unpublished observation). Moreover, its transcription is not responsive to a reduced oxygen concentration both in vitro and in vivo (H. Kalinski, A. Faerman, and E. Feinstein, unpublished observation). Overall, the data suggest that the product of the newly cloned RTP801 cDNA belongs to a protein family not previously described that participates in various cellular processes.
The predicted ORF of RTP801 codes for a protein of 25 kDa. However, all utilized expression systems gave rise to a recombinant protein that migrated on gels with a mobility corresponding to ~35 kDa. This size discrepancy is unlikely to be due to posttranslational phosphorylation or glycosylation since the 35-kDa protein was detected not only in mammalian cells but also when the RTP801 ORF (from both rats and humans) was either translated in vitro or expressed in bacteria. The reason for the observed gel mobility shift is thus unclear.
Our initial experiments strongly suggested that RTP801
is a HIF-1-responsive gene, since mouse ES HIF-1α−/−
cells failed to induce its expression in response to hypoxia. This suggestion was also supported by the presence of three evolutionarily conserved HREs in close vicinity (as judged by the position of the TATA box) to the initiation sites of both human and mouse mRNAs. Moreover, when the oligonucleotide containing one of these potential HRE elements was used in an EMSA, it promoted the formation of a specific complex (complex A in Fig. ). The formation of this complex was dependent on the presence of (i) a core HRE sequence within the labeled oligonucleotide, (ii) hypoxic conditions of cell cultivation, and (iii) the presence of the HIF-1
α gene in the genomes of the cells used for the preparation of nuclear extracts. Overall, the experimental data confirm that RTP801
is a new member of the growing family of direct HIF-1 target genes. HIF-1α knockout in mice leads to embryonic lethality at the late stages of fetal development (19
). Therefore, it will be interesting to evaluate the potential modifications in the RTP801
expression pattern in early, still-viable, HIF-1α null embryos. In addition, such an analysis will shed light on whether embryonic expression of RTP801
is regulated solely by HIF-1α or whether other regulatory routes (e.g., homeobox genes) are also involved.
As is evident from Fig. , HIF-1α−/−
ES cells still express the residual hypoxia-regulated levels of RTP801
, potentially indicating involvement of additional transcription factors in the regulation of RTP801
expression. These may be other members of the HIF family capable of binding to the same HREs as HIF-1α. In this regard, it is worth noting that, in the EMSA experiments, the utilized RTP801-HRE formed additional specific complexes (migrating faster than complex B) that were abolished by mutating the core HRE sequence (Fig. , lane 10). It is also possible that transcription factors belonging to other families contribute to regulation of the RTP801
gene as well. For example, we have found a conserved binding site for Egr-1 in the upstream regions of both mouse and human RTP801
orthologues (Fig. ). This factor was recently demonstrated to mediate the ischemic stress induction of numerous cytokines and chemokines (44
). Egr-1 binding sites were found in the regulatory sequences of a range of genes relevant to vascular homeostasis and dysfunction (20
); this finding may be in agreement with the observed increased expression of RTP801
in endothelial cells within the stroke core.
The potential complexity of transcriptional regulation of RTP801
is further underscored by its complex temporal and spatial expression pattern in the MCAO model. It seems conceivable that the early (30 min) peri-infarction activation of RTP801
stems not from the reduced oxygen supply but rather from the developing excitotoxicity and spreading depression. The latter is known to induce transcription of immediate-early genes (9
), in agreement with the observed colocalization of RTP801- and c-fos-specific hybridization signals and the absence of up-regulation of hypoxia-specific marker VEGF (31
) at this time point. Only later on did the RTP801 hybridization signal become spatially colocalized with that of VEGF, suggesting a hypoxia-dependent regulation.
HIF-1 has a dual role in the cellular response to hypoxia. It is an important mediator of hypoxia-induced cell death (6
), and it seems to play an equally important role in mediating hypoxia-induced ischemic tolerance (3
). Like HIF-1 itself, RTP801 had a dual effect on the target cells. We have demonstrated that the prior expression of RTP801 in MCF7 and PC12 cells prevented apoptosis triggered by hypoxia and no glucose or H2
treatments via complete abolishment of the generation of ROS that would otherwise be caused by both types of treatment. It is not clear at this stage whether RTP801 has an antioxidant activity by itself or whether it somehow stimulates this adaptive response. The latter possibility seems more plausible, since (i) RTP801 does not have conserved cysteine residues, whose presence is typical for proteins with direct antioxidant activity, and (ii) its protective effect is transient. Other products of HIF-1 target genes, e.g., erythropoietin (22
) and heme-oxygenase 1 (23
), were also shown to elicit a protective effect in target cells against similar cytotoxic stimuli. However, under other conditions tested, overexpression of RTP801 turned out to be detrimental. First, nondividing neuron-like differentiated PC12 cells were killed by the very fact of enhanced expression of RTP801 through activation of caspases (moreover, these cells became much more sensitive to hypoxia and H2
cytotoxicity). Second, both MCF7 and PC12 cells overexpressing RTP801 died from serum starvation unlike their control counterparts. And third, the liposomal delivery of RTP801
to mouse lungs elicited a prominent apoptotic response on the target cells. The observed proapoptotic activity of RTP801 seems to be in line with the indirect correlative evidence obtained from the in situ hybridization studies of RTP801 using the MCAO model. At the brain infarct boundary in the MCAO model of stroke, the RTP801-specific mRNA was concentrated within the so-called eosinophilic, or “red,” neurons, located close to the necrotic core (Fig. and ). These cells are not shrunken, and their nuclei are not pyknotic although they contain a clumped chromatin. The pathogenesis and the fate of red neurons remain uncertain, but they are regarded as possibly representing early apoptotic neurons or neurons suffering from hypoxia (14
Currently, the intracellular pathways affected by RTP801 overexpression are unknown. The only common feature that could be traced is that RTP801 overexpression caused the apoptosis-resistant phenotype in cycling cells and apoptosis sensitivity in growth arrested cells. Indeed, serum starvation is known to synchronize MCF7 cells in G0
), and differentiated PC12 and lung parenchyma cells are also nondividing. The observed differences in the consequences of RTP801 overexpression in cycling and resting cells may be potentially explained by the underlying differences in their energy metabolism and demand. Specifically, the proliferative state is associated with a significant increase in aerobic glycolysis, and these cells are able to suppress phorbol myristate acetate-induced generation of ROS, probably due to increased concentrations of pyruvate, an effective ROS scavenger (4
). The fact that overproduction of RTP801 in serum-starved neuron-like PC12 cells confers their increased sensitivity to hypoxic or oxidative injury may have certain implications for stroke, where due to the lack of blood supply the brain neurons suffer not only from ischemia but also from deprivation of growth factors.
Overall, the data lead us to conclude that newly identified direct HIF-1α target gene RTP801 participates not only in protective HIF-1-dependent molecular pathways but also in its proapoptotic effects. Accordingly, it will be important to monitor the changes in gene expression in general and changes in the expression of pro- and antiapoptotic genes specifically following the tetracycline induction of RTP801 in cycling and arrested cells. It will also be interesting to study the response of RTP801 null and transgenic mice to ischemic injury. Experiments with transgenic mice overexpressing exogenous RTP801 in brain, retina, and heart are ongoing. We believe that further investigation of RTP801 function will lead to the opening of new avenues in the treatment of hypoxia- and ischemia-associated diseases.