We observed that during the first 24 h of hypoxia HIF‐1α protein expression in ARPE‐19 cells showed an increasing trend, and returned to baseline levels afterwards. HIF‐1α mRNA levels, on the other hand, showed significant fluctuation during the first 24 h. These observations suggest that both transcriptional and translational mechanisms may be responsible for regulating HIF‐1α levels in ARPE‐19 cells, and that these mechanisms are preferentially acting at different times.
PHD1 protein levels remained essentially unchanged during our experiments. However, PHD2 and PHD3 proteins steadily rose over the time course of our experiments. PHD2 and PHD3 levels peaked when HIF‐1α protein returned to baseline levels. It has been shown that under hypoxic conditions HIF‐1α regulates its own degradation through a feedback loop that involves either upregulation of PHD2 and PHD3 enzymes21,22
or transcription‐dependent (PHD independent) depletion.23
Our results support the hypothesis that both of these factors are regulating HIF‐1α protein levels in ARPE‐19 cells under hypoxic conditions. These two mechanisms are likely acting together as a homeostatic negative feedback loop to keep HIF‐1α protein levels in check.
Based on our observations, it would appear that during acute hypoxia (24 h) HIF‐1α protein levels are regulated mainly by transcription‐dependent mechanisms. HIF‐1α protein is more stable under hypoxia and accumulates under these conditions.14
The decreased HIF‐1α mRNA that we observed in ARPE‐19 cells after 12 h of hypoxia may represent a mechanism for keeping HIF‐1α protein levels in check and preventing excessive production and accumulation. Transcription‐mediated downregulation of HIF‐1α has been shown to occur during the first 24 h of hypoxia.23
Under chronic conditions (more than 24 h) these mechanisms likely no longer function, and PHD2 and PHD3 enzymes are upregulated to bring HIF‐1α down to baseline levels. In this case, PHD2 and PHD3 act as a ‘thermostat' mechanism to turn down HIF‐1α levels under chronic hypoxia. Upregulation of PHD2 and PHD3 as a self‐regulatory mechanism for reducing HIF‐1α protein levels in chronic hypoxia has been described in other systems.22
Furthermore, it has been shown that PHD2 and PHD3 are under transcriptional control of HIF‐1α, suggesting a possible feedback loop between these proteins.24,25
RNA interference using siRNA was effective at significantly reducing HIF‐1α protein levels during 36 h of hypoxia (72 h after transfection). In parallel to this, secreted VEGF protein levels were also significantly reduced over 36 h of hypoxia. Although secreted VEGF levels were reduced in the cells treated with siRNA against HIF‐1α, they were not completely depleted. Furthermore, secreted VEGF levels continued to rise despite a significant decrease in VEGF mRNA levels at 12 h. Increased stability of VEGF mRNA under hypoxic conditions26
and improved efficiency VEGF mRNA translation under hypoxia27,28
could explain these observations. We did not detect significant amounts of the HIF‐2α and HIF‐3α isoforms, even after HIF‐1α silencing, suggesting that the upregulation of alternate isoforms of HIF‐α is not responsible for VEGF production by ARPE‐19 cells. Expression of HIF‐1α and its relation to VEGF expression in ARPE‐19 cells has previously been reported.29
Our data suggest that the 1α isoform is the main isoform of HIF‐α in human RPE cells, and that loss of HIF‐1α function is not accompanied by upregulation of other isoforms. While HIF‐1α/β is the most widely studied isoform of HIF and considered the primary mediator of hypoxia‐induced gene expression, less is understood about HIF‐2α/β and HIF‐3α/β.13,30
Animal models and in vitro
studies have shown that both HIF‐2α and HIF‐3α are upregulated in hypoxia, suggesting a role that is complementary rather than redundant with HIF‐1α.31,32
Whatever the function of HIF‐2α and HIF‐3α may be in other systems, it appears that these isoforms play little (if any) role in the hypoxic response of ARPE‐19 cells.
We were unable to detect EPO production from RPE cells at the protein level. Furthermore, EPO mRNA levels were barely detectable in RPE cells under hypoxia. It has previously been demonstrated that retina explants produce EPO under hypoxic conditions.6,33
A recent study34
has shown that in the murine liver, which expresses both HIF‐1α and HIF‐2α, EPO production in preferentially regulated by HIF‐2α and not HIF‐1α. We did not detect any significant HIF‐2α production in ARPE‐19 cells, even after HIF‐1α silencing, indicating that HIF‐1α is the major isoform in these cells. The lack of significant HIF‐2α and EPO expression in these cells suggests that retinal EPO production may also be regulated by HIF‐2α. The cellular elements responsible for EPO production in the retina remains elusive, and deserves further investigation.
In this report we have described HIF expression in human RPE cells. Similar to other systems, HIF levels appear to be self‐regulated in RPE cells by feedback mechanisms which operate at the transcriptional and post‐translational level. Furthermore, HIF‐1α appears to be the major isoform of HIF in human RPE cells and HIF‐1α regulates VEGF expression in these cells. EPO is not produced by human RPE cells under hypoxia, an effect which may be secondary to the fact that these cells do not express significant HIF‐2α. Although RPE cells are an important source of angiogenic factors in the retina, our results indicate that other retinal elements are likely also involved in the production of angiogenic factors like EPO. Our study was thus limited by its in vitro nature, and the fact that only RPE cells were studied. Examination of the role of these retinal elements as well as alternate isoforms of HIF in regulating the expression of angiogenic factors in vivo will likely unveil more clues to the molecular mechanisms underlying retinal ischaemic disease.