This work comprises the first direct analysis of the differential regulation of sites of HIF prolyl and asparaginyl hydroxylation in cells. Hydroxy residue-specific antibodies, whose performance was validated by MS, were used to analyze hydroxylation levels at each of the three known sites of hydroxylation in human HIF-1α (Pro402, Pro564, and Asn803). The findings reveal major differences in the regulation of these hydroxylations by hypoxia and in their sensitivity to supposedly nonspecific hydroxylase inhibitors.
Using VHL-defective RCC4 cells to avoid confounding bias from selective degradation of prolyl-hydroxylated HIF-α polypeptides, we were able to directly compare changes in prolyl versus asparaginyl hydroxylation. These experiments demonstrated that in normoxic RCC4 cells HIF-1α is heavily or even completely hydroxylated at all three sites. They reveal that HIF-1α asparaginyl hydroxylation is substantially more resistant to hypoxia than either site of prolyl hydroxylation. Similar differential sensitivity of prolyl and asparaginyl hydroxylation was observed over a range of short hypoxic exposures and maintained during chronic hypoxia.
Reported apparent Km
measurements for oxygen for the HIF hydroxylases (21
) are all very much higher than the oxygen concentrations used in the graded hypoxia experiment in the current work and those previously measured in studies of human tissues (37
). Therefore, whereas differences in Km
values for oxygen have been reported for the PHDs versus
), they cannot account for our in vivo
findings, and the precise kinetic parameters underlying the differential sensitivity to hypoxia that we have observed in vivo
are not yet clear. Such analyses are also likely to be complicated by the existence of alternative non-HIF substrates, particularly for FIH. In addition to HIF, FIH catalyzes hydroxylation of specific asparaginyl residues in a wide range of ankyrin repeat domain proteins (34
). Some of these, such as the Notch-1 receptor are very effective competitors of HIF hydroxylation in vitro
). Given that many ankyrin repeat domain proteins that are subject to FIH-catalyzed hydroxylation such as cytoskeletal ankyrins (43
) are also expressed at relatively high abundance across a wide range of cells, our finding of high levels of hydroxylation of HIF-1α Asn803
except in cells exposed to severe hypoxia or chemical inhibitors was somewhat surprising and suggests that additional factors operate in vivo
to direct FIH-mediated catalysis to particular molecular targets.
Our studies did not reveal any evidence for a compensatory increase in the specific activity of the HIF hydroxylases in chronic hypoxia. Although an increased specific activity of the PHDs during chronic hypoxia has been reported in VHL-competent cells that manifest intact HIF transcriptional responses, this was potentially attributed to HIF-dependent reductions in mitochrondrial oxygen consumption that could reduce the severity of cellular hypoxia (44
). In our studies of chronic hypoxia in VHL-defective cells, such HIF-dependent effects would not be anticipated and our finding of no increase in specific activity of the HIF hydroxylases in this setting is therefore consistent with the authors' interpretation of the previous work.
Although the use of VHL-defective cells was necessary to enable direct comparison of interventions on prolyl and asparaginyl hydroxylation, up-regulation of HIF pathways in these cells leads to constitutive up-regulation of HIF prolyl hydroxylases, PHD2 and PHD3 (12
). In contrast, expression of FIH is unaffected by the VHL status. Thus it is likely that VHL-competent cells, which therefore express lower levels of PHD2 and PHD3 relative to FIH, effectively manifest even greater differential sensitivity of prolyl to asparaginyl hydroxylation at least during the acute phase of hypoxic exposure. Operationally, however, the key consideration in defining the response to hypoxia in VHL-competent cells is the extent to which HIF asparaginyl hydroxylation persists on HIF-1α molecules that escape the prolyl hydroxylation/VHL degradation pathway. Our findings reveal that in moderately hypoxic cells, tissues from hypoxic animals, and human tumors, HIF-1α remains substantially strongly hydroxylated on asparagine. The levels of persistent asparaginyl hydroxylation were cell type-specific and strongly dependent on the severity of hypoxic exposure. These findings are consistent with reports that suppression of FIH can increase HIF target gene expression in moderately hypoxic cells (27
) and they indicate that monitoring of HIF asparaginyl hydroxylation is likely to be important in future physiological analyses of the HIF transcriptional pathway (46
Analysis of all three sites of HIF hydroxylation also permitted comparison of the two sites of prolyl hydroxylation. Consistent with previous work we observed that hydroxylation at Pro402
apparently occurred after that at Pro564
and was more sensitivity to hypoxia. Whether this is due to intrinsic differences in the sensitivity of each site to hypoxia or competition between the two sites as has been demonstrated in vitro
) is unclear. However, differences in the behavior of the two sites in vivo
were marked. For instance, upon exposure to moderate hypoxia, striking accumulation of HIF-1α that was hydroxylated at Pro564
but not Pro402
was observed, which was again cell type-specific. Interestingly, accumulation of HIF-1α that was hydroxylated on Pro564
correlated with increased expression of PHD3, the enzyme that preferentially hydroxylates this site (12
), suggesting that cell-type differences in expression of PHD3 may contribute to these differences. Mutational studies of HIF-1α have indicated that each site of prolyl hydroxylation in HIF-1α can independently mediate VHL-dependent degradation of HIF-α (7
). Nevertheless, the current findings would suggest that the preferential hydroxylation of Pro564
that is observed in moderately hypoxic cells might not on its own be sufficient to cause VHL-mediated degradation of HIF-1α at a rate that is adequate to overcome synthesis. It is consistent with the proposal that efficient operation of the prolyl hydroxylation-VHL degradation pathway is dependent on some form of cooperative interaction between the 2 sites of prolyl hydroxylation that promotes more rapid, or more complete VHL-dependent degradation (25
Dependence of rapid HIF-1α degradation on hydroxylation at both sites would be predicted to further enhance the sensitivity of the prolyl hydroxylation-VHL degradation pathway to hypoxia relative to that of asparaginyl hydroxylation. Furthermore, because Pro564
, like Asn803
is sited within a HIF-α transactivation domain, the hydroxylation status at Pro564
may have additional regulatory effects on HIF activity (48
The use of hydroxy residue-specific antibodies also permitted comparative analysis of different modes of HIF hydroxylase inhibition on different sites of HIF hydroxylation. Striking differences were observed among agents that are often regarded as being nonspecific hydroxylase inhibitors and which are commonly applied to cells as hypoxia mimetics. In particular, at concentrations that had marked effects on HIF prolyl hydroxylation, iron chelators were much less effective, and transition metals ions such as Co(II) were almost completely ineffective in inhibiting asparaginyl hydroxylation of HIF-1α. In contrast, the generic 2-OG analogue DMOG was found to be even more effective at suppressing HIF asparaginyl hydroxylation than HIF prolyl hydroxylation.
Interestingly, findings on the differential sensitivity of prolyl and asparaginyl hydroxylation to transition metals ions and iron chelators in cells are very different from effects of these inhibitors on purified or partially purified preparations of these enzymes in vitro
). FIH has been reported to be much more sensitive than PHDs to inhibition by metal ions in vitro
), whereas we observed the opposite in vivo
. Similarly PHDs (particularly PHD2) bind Fe(II) tightly in vitro
and are much less sensitive to inhibition by DFO than FIH (49
). However, in vivo
we observed the opposite; greater sensitivity of prolyl versus
asparaginyl hydroxylation to inhibition by iron chelators. The reasons for these differences are not clear and strongly suggest that the mechanism(s) of inhibition are more complex than facile exchange of iron at the catalytic center of the HIF hydroxylases with a chelatable iron pool.
Exposure of cells to several different small molecule HIF hydroxylase inhibitors revealed greatly differing levels of specificity for the two types of HIF hydroxylation. Some inhibitors, such as ethyl-3,4-dihydroxybenzoate and 4-oxo-1,4-dihydro-1,10-phenanthroline-3-carboxylic acid, inhibited both types of hydroxylation, in keeping with in vitro
data on these compounds (21
). In contrast, the bicyclic isoquinolinyl compounds A and B (29
) revealed almost complete selectivity for inhibition of prolyl versus
asparaginyl hydroxylation, whereas the N
-oxalyl phenylalanine derivative, compound E (30
), was strongly selective for HIF asparaginyl over HIF prolyl hydroxylation, clearly demonstrating the feasibility of specifically inhibiting each pathway in cells.
The HIF pathway plays a central role in directing cellular responses to hypoxia and is currently the focus of attempts at therapeutic manipulation in a range of disease settings that are associated with tissue hypoxia (18
). Major differences in the regulation of HIF prolyl and asparaginyl hydroxylation described above are therefore important in understanding biological responses to hypoxia. These insights, together with the existence of validated reagents for further study, should assist in guiding attempts at pharmacological modulation of hypoxia pathways.