The strategy of constitutive expression of a pool of HIF-α that will be almost instantaneously available in the rare times of need (e.g. hypoxia) is apparently wasteful. Moreover, the presence of functional HIF-α at other times is likely to be deleterious and the cells not only rapidly dispose of unwanted HIF-α in the proteasome but also make sure that HIF-α that escapes proteasomal degradation will be transcriptionally inactivated by FIH-1. This ingenious two-tier regulatory mechanism of controlling activity by regulating HIF-α stability (PHDs-VHL-proteasome) and its transcriptional activity (FIH-1) accounts for the exceptionally tight control of the HIF pathway. On the other hand, the existence of these two tiers also means that for proper induction of HIF activity both have to be inactivated. Because both PHDs and FIH-1 require O2 for function, hypoxia, the physiological stimulator of the HIF pathway, simultaneously inhibits proline and asparagine hydroxylation. Hypoxia thus allows concomitant accumulation of HIF-α and its transcriptional activation, leading to a robust transcriptional response.
Although the HIF system originally evolved for the adaptation of organisms to hypoxia, it has become evident that both HIF-α protein abundance and its transactivation potential can be modulated under apparently normoxic conditions in response to various stimuli. A number of agents or genetic factors co-inhibiting PHDs and FIH-1 have been described (reviewed in [16
]. The requirement for the same co-substrate (2OG) and co-factor Fe(II) [16
] suggests that activities of PHDs and FIH-1 could be regulated by their availability. Indeed, iron chelation and divalent metal cations (Co(II), Ni(II), and Mn(II)) have hypoxia-mimicking effects [40
]. Analogs of 2OG dimethyloxalylglycine and citric acid cycle intermediates (pyruvate and oxaloacetate) act as competitive inhibitors of PHDs and FIH-1 and induce the HIF system [41
]. Interestingly, in the hereditary cancer syndromes, hereditary paraganglioma (HPGL), and hereditary leiomyomatosis and renal cell carcinoma (HLRCC), accumulation of succinate and fumarate, respectively, due to inactivating mutations of various subunits of succinate dehydrogenase [42
] and fumarate hydratase [43
], results in reduced PHD activity and upregulation of the HIF pathway. Nitric oxide also regulates the HIF system, presumably because it can act, at sufficiently high concentrations, as an analogue of molecular O2
and inhibit 2OG-dependent oxygenases [44
]. Activation of HIF by reactive oxygen species has been explained in terms of inactivation of 2OG-dependent oxygenases by oxidative damage through hydroxylation of the active site, fragmentation, and conversion of catalytic Fe(II) to inactive Fe(III) [45
]. However, the relation between oxidative stress and HIF function is apparently more complex than this, as it was reported that attenuation of oxidative stress by antioxidants can also stabilize HIF-1α [46
]. In all of the described cases, co-inhibition of PHDs and FIH-1 result in HIF-α accumulation in a transcriptionally active form.
In the classical VHL-associated hereditary cancer syndrome, affected individuals are heterozygous for a germline VHL mutation that predisposes to specific types of tumors: clear cell renal carcinomas, hemangiomas of the retina, or hemangioblastomas of the retina and central nervous system [19
]. Following somatic inactivation of the second allele, disruption of hypoxic signaling and clinical manifestations are confined to tumors. Unable to target HIF-α for proteasomal degradation, cells in these tumors overproduce angiogenic factors and other HIF-activated transcripts, even under normoxic conditions [19
]. Interestingly, erythrocytosis due to overproduction of erythropoietin (EPO), one of the HIF target genes (), is not a characteristic feature of the VHL disease. In contrast, erythrocytosis is observed in the Chuvash variant of familial polycythemia, a rare autosomal recessive condition linked to a C598T mutation in VHL
that impairs but does not ablate HIF regulation [47
]. Introduction of the wild-type VHL restores the O2
-dependent regulation of HIF-α and accordingly down-regulates the expression of hypoxia-inducible genes [19
]. Furthermore, VHL behaves as a bona fide
tumor suppressor in that restoration of wild type VHL expression results in inhibition of tumor growth in vivo
]. The presence of defective VHL has no effect on PHD activity and HIF-α accumulates in a proline-hydroxylated form. Nevertheless, this form is fully functional because the loss of VHL function also adversely affects FIH-1 and compromises its capacity to inhibit the HIF-α CAD [48
Selected HIF target genes grouped according to their function
Although in most cases the stability and transcriptional activity of HIF-α are co-regulated, there are several notable exceptions. Measurements of the Km
of FIH-1 for O2
revealed that it is less than half that of the PHD family members [49
], suggesting that a hypoxic window could exist in which HIF-1α would be stable due to the absence of prolyl hydroxylation and yet would be transcriptionally inactive due to hydroxylation of N803. In another example, proteasomal inhibitors, despite having a strong positive effect on HIF-1α stability, not only do not activate HIF under normoxia but they considerably interfere with hypoxia-induced HIF-1 activity [50
]. Although some theories have been put forward (reviewed in [52
]), the mechanism by which proteasomal inhibitors inactivate HIF is not known at present.
In this section, we outlined the importance of co-regulation of HIF-α stability and its transcriptional activation, each regulated by different but related entities. In most cases, stability and transcriptional activity of HIF-α are co-regulated, leading to maximal activation of the HIF pathway. There are, however, situations in which regulation of stability and transcriptional activity are uncoupled, leading to accumulation of a transcriptionally inactive HIF-α. Theoretically, it should be also possible to de-repress transcriptional activity of HIF-α without significantly affecting its stability. The observation that even wild-type VHL-containing cells express variable levels of HIF-1α in the basal state [53
] provides the groundwork for this possibility. In this case, selective inhibition of FIH-1 would result in low to intermediate activation of the HIF pathway. To this end, it has been reported that moderate induction of the hypoxia marker CAIX by pericellular hypoxia generated in dense cultures of certain transformed cells is HIF-1α-dependent, yet it occurs without appreciable accumulation of HIF-1α [54
]. Although this phenomenon could be cell-type specific and the mechanism is not fully understood, it would suggest that it is possible to selectively up-regulate HIF transcriptional activity (presumably via inhibition of FIH-1) without significantly affecting the stability of HIF-α (activity of PHDs).