EDITORIALS

aHIF: the Missing Link Between HIF-1 and VHL?

Leonard M. Neckers

Affiliation of author: L. M. Neckers, Cell and Cancer Biology Department, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD.

Correspondence to: Leonard M. Neckers, Ph.D., National Institutes of Health, 9610 Medical Center Drive, Suite 300, Rockville, MD 20850 (e-mail: len{at}helix.nih.gov).

Response to hypoxia at the cellular level is composed of two components. Investigation of erythropoietin (EPO) gene regulation led to the discovery several years ago of a novel, hypoxia-inducible transcription factor, called hypoxia-inducible factor-1 (HIF-1), that stimulates transcription of a number of cellular hypoxia-survival genes, including EPO, vascular endothelial growth factor (VEGF), certain glycolytic enzymes, and the glucose transport protein GLUT-1. HIF-1 itself is heterodimeric, consisting of a constitutively expressed subunit (HIF-1ß or ARNT [aryl hydrocarbon nuclear translocator protein]) and a hypoxia-inducible subunit (HIF-1{alpha}). Hypoxia appears to regulate HIF-1{alpha} not at the transcriptional level but by post-transcriptional protein stabilization (1).

The second component of the cellular response to hypoxia involves the stabilization of certain messenger RNAs (mRNAs), usually the very ones whose genes are transcriptionally activated by HIF-1, including EPO, GLUT-1, and VEGF (2). This mechanism is HIF-1 independent and appears to be mediated by protein binding to adenylate-uridylate-rich elements (AREs) in the 3'-untranslated (UTR) region of target RNAs. When these AREs are deleted, RNA stability is markedly increased. Conversely, when these AREs are inserted at the 3' end of otherwise stable mRNAs, these transcripts become labile under normoxic conditions (3). Several groups recently have identified a family of high affinity, ARE-binding proteins, leading to the hypothesis that hypoxia-induced binding of one or more of these proteins to specific AREs masks these destabilizing motifs. In recent studies, Levy et al. (4,5) have identified three AREs in the 3'-UTR of VEGF mRNA and similar motifs in GLUT-1 mRNA, which bind to such a protein complex in a hypoxia-inducible manner. Intriguingly, these RNA-protein complexes are constitutively elevated in cells lacking wild-type VHL protein (4).

The von Hippel-Lindau (VHL) syndrome is autosomally dominant and is characterized by the familial occurrence of several cancers, including those of the retina, central nervous system, and kidney (6). The VHL gene is mutated in at least 75% of affected families, and development of cancers in these individuals is associated with somatic loss or inactivation of the remaining wild-type VHL allele. VHL-associated cancers are generally hypervascularized, and molecular studies aimed at understanding the function of the VHL protein (pVHL) have found that the wild-type protein negatively regulates the stability of hypoxia-inducible mRNAs, including VEGF (7). Cells lacking wild-type pVHL contain elevated levels of hypoxia-inducible RNAs under normoxic conditions, and subjection of such cells to hypoxia does not further increase the level of these RNAs. Re-introduction of wild-type pVHL to these cells specifically reduces the level of these RNAs under normoxic conditions and restores the normal response of these genes to hypoxia. Thus, although tumors in which VHL has been inactivated do not generally demonstrate hypoxia-inducible responses, a battery of hypoxia-inducible genes are constitutively expressed at levels characteristic of the hypoxic state.

Hypoxic induction of HIF-1{alpha} protein occurs primarily via protein stabilization and not by hypoxia-dependent stabilization or induction of HIF-1{alpha} mRNA (8). Thus, pVHL is probably not involved in regulating the HIF-1{alpha} mRNA level in normoxic cells. Interestingly, prolonged exposure to hypoxia, both in tissue culture cells and in the whole organism, has been reported to decrease HIF-1{alpha} mRNA (9,10). How might this be accomplished in otherwise hypoxia-responsive cells? In addition, why does hypoxia fail to further up-regulate hypoxia-inducible genes in cells that lack wild-type pVHL, even though transcriptional activation by HIF-1 appears to be independent of VHL? The study by Thrash-Bingham and Tartof (11), appearing in this issue of the Journal, may supply an intriguing answer to these questions.

Loss of pVHL function can be demonstrated in at least 80% of all sporadic nonpapillary clear cell renal carcinomas. In these cancers, Thrash-Bingham and Tartof (11) have identified an endogenously occurring and highly abundant HIF-1{alpha} antisense transcript (referred to by the authors as aHIF) that arises from the 3'-UTR of the HIF-1{alpha} mRNA itself. This transcript is not overexpressed in lymphocytes or in seven of seven papillary renal carcinomas (which have normal VHL function) but is markedly elevated in 10 of 10 nonpapillary renal carcinoma specimens examined by the authors. The authors speculate that aHIF, by hybridizing to the 3' end of HIF-1{alpha} mRNA, prevents its translation. This inhibition could be mediated in several ways, including trapping and modification of HIF-1{alpha}/aHIF double-stranded RNAs in the nucleus (12).

aHIF RNA fails to respond to hypoxia in cells lacking functional pVHL, but, unlike the sense HIF-1{alpha} transcript, aHIF RNA is markedly induced by hypoxia in lymphocytes. Thus, aHIF behaves like a VHL-responsive gene. It remains to be seen whether aHIF contains hypoxia-sensitive AREs of similar sequence to those in the 3'-UTRs of VEGF and GLUT-1 mRNAs. Induction of aHIF in lymphocytes associates temporally with a decline in HIF-1{alpha} message that is noticeable 4-6 hours after hypoxic exposure. Thrash-Bingham and Tartof speculate that aHIF induction is responsible for the observed decrease in HIF-1{alpha} mRNA. Is aHIF transcription induced by HIF-1? If so, then HIF-1 induction of aHIF may represent an autofeedback mechanism to regulate HIF-1 activity, similar to the down-regulation of p53 protein by Mdm2, itself the product of a p53-induced gene. Such an autofeedback mechanism would explain those instances of HIF-1{alpha} mRNA decrease after prolonged exposure of cells or animals to hypoxia. The HIF-1-responsiveness of aHIF can be readily tested. Embryonic stem cells lacking HIF-1ß, the dimerization partner of HIF-1{alpha}, do not induce HIF-1{alpha} protein after hypoxic stimulation, and many hypoxia-inducible genes remain at baseline levels when these cells are exposed to low oxygen tension, even though HIF-1{alpha} mRNA is expressed normally (13). The failure of hypoxia to induce aHIF transcripts in HIF-1ß-/-cells would be strong evidence that aHIF is HIF-1 responsive.

Likewise, if aHIF were a VHL-responsive RNA, its marked overexpression in the absence of VHL might explain why HIF-1-responsive genes are not further induced by hypoxia in cells lacking functional pVHL. Elevated aHIF levels would be expected to greatly depress the steady-state level of HIF-1{alpha} mRNA in such cells, thereby counteracting any hypoxic effect on stabilization of HIF-1{alpha} protein. To determine if aHIF levels are elevated in nonpapillary renal carcinoma due to loss of function of pVHL, one could simply reintroduce a wild-type VHL gene into such cells and observe the resultant effects on aHIF expression and the subsequent response of aHIF levels to hypoxia.

What benefit would clear cell renal carcinomas gain by disabling the VHL gene? Based on the current study of Thrash-Bingham and Tartof, this may be an alternative mechanism for up-regulating hypoxia-survival genes, while at the same time preventing hypoxic induction of HIF-1{alpha}. Two recent reports demonstrated that hypoxic stabilization of wild-type p53 requires HIF-1{alpha} protein and that hypoxia-induced p53 suppresses further HIF-1-mediated transactivation and promotes apoptosis (14,15). Although p53 itself is frequently mutated or deleted in many solid tumors, p53 mutation does not appear to play a major role in the development of the majority of cases of renal cell carcinoma (16,17). Thus, it would certainly be beneficial for this type of carcinoma to find a means of surviving hypoxia (as all solid tumors must) without inducing p53. Inactivation of pVHL, by stabilizing hypoxia-survival RNAs while concomitantly inhibiting HIF-1{alpha} expression, would serve this purpose.

In conclusion, aHIF can be considered to link HIF-1{alpha}, which is responsible for the transcriptional activation of hypoxia-response genes, and pVHL, which prevents expression of these same genes under normoxic conditions. Hypoxia, by releasing aHIF from VHL-mediated suppression, normally sets in motion its own negative-feedback signal. One must continue to marvel at the ingenuity of cancer cells in their ability to subvert this pathway to find yet another means of circumventing the deleterious effects of uncontrolled proliferation.

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