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). Hypoxia
appears to regulate HIF-1
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 protein occurs primarily via protein
stabilization and not by hypoxia-dependent stabilization or induction
of HIF-1
mRNA (8). Thus, pVHL is probably not involved in
regulating the HIF-1
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
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
antisense transcript (referred to by the authors as aHIF) that arises
from the 3'-UTR of the HIF-1
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
mRNA, prevents its translation. This inhibition could
be mediated in several ways, including trapping and modification of
HIF-1
/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 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
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
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
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
, do not induce HIF-1
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
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 mRNA in such cells, thereby counteracting any hypoxic
effect on stabilization of HIF-1
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. Two recent reports demonstrated that hypoxic
stabilization of wild-type p53 requires HIF-1
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
expression, would serve this purpose.
In conclusion, aHIF can be considered to link HIF-1, 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.
REFERENCES
1
Ratcliffe PJ, O'Rourke JF, Maxwell PH, Pugh CW.
Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression. J Exp Biol 1998;201:1153-62.
2 Levy AP, Levy NS, Iliopoulos O, Jiang C, Kaplin WG Jr, Goldberg MA. Regulation of vascular endothelial growth factor by hypoxia and its modulation by the von Hippel-Lindau tumor suppressor gene. Kidney Int 1997;51:575-8.[Medline]
3
Levy AP, Levy NS, Goldberg MA. Post-transcriptional regulation
of vascular endothelial growth factor by hypoxia. J Biol Chem 1996;271:2746-53.
4
Levy AP, Levy NS, Goldberg MA. Hypoxia-inducible protein
binding to vascular endothelial growth factor mRNA and its modulation by the von
Hippel-Lindau protein. J Biol Chem 1996;271:25492-7.
5
Levy NS, Chung S, Furneaux H, Levy AP. Hypoxic stabilization
of vascular endothelial growth factor mRNA by the RNA-binding protein HuR. J Biol
Chem 1998;273:6417-23.
6 Iliopoulos O, Kaelin WG Jr. The molecular basis of von Hippel-Lindau disease. Mol Med 1997;3:289-93.[Medline]
7
Iliopoulos O, Levy AP, Jiang C, Kaelin WG Jr, Goldberg MA.
Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc
Natl Acad Sci U S A 1996;93:10595-9.
8
Huang LE, Gu J, Schau M, Bunn HF. Regulation of
hypoxia-inducible factor 1 is mediated by an O2-dependent degradation domain
via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A 1998;95:7987-92.
9
Wenger RH, Kvietikova I, Rolfs A, Gassmann M, Marti HH.
Hypoxia-inducible factor 1 is regulated at the post-mRNA level. Kidney Int 1997;51:560-3.[Medline]
10
Wenger RH, Rolfs A, Spielmann P, Zimmermann DR,
Gassmann M. Mouse hypoxia-inducible factor-1 is encoded by two different mRNA
isoforms: expression from a tissue-specific and a housekeeping-type promoter. Blood 1998;91:3471-80.
11 Thrash-Bingham CA, Tartof KD. aHIF: a natural antisense transcript overexpressed in human renal cancer and during hypoxia. J Natl Cancer Inst 1999;91:143-51.[Medline]
12
Kumar M, Carmichael GG. Nuclear antisense RNA induces
extensive adenosine modifications and nuclear retention of target transcripts. Proc Natl
Acad Sci U S A 1997;94:3542-7.
13 Maltepe E, Schmidt JV, Baunoch D, Bradfield CA, Simon MC. Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature 1997;386:403-7.[Medline]
14
An WG, Kanekal M, Simon MC, Maltepe E, Blagosklonny MV,
Neckers LM. Stabilization of wild-type p53 by hypoxia-inducible factor 1. Nature 1998;392:405-8.[Medline]
15
Blagosklonny MV, An WG, Romanova LY, Trepel J, Fojo T,
Neckers L. p53 inhibits hypoxia-inducible factor-stimulated transcription. J Biol Chem 1998;273:11995-8.
16 Uchida T, Wada C, Shitara T, Egawa S, Mashimo S, Koshiba K. Infrequent involvement of p53 mutations and loss of heterozygosity of 17p in the tumorigenesis of renal cell carcinoma. J Urol 1993;150:1298-301.[Medline]
17 Vignoli GC, Martorana G. Molecular genetics of renal cell carcinoma. Arch Ital Urol Androl 1997;69:265-9.[Medline]
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |