Hypoxia-inducible factor-1 (HIF-1) is a master regulator of mammalian oxygen homeostasis. HIF-1 consists of two subunits, HIF-1alpha and the aryl hydrocarbon receptor nuclear translocator (ARNT). Whereas hypoxia prevents proteasomal degradation of HIF-1alpha, ARNT expression is thought to be oxygen-independent. We and others previously showed that ARNT is indispensable for HIF-1 DNA-binding and transactivation function. Here, we have used ARNT-mutant mouse hepatoma and embryonic stem cells to examine the requirement of ARNT for accumulation and nuclear translocation of HIF-1alpha in hypoxia. As shown by immunofluorescence, HIF-1alpha accumulation in the nucleus of hypoxic cells was independent of the presence of ARNT, suggesting that nuclear translocation is intrinsic to HIF-1alpha. Co-immunoprecipitation of HIF-1alpha together with ARNT could be performed in nuclear extracts but not in cytosolic fractions, implying that formation of the HIF-1 complex occurs in the nucleus. A proteasome inhibitor and a thiol-reducing agent could mimic hypoxia by inducing HIF-1alpha in the nucleus, indicating that escape from proteolytic degradation is sufficient for accumulation and nuclear translocation of HIF-1alpha. During biochemical separation, both HIF-1alpha and ARNT tend to leak from the nuclei in the absence of either subunit, suggesting that heterodimerization is required for stable association within the nuclear compartment. Nuclear stabilization of the heterodimer might also explain the hypoxically increased total cellular ARNT levels observed in some of the cell lines examined.

REFERENCES

An
W. G.
,
Kanekal
M.
,
Simon
M. C.
,
Maltepe
E.
,
Blagosklonny
M. V.
,
Neckers
L. M.
(
1998
).
Stabilization of wild-type p53 by hypoxia-inducible factor 1.
Nature
392
,
405
408
Bunn
H. F.
,
Poyton
R. O.
(
1996
).
Oxygen sensing and molecular adaptation to hypoxia.
Physiol. Rev.
76
,
839
885
Camenisch
G.
,
Tini
M.
,
Chilov
D.
,
Kvietikova
I.
,
Srinivas
V.
,
Caro
J.
,
Spielmann
P.
,
Wenger
R. H.
,
Gassmann
M.
(
1999
).
General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against the hypoxia-inducible factor 1.
FASEB J
13
,
81
88
Carmeliet
P.
,
Dor
Y.
,
Herbert
J. M.
,
Fukumura
D.
,
Brusselmans
K.
,
Dewerchin
M.
,
Neeman
M.
,
Bono
F.
,
Abramovitch
R.
,
Maxwell
P.
, et al. 
(
1998
).
Role of HIF-1in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis.
Nature
394
,
485
490
Eguchi
H.
,
Ikuta
T.
,
Tachibana
T.
,
Yoneda
Y.
,
Kawajiri
K.
(
1997
).
A nuclear localization signal of human aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor 1is a novel bipartite type recognized by the two components of nuclear pore-targeting complex.
J. Biol. Chem
272
,
17640
17647
Forsythe
J. A.
,
Jiang
B. H.
,
Iyer
N. V.
,
Agani
F.
,
Leung
S. W.
,
Koos
R. D.
,
Semenza
G. L.
(
1996
).
Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.
Mol. Cell. Biol
16
,
4604
4613
Gassmann
M.
,
Kvietikova
I.
,
Rolfs
A.
,
Wenger
R. H.
(
1997
).
Oxygen-and dioxin-regulated gene expression in mouse hepatoma cells.
Kidney Int
51
,
567
574
Gradin
K.
,
McGuire
J.
,
Wenger
R. H.
,
Kvietikova
I.
,
Whitelaw
M. L.
,
Toftgård
R.
,
Tora
L.
,
Gassmann
M.
,
Poellinger
L.
(
1996
).
Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor.
Mol. Cell. Biol
16
,
5221
5231
Hankinson
O.
(
1995
).
The aryl hydrocarbon receptor complex.
Annu. Rev. Pharmacol. Toxicol
35
,
307
340
Hoffman
E. C.
,
Reyes
H.
,
Chu
F. F.
,
Sander
F.
,
Conley
L. H.
,
Brooks
B. A.
,
Hankinson
O.
(
1991
).
Cloning of a factor required for activity of the Ah (dioxin) receptor.
Science
252
,
954
958
Hord
N. G.
,
Perdew
G. H.
(
1994
).
Physicochemical and immunocytochemical analysis of the aryl hydrocarbon receptor nuclear translocator: characterization of two monoclonal antibodies to the aryl hydrocarbon receptor nuclear translocator.
Mol. Pharmacol
46
,
618
626
Huang
E. L.
,
Gu
J.
,
Schau
M.
,
Bunn
H. F.
(
1998
).
Regulation of hypoxia-inducible factor 1is mediated by an oxygen-dependent domain via the ubiquitin-proteasome pathway.
Proc. Natl. Acad. Sci. USA
95
,
7987
7992
Huang
L. E.
,
Arany
Z.
,
Livingston
D. M.
,
Bunn
H. F.
(
1996
).
Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of itssubunit.
J. Biol. Chem
271
,
32253
32259
Ikuta
T.
,
Eguchi
H.
,
Tachibana
T.
,
Yoneda
Y.
,
Kawajiri
K.
(
1998
).
Nuclear localization and export signals of the human aryl hydrocarbon receptor.
J. Biol. Chem
273
,
2895
2904
Iyer
N. V.
,
Kotch
L. E.
,
Agani
F.
,
Leung
S. W.
,
Laughner
E.
,
Wenger
R. H.
,
Gassmann
M.
,
Gearhart
J. D.
,
Lawler
A. M.
,
Yu
A. Y.
, et al. 
(
1998
).
Cellular and developmental control of O2homeostasis by hypoxia-inducible factor 1.
Genes Dev
12
,
149
162
Jiang
B. H.
,
Agani
F.
,
Passaniti
A.
,
Semenza
G. L.
(
1997
).
V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression.
Cancer Res
57
,
5328
5335
Jiang
B. H.
,
Rue
E.
,
Wang
G. L.
,
Roe
R.
,
Semenza
G. L.
(
1996
).
Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1.
J. Biol. Chem
271
,
17771
17778
Jiang
B. H.
,
Semenza
G. L.
,
Bauer
C.
,
Marti
H. H.
(
1996
).
Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2tension.
Am. J. Physiol
271
,
1172
1180
Jiang
B. H.
,
Zheng
J. Z.
,
Leung
S. W.
,
Roe
R.
,
Semenza
G. L.
(
1997
).
Transactivation and inhibitory domains of hypoxia-inducible factor 1. Modulation of transcriptional activity by oxygen tension.
J. Biol. Chem
272
,
19253
19260
Kallio
P. J.
,
Okamoto
K.
,
O'Brien
S.
,
Carrero
P.
,
Makino
Y.
,
Tanaka
H.
,
Poellinger
L.
(
1998
).
Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1.
EMBO J
17
,
6573
6586
Kallio
P. J.
,
Pongratz
I.
,
Gradin
K.
,
McGuire
J.
,
Poellinger
L.
(
1997
).
Activation of hypoxia-inducible factor 1: posttranscriptional regulation and conformational change by recruitment of the Arnt transcription factor.
Proc. Natl. Acad. Sci. USA
94
,
5667
5672
Kozak
K. R.
,
Abbott
B.
,
Hankinson
O.
(
1997
).
Arnt-deficient mice and placental differentiation.
Dev. Biol
191
,
297
305
Kvietikova
I.
,
Wenger
R. H.
,
Marti
H. H.
,
Gassmann
M.
(
1995
).
The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site.
Nucl. Acids Res
23
,
4542
4550
Lee
P. J.
,
Jiang
B. H.
,
Chin
B. Y.
,
Iyer
N. V.
,
Alam
J.
,
Semenza
G. L.
,
Choi
A. M.
(
1997
).
Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia.
J. Biol. Chem
272
,
5375
5381
Liu
Y. X.
,
Christou
H.
,
Morita
T.
,
Laughner
E.
,
Semenza
G. L.
,
Kourembanas
S.
(
1998
).
Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial growth factor gene via the 5enhancer.
J. Biol. Chem
273
,
15257
15262
Maltepe
E.
,
Schmidt
J. V.
,
Baunoch
D.
,
Bradfield
C. A.
,
Simon
M. C.
(
1997
).
Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT.
Nature
386
,
403
407
Martin
C.
,
Yu
A. Y.
,
Jiang
B. H.
,
Davis
L.
,
Kimberly
D.
,
Hohimer
A. R.
,
Semenza
G. L.
(
1998
).
Cardiac hypertrophy in chronically anemic fetal sheep—increased vascularization is associated with increased myocardial expression of vascular endothelial growth factor and hypoxia-inducible factor 1.
Am. J. Obst. Gynecol
178
,
527
534
Numayama-Tsuruta
K.
,
Kobayashi
A.
,
Sogawa
K.
,
Fujii-Kuriyama
Y.
(
1997
).
A point mutation responsible for defective function of the aryl-hydrocarbon-receptor nuclear translocator in mutant Hepa-1c1c7 cells.
Eur. J. Biochem
246
,
486
495
Pollenz
R. S.
(
1996
).
The aryl-hydrocarbon receptor, but not the aryl-hydrocarbon receptor nuclear translocator protein, is rapidly depleted in hepatic and nonhepatic culture cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Mol. Pharmacol
49
,
391
398
Pollenz
R. S.
,
Sattler
C. A.
,
Poland
A.
(
1994
).
The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy.
Mol. Pharmacol
45
,
428
438
Ryan
H. E.
,
Lo
J.
,
Johnson
R. S.
(
1998
).
HIF-1is required for solidtumor formation and embryonic vascularization.
EMBO J
17
,
3005
3015
Salceda
S.
,
Beck
I.
,
Caro
J.
(
1996
).
Absolute requirement of aryl hydrocarbon receptor nuclear translocator protein for gene activation by hypoxia.
Arch. Biochem. Biophys
334
,
389
394
Salceda
S.
,
Caro
J.
(
1997
).
Hypoxia-inducible factor 1(HIF-1) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes.
J. Biol. Chem
272
,
22642
22647
Schmidt
J. V.
,
Bradfield
C. A.
(
1996
).
Ah receptor signaling pathways. Ann. Rev. Cell.
Dev. Biol
12
,
55
89
Semenza
G. L.
(
1998
).
Hypoxia-inducible factor 1: master regulator of O2homeostasis.
Curr. Opin. Genet. Dev
8
,
588
594
Tian
H.
,
Hammer
R. E.
,
Matsumoto
A. M.
,
Russell
D. W.
,
McKnight
S. L.
(
1998
).
The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development.
Genes Dev
12
,
3320
3324
Wang
G. L.
,
Jiang
B. H.
,
Rue
E. A.
,
Semenza
G. L.
(
1995
).
Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2tension.
Proc. Natl. Acad. Sci. USA
92
,
5510
5514
Wang
G. L.
,
Jiang
B. H.
,
Semenza
G. L.
(
1995
).
Effect of altered redox states on expression and DNA-binding activity of hypoxia-inducible factor 1.
Biochem. Biophys. Res. Commun
212
,
550
556
Wang
G. L.
,
Jiang
B. H.
,
Semenza
G. L.
(
1995
).
Effect of protein kinase and phosphatase inhibitors on expression of hypoxia-inducible factor 1.
Biochem. Biophys. Res. Commun
216
,
669
675
Wenger
R. H.
,
Camenisch
G.
,
Desbaillets
I.
,
Chilov
D.
,
Gassmann
M.
(
1998
).
Up-regulation of hypoxia-inducible factor-1is not sufficient for hypoxic/anoxic p53 induction.
Cancer Res
58
,
5678
5680
Wenger
R. H.
,
Gassmann
M.
(
1997
).
Oxygen(es) and the hypoxia-inducible factor-1.
Biol. Chem
378
,
609
616
Wenger
R. H.
,
Kvietikova
I.
,
Rolfs
A.
,
Gassmann
M.
,
Marti
H. H.
(
1997
).
Hypoxia-inducible factor-1is regulated at the post-mRNA level.
Kidney Int
51
,
560
563
Wood
S. M.
,
Gleadle
J. M.
,
Pugh
C. W.
,
Hankinson
O.
,
Ratcliffe
P. J.
(
1996
).
The role of the aryl hydrocarbon receptor nuclear translocator (ARNT) in hypoxic induction of gene expression. Studies in ARNT-deficient cells.
J. Biol. Chem
271
,
15117
15123
This content is only available via PDF.