Abstract
The hypoxia-inducible transcription factor (HIF) controls (in an oxygen-dependent manner) the expression of a large number of genes whose products are involved in the response of cells to hypoxia. HIF is an αβ dimer that binds to hypoxia response elements (HREs) in its target genes. Human HIF-α has three isoforms, HIF-1α, HIF-2α and HIF-3α, of which the roles of HIF-3α are largely unknown, although it is usually regarded as a negative regulator of HIF-1α and HIF-2α. The human HIF-3α locus is subject to extensive alternative splicing, leading to at least seven variants. We analyzed here the effects of the long variants and the short variant HIF-3α4 on the hypoxia response. All these variants were found to interact with HIF-β, HIF-1α and HIF-2α. The long HIF-3α variants were localized in the nucleus in hypoxia, while HIF-3α4 was cytoplasmic. Interaction of the HIF-3α variants with HIF-1α inhibited the nuclear translocation of both. None of the long HIF-3α variants was capable of efficient induction of an HRE reporter in overexpression experiments, but instead inhibited the transcriptional activation of the reporter by HIF-1 and HIF-2. Unexpectedly, siRNA knock-down of the endogenous HIF-3α variants led to downregulation of certain HIF target genes, while overexpression of individual long HIF-3α variants upregulated certain HIF target genes in a variant and target gene-specific manner under conditions in which HIF-β was not a limiting factor. These data indicate that the HIF-3α variants may have more versatile and specific roles in the regulation of the hypoxia response than previously anticipated.
Similar content being viewed by others
Abbreviations
- HIF:
-
Hypoxia-inducible factor
- ODDD:
-
Oxygen-dependent degradation domain
- P4H:
-
Prolyl 4-hydroxylase
- C-TAD:
-
C-terminal transactivation domain
- bHLH:
-
Basic helix-loop-helix
- PAS:
-
Per-ARNT-Sim
- HRE:
-
Hypoxia response element
- IPAS:
-
Inhibitory PAS domain protein
- LZIP:
-
Leucine zipper
- qPCR:
-
Quantitative real-time RT-PCR
References
Semenza GL (2010) HIF-1: upstream and downstream of cancer metabolism. Curr Opin Genet Dev 20:51–56
Kaelin WG Jr, Ratcliffe PJ (2008) Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 30:393–402
Weidemann A, Johnson RS (2008) Biology of HIF-1α. Cell Death Differ 15:621–627
Myllyharju J (2008) Prolyl 4-hydroxylases, key enzymes in the synthesis of collagens and regulation of the response to hypoxia, and their roles as treatment targets. Ann Med 40:402–417
Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lanie WS, Kaelin WG Jr (2001) HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292:464–468
Jaakkola P, Mole DR, Tian Y-M, Wilson MI, Gielbert J, Gaskell SJ, Kriegsheim AV, Hebestreit HF, Mukherji M, Schofield CJ, Maxwell PH, Pugh CW, Ratcliffe PJ (2001) Targeting of HIF-α to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292:468–472
Yu F, White SB, Zhao Q, Lee FS (2001) HIF-1α binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci USA 98:9630–9635
Bruick RK, McKnight SL (2001) A conserved family of prolyl-4-hydroxylases that modify HIF. Science 294:1337–1340
Epstein ACR, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian Y-M, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ (2001) C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107:43–54
Ivan M, Haberberger T, Gervasi DC, Michelson KS, Günzler V, Kondo K, Yang H, Sorokina I, Conaway RC, Conaway JW, Kaelin WG Jr (2002) Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor. Proc Natl Acad Sci USA 99:13459–13464
Hirsilä M, Koivunen P, Günzler V, Kivirikko KI, Myllyharju J (2003) Characterization of the human prolyl 4-hydroxylases that modify the hypoxia-inducible factor. J Biol Chem 278:30772–30780
Myllyharju J (2009) HIF prolyl 4-hydroxylases and their potential as drug targets. Curr Pharm Des 15:3878–3885
Lando D, Peet DJ, Whelan DA, Gorman JJ, Whitelaw ML (2002) Asparagine hydroxylation of the HIF transactivation domain: a hypoxic switch. Science 295:858–861
Hewitson KS, McNeill LA, Riordan MV, Tian Y-M, Bullock AN, Welford RW, Elkins JM, Oldham NJ, Bhattacharya S, Gleadle JM, Ratcliffe PJ, Pugh CW, Schofield CJ (2002) Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family. J Biol Chem 277:26351–26355
Lando D, Peet DJ, Gorman JJ, Whelan DA, Whitelaw ML, Bruick RK (2002) FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor. Genes Dev 16:1466–1471
Patel SA, Simon MC (2008) Biology of hypoxia-inducible factor-2α in development and disease. Cell Death Differ 15:628–634
Wiesener MS, Turley H, Allen WE, Willam C, Eckardt K-U, Talks KL, Wood SM, Gatter KC, Harris AL, Pugh CW, Ratcliffe PJ, Maxwell PH (1998) Induction of endothelial PAS domain protein-1 by hypoxia: characterization and comparison with hypoxia-inducible factor-1α. Blood 92:2260–2268
Wenger RH (2002) Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J 16:1151–1162
Wenger RH, Stiehl DP, Camenisch G (2005) Integration of oxygen signaling at the consensus HRE. Sci. STKE 306:re12
Mole DR, Blancher C, Copley RR, Pollard PJ, Gleadle JM, Ragoussis J, Ratcliffe PJ (2009) Genome-wide association of hypoxia-inducible factor (HIF)-1α and HIF-2α DNA binding with expression profiling of hypoxia-inducible transcripts. J Biol Chem 284:16767–16775
Gu Y-Z, Moran SM, Hogenesch JB, Wartman L, Bradfield CA (1998) Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3α. Gene Expr 7:205–213
Hara S, Hamada J, Kobayashi C, Kondo Y, Imura N (2001) Expression and characterization of hypoxia-inducible factor (HIF)-3α in human kidney: suppression of HIF-mediated gene expression by HIF-3α. Biochem Biophys Res Commun 287:808–813
Makino Y, Cao R, Svensson K, Bertilsson G, Åsman M, Tanaka H, Cao Y, Berkenstam A, Poellinger L (2001) Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 414:550–554
Makino Y, Kanopka A, Wilson WJ, Tanaka H, Poellinger L (2002) Inhibitory PAS domain protein (IPAS) is a hypoxia-inducible splicing variant of the hypoxia-inducible factor-3α locus. J Biol Chem 277:32405–32408
Yamashita T, Ohneda O, Nagano M, Iemitsu M, Makino Y, Tanaka H, Miyauchi T, Goto T, Ohneda K, Fujii-Kuriyama Y, Poellinger L, Yamamoto M (2008) Abnormal heart development and lung remodeling in mice lacking the hypoxia-inducible factor-related basic helix-loop-helix PAS protein NEPAS. Mol Cell Biol 28:1285–1297
Maynard MA, Qi H, Chung J, Lee EHL, Kondo Y, Hara S, Conaway RC, Conaway JW, Ohh M (2003) Multiple splice variants of the human HIF-3α locus are targets of the von Hippel-Lindau E3 ubiquitin ligase complex. J Biol Chem 278:11032–11040
Pasanen A, Heikkilä M, Rautavuoma K, Hirsilä M, Kivirikko KI, Myllyharju J (2010) Hypoxia-inducible factor (HIF)-3α is subject to extensive alternative splicing in human tissues and cancer cells and is regulated by HIF-1 but not HIF-2. Int J Biochem Cell Biol 42:1189–1200
Jang MS, Park JE, Lee JA, Park SG, Myung PK, Lee DH, Park BC, Cho S (2005) Binding and regulation of hypoxia-inducible factor-1 by the inhibitory PAS proteins. Biochem Biophys Res Commun 337:209–215
Maynard MA, Evans AJ, Hosomi T, Hara S, Jewett MAS, Ohh M (2005) Human HIF-3α4 is a dominant-negative regulator of HIF-1 and is down-regulated in renal cell carcinoma. FASEB J 19:1396–1406
Besson A, Wilson TL, Yong VW (2002) The anchoring protein RACK1 links protein kinase Cε to integrin β chains. Requirements for adhesion and motility. J Biol Chem 277:22073–22084
Koivunen P, Hirsilä M, Kivirikko KI, Myllyharju J (2006) The length of peptide substrates has a marked effect on hydroxylation by the hypoxia-inducible factor prolyl 4-hydroxylases. J Biol Chem 281:28712–28720
Tanaka T, Wiesener M, Bernhardt W, Eckardt K-U, Warnecke C (2009) The human HIF (hypoxia-inducible factor)-3α gene is a HIF-1 target gene and may modulate hypoxic gene induction. Biochem J 424:143–151
Berra E, Benizri E, Ginouvès A, Volmat V, Roux D, Pouysségur J (2003) HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1α in normoxia. EMBO J 22:4082–4090
Appelhoff RJ, Tian Y-M, Raval RR, Turley H, Harris AL, Pugh CW, Ratcliffe PJ, Gleadle JM (2004) Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. J Biol Chem 279:38458–38465
Takeda K, Ho VC, Takeda H, Duan L-J, Nagy A, Fong G-H (2006) Placental but not heart defects are associated with elevated hypoxia-inducible factor alpha levels in mice lacking prolyl hydroxylase domain protein 2. Mol Cell Biol 26:8336–8346
Chan DA, Sutphin PD, Yen S-E, Giaccia AJ (2005) Coordinate regulation of the oxygen-dependent degradation domains of hypoxia-inducible factor 1α. Mol Cell Biol 25:6415–6426
Lee PJ, Jiang B-H, Yoke Chin B, Iyer NV, Alam J, Semenza GL, Choi AMK (1997) Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J Biol Chem 272:5375–5381
Kitamuro T, Takahashi K, Ogawa K, Udono-Fujimori R, Takeda K, Furuyama K, Nakayama M, Sun J, Fujita H, Hida W, Hattori T, Shirato K, Igarashi K, Shibahara S (2003) Bach1 functions as a hypoxia-inducible repressor for the heme oxygenase-1 gene in human cells. J Biol Chem 278:9125–9133
Acknowledgments
We thank Anne Kokko and Raija Juntunen for their expert technical assistance. This work was supported by the Academy of Finland (Grants 1114344 and 1211128 to JM and 121789 to MH), the Sigrid Juselius Foundation and FibroGen Inc.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Heikkilä, M., Pasanen, A., Kivirikko, K.I. et al. Roles of the human hypoxia-inducible factor (HIF)-3α variants in the hypoxia response. Cell. Mol. Life Sci. 68, 3885–3901 (2011). https://doi.org/10.1007/s00018-011-0679-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00018-011-0679-5