HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/12/1462 04-1640fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WARNECKE, C. Articles by ECKARDT, K.-U. Search for Related Content PubMed PubMed Citation Articles by WARNECKE, C. Articles by ECKARDT, K.-U.

Differentiating the functional role of hypoxia-inducible factor (HIF)-1 and HIF-2 (EPAS-1) by the use of RNA interference: erythropoietin is a HIF-2 target gene in Hep3B and Kelly cells

CHRISTINA WARNECKE^*,1, ZANETA ZABOROWSKA, JENS KURRECK, VOLKER A. ERDMANN, ULRICH FREI^*, MICHAEL WIESENER^*,2 and KAI-UWE ECKARDT^*,2

^* Department of Nephrology and Medical Intensive Care, Virchow Clinic, Charité University Medicine, Berlin, Germany; and
Institute for Biochemistry, Free University Berlin, Germany

¹ Correspondence: Nephrologische Forschungslaboratorien der Medizinischen Klinik IV, Universität Erlangen-Nürnberg, Loschgestrasse 8 1/2, D-91054 Erlangen, Germany. E-mail: christina.warnecke{at}med4.imed.uni-erlangen.de

SPECIFIC AIMS

Cellular adaptation to hypoxia is achieved by a transcriptional response system mediated by the hypoxia-inducible factor (HIF). Since the individual roles of its two alternative oxygen-regulated subunits, HIF-1 and HIF-2, are not fully understood, our aim was to determine functional differences between HIF-1 and HIF-2 in different human cell lines by using RNA interference (RNAi) and to compare the results with HIF overexpression experiments.

PRINCIPAL FINDINGS

1. Specificity and efficiency of HIF-1 and HIF-2 siRNAs
The transfection of HIF-1 and HIF-2 siRNAs in HeLa and Hep3B cells selectively and reproducibly reduced mRNA expression of the respective HIF isoform by >80%, whereas a luciferase control siRNA had no effect. Concordantly, knockdown of HIF mRNA levels led to substantially diminished HIF protein expression under inducing conditions.

2. Differential effects of HIF-1 and HIF-2 siRNAs on HIF target gene mRNA induction
RNase protection assays revealed that HIF-1 knockdown in HeLa cells reduced hypoxia- (0.5% oxygen) or iron chelator- (2,2 ipyridyl=DP) stimulated mRNA induction of glucose transporter 1 (GLUT-1), lactate dehydrogenase A (LDH-A), vascular endothelial growth factor (VEGF), carbonic anhydrase IX (CA IX), and HIF prolyl hydroxylase 2 (PHD2) by 40–60% (Fig. 1 A–F); HIF-2 knockdown had no effect on these HIF target genes. The same applied for Hep3B cells, although the suppression of mRNA induction was less pronounced than in HeLa cells, possibly due to the lower abundance of HIF-1 in these cells and/or the contribution of other hypoxia-activated signaling pathways in regulating these genes (Fig. 1G ).

View larger version (44K): [in this window] [in a new window]   Figure 1. Reduction of HIF target gene mRNA induction by HIF-1 siRNA in HeLa and Hep3B cells. A) RNase protection assay demonstrating the influence of HIF-1 knockdown on GLUT-1, LDH-A, and VEGF mRNA expression in HeLa cells. U6 small nuclear RNA (U6sn) served as loading control. HIF-1 and -2 mRNA levels were shown to confirm HIF knockdown; luc = luciferase control siRNA. B–D) Quantification of siRNA effects on GLUT-1, LDH-A, and VEGF mRNA levels in HeLa cells; data are means of 3–4 independent experiments ± SD. E) Effects of the siRNAs on VEGF and GLUT-1 were similar in hypoxic (H=0.5% O2), desferrioxamine- (DFO), and cobalt chloride- (Cob) stimulated cells (each 100 µM). F) Induction of CA IX and PHD2 mRNA was reduced by the HIF-1 siRNA but not affected by HIF-2 siRNA. G) In Hep3B cells VEGF, LDH-A, and CA IX were dependent on HIF-1.

3. Suppression of HIF-dependent reporter gene activation by siRNAs
In HeLa cells transfected with a luciferase reporter containing six copies of the phosphoglycerate kinase (PGK) hypoxia-responsive element (HRE), hypoxia- or DP-stimulated luciferase expression was reduced from 11- to 2-fold by HIF-1 knockdown whereas HIF-2 knockdown had no effect. Identical results were achieved with Hep3B cells.

4. Erythropoietin is a HIF-2 target gene in Hep3B and Kelly cells
Surprisingly, mRNA induction of erythropoietin (EPO) after exposure of Hep3B cells to hypoxia or hypoxia mimetics was almost abolished by the HIF-2 siRNA whereas HIF-1 knockdown had no effect (Fig. 2 A–C). This result was confirmed in an unrelated cell line, neuroblastoma Kelly cells, which in hypoxia expressed EPO at high levels (Fig. 2D ).

View larger version (46K): [in this window] [in a new window]   Figure 2. Reduction of EPO mRNA induction by the HIF-2 siRNA in Hep3B and Kelly cells. A) RNase protection assays revealed that EPO is a HIF-2 target gene in Hep3B cells (40 µg RNA/lane). B) Quantification of the siRNA effects on EPO mRNA levels in Hep3B cells (n=3). C) Effect of the HIF-2 siRNA on EPO mRNA expression in Hep3B cells was independent of the stimulus; N = normoxia, H = hypoxia (0.5% O2) and the hypoxia mimetics desferrioxamine (DFO) and cobalt chloride (Cob). D) RNase protection assays with human neuroblastoma Kelly cells (80 µg RNA/lane) confirmed the HIF-2 dependency of EPO induction in an unrelated cell line.

5. Transactivation of the EPO enhancer by HIF-2 requires cis-active elements adjacent to the HRE
Insights into the regulatory pathway underlying the HIF-2–specific effect were obtained by the use of luciferase reporter assays. A reporter containing five copies of the EPO HRE was merely induced 2- to 3-fold in HeLa and Hep3B cells and responded only to HIF-1 knockdown. In contrast, luciferase expression from a reporter plasmid containing the full-length 223 bp EPO enhancer was activated in a cell type-dependent manner (2.9-fold in DP-stimulated or hypoxic HeLa cells and 10-fold in Hep3B cells). In Hep3B cells, induction of luciferase activity was approximately halved by both HIF-1 and HIF-2 knockdown. These data indicate that reduction of the EPO enhancer to the 25 bp EPO HRE leads to a loss of the cell type-specific induction pattern and to a shift in response to the HIF isoforms.

6. Target gene specificity can be overcome by forced expression of HIF subunits
When wild-type human HIF-1 or HIF-2 expression plasmids were cotransfected with the 6x PGK HRE reporter in HeLa cells, luciferase expression was increased 10.1-fold by overexpression of HIF-2 even under baseline conditions; this increase responded to HIF-2 but not to HIF-1 knockdown. Similarly, the reporter could be induced by HIF-1 overexpression, although less efficiently. Thus, target gene specificity can be overcome by forced expression of the HIF subunits.

To confirm this, we cotransfected stable mouse HIF-1 and HIF-2 mutants with inactivated prolyl and asparagyl hydroxylation sites together with a human VEGF promoter or a mouse LDH-A promoter construct in HeLa cells. We observed that both promoters were inducible by HIF-1 as well as by HIF-2, albeit to different degrees.

Finally, we overexpressed the stable mouse HIF mutants in Hep3B cells and determined endogenous HIF target gene mRNA levels. EPO mRNA levels were increased only 11.3-fold by HIF-1- but 203.2-fold by HIF-2-overexpression, supporting the concept that EPO is a HIF-2 target gene. HIF-2 overexpression resulted in mRNA induction of CA IX and VEGF but, in contrast to EPO, the amplitude of induction did not reach that achieved by activation of endogenous HIF.

7. In 786-0 renal carcinoma cells HIF-2 compensates for the loss of functional HIF-1 protein
In 786-0 renal carcinoma cells lacking functional von Hippel-Lindau protein (pVHL) and HIF-1, HIF target gene expression is supposed to depend on HIF-2. Indeed, knockdown of HIF-2, but not HIF-1, reduced the mRNA levels of GLUT-1, LDH-A, and VEGF in 786-0 cells and hypoxic induction of these mRNAs in 786-0 cells stably retransfected with functional pVHL. Thus, similar to HIF overexpression, in 786-O cells target gene specificity is lost due to unknown mechanisms.

CONCLUSIONS AND SIGNIFICANCE

Until now, comparative studies aiming at a functional differentiation of the two HIF subunits were hampered by the fact that in vitro most cell lines express both HIF isoforms. In contrast, immunohistochemistry revealed differential expression of HIF-1 and HIF-2 in most cell types in vivo. Targeted deletion of the HIF subunits in mice resulted in divergent—in most cases, lethal—phenotypes. On the other hand, forced expression of the subunits in cell cultures suggested at least partial functional redundancy. In the present study, we show by use of the novel technique of RNAi that HIF-1 and HIF-2 have clearly defined and non-overlapping target gene specificities in different human cell lines, independent of the relative abundance of either HIF isoform. Furthermore, we show for the first time that this specificity can be overcome by forced expression of the HIF subunits from expression vectors and in tumor cells with severe perturbations in the HIF/pVHL axis. In Fig. 3 we summarize and interpret our findings.

View larger version (29K): [in this window] [in a new window]   Figure 3. Predicted model of the molecular mechanism of HIF isoform specificity and its loss by HIF overexpression. A) In mammalian cells with a functional HIF/pVHL system, each HIF isoform exhibits high affinity binding only to the HREs of its specific target genes, resulting in maximum hypoxic gene induction. Of course, HIF-1 also acts in cooperation with other transcription factors and transcriptional coactivators (not depicted) beyond CBP/p300, but for binding of HIF-2 to the EPO enhancer and full transactivation of the EPO gene the interaction with another transcription factor that binds to cis-active elements in the vicinity of the HRE is an absolute requirement (CBS=cofactor binding site). A candidate transcription factor for this interaction is hepatocyte nuclear factor 4 (HNF-4), which binds to a direct repeat of a steroid/thyroid nuclear hormone receptor response element half-site separated by a 2 bp spacer and was shown to be essential for the full hypoxic induction of the EPO gene. Formation of the stable DNA binding complex is possibly subject to redox control. B) HIF isoform overexpression induced by gene transfer or tumorigenic events affecting the HIF/pVHL system may lead to a loss of target gene specificity in that HIF-1 is now capable of binding to the HREs of HIF-2 targets, and vice versa. However, this promiscuous binding may be of lower affinity and the resulting gene induction less robust than the induction by the respective legitimate HIF isoform.

The fact that the genes involved in glucose metabolism, as well as CA IX and PHD2, were HIF-1 target genes was not entirely unexpected and agrees with other gene targeting studies. Concerning the previously proposed HIF-2 dependency of VEGF, we provide evidence that these conflicting results presumably were due to the experimental approach, i.e., overexpression of HIF-2, which may override target gene specificity.

Thus, overexpression experiments do not seem to be a useful approach for the characterization of HIF target gene specificities.

The most significant finding of the present study was the exclusive and unequivocal HIF-2 dependency of the hypoxic EPO mRNA induction in Hep3B and Kelly cells. This finding was surprising, since HIF-1 was identified as the nuclear factor that bound to the EPO HRE. On the other hand, immunohistochemical studies provided the first evidence for EPO as an HIF-2 target: renal peritubular fibroblasts, known to produce EPO, express HIF-2 but not HIF-1. Finally, recent HIF-2 gene targeting studies have supported our finding.

We also provide insight into the regulatory mechanism underlying the specificity of the endogenous EPO gene for transactivation by HIF-2. Reporter assays revealed that HIF-2 activates the EPO enhancer only in conjunction with other regulatory sequences in the vicinity of the HRE; the nuclear factors presumably binding to these cis-active elements remain to be determined. Since the 25 bp EPO HRE oligonucleotide has often been used as a probe in gel shift experiments, the difficulties encountered with HIF-2 gel shifts are now comprehensible.

In light of the increasing significance of recombinant EPO as a therapeutic drug not only in treating anemia, but possibly in the therapy of stroke and heart failure, the knowledge that EPO is a physiological HIF-2 target gene and that HIF-1 and HIF-2 signaling differs will be an important prerequisite for attempts to design specific therapeutic approaches to stimulate endogenous EPO production.

FOOTNOTES

² The two senior authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1640fje; doi: 10.1096/fj.04-1640fje

This article has been cited by other articles:

				A. J. Filiano, C. D. C. Bailey, J. Tucholski, S. Gundemir, and G. V. W. Johnson Transglutaminase 2 protects against ischemic insult, interacts with HIF1{beta}, and attenuates HIF1 signaling FASEB J, August 1, 2008; 22(8): 2662 - 2675. [Abstract] [Full Text] [PDF]

				D. P. Gale, S. K. Harten, C. D. L. Reid, E. G. D. Tuddenham, and P. H. Maxwell Autosomal dominant erythrocytosis and pulmonary arterial hypertension associated with an activating HIF2{alpha} mutation Blood, August 1, 2008; 112(3): 919 - 921. [Full Text] [PDF]

				M. Puppo, F. Battaglia, C. Ottaviano, S. Delfino, D. Ribatti, L. Varesio, and M. C. Bosco Topotecan inhibits vascular endothelial growth factor production and angiogenic activity induced by hypoxia in human neuroblastoma by targeting hypoxia-inducible factor-1{alpha} and -2{alpha} Mol. Cancer Ther., July 1, 2008; 7(7): 1974 - 1984. [Abstract] [Full Text] [PDF]

				Y. Maeda, T. Suzuki, X. Pan, G. Chen, S. Pan, T. Bartman, and J. A. Whitsett CUL2 Is Required for the Activity of Hypoxia-inducible Factor and Vasculogenesis J. Biol. Chem., June 6, 2008; 283(23): 16084 - 16092. [Abstract] [Full Text] [PDF]

				K. Takeda, H. L. Aguila, N. S. Parikh, X. Li, K. Lamothe, L.-J. Duan, H. Takeda, F. S. Lee, and G.-H. Fong Regulation of adult erythropoiesis by prolyl hydroxylase domain proteins Blood, March 15, 2008; 111(6): 3229 - 3235. [Abstract] [Full Text] [PDF]

				A. Weidemann, W. M. Bernhardt, B. Klanke, C. Daniel, B. Buchholz, V. Campean, K. Amann, C. Warnecke, M. S. Wiesener, K.-U. Eckardt, et al. HIF Activation Protects From Acute Kidney Injury J. Am. Soc. Nephrol., March 1, 2008; 19(3): 486 - 494. [Abstract] [Full Text] [PDF]

				P. Lechler, X. Wu, W. Bernhardt, V. Campean, S. Gastiger, T. Hackenbeck, B. Klanke, A. Weidemann, C. Warnecke, K. Amann, et al. The Tumor Gene Survivin Is Highly Expressed in Adult Renal Tubular Cells: Implications for a Pathophysiological Role in the Kidney Am. J. Pathol., November 1, 2007; 171(5): 1483 - 1498. [Abstract] [Full Text] [PDF]

				C.-J. Hu, A. Sataur, L. Wang, H. Chen, and M. C. Simon The N-Terminal Transactivation Domain Confers Target Gene Specificity of Hypoxia-inducible Factors HIF-1{alpha} and HIF-2{alpha} Mol. Biol. Cell, November 1, 2007; 18(11): 4528 - 4542. [Abstract] [Full Text] [PDF]

				O. Baranova, L. F. Miranda, P. Pichiule, I. Dragatsis, R. S. Johnson, and J. C. Chavez Neuron-Specific Inactivation of the Hypoxia Inducible Factor 1{alpha} Increases Brain Injury in a Mouse Model of Transient Focal Cerebral Ischemia J. Neurosci., June 6, 2007; 27(23): 6320 - 6332. [Abstract] [Full Text] [PDF]

				L. K. Martens, K. M. Kirschner, C. Warnecke, and H. Scholz Hypoxia-inducible Factor-1 (HIF-1) Is a Transcriptional Activator of the TrkB Neurotrophin Receptor Gene J. Biol. Chem., May 11, 2007; 282(19): 14379 - 14388. [Abstract] [Full Text] [PDF]

				I. Kojima, T. Tanaka, R. Inagi, H. Kato, T. Yamashita, A. Sakiyama, O. Ohneda, N. Takeda, M. Sata, T. Miyata, et al. Protective Role of Hypoxia-Inducible Factor-2{alpha} against Ischemic Damage and Oxidative Stress in the Kidney J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1218 - 1226. [Abstract] [Full Text] [PDF]

				W. M. Bernhardt, M. S. Wiesener, A. Weidemann, R. Schmitt, W. Weichert, P. Lechler, V. Campean, A. C. M. Ong, C. Willam, N. Gretz, et al. Involvement of Hypoxia-Inducible Transcription Factors in Polycystic Kidney Disease Am. J. Pathol., March 1, 2007; 170(3): 830 - 842. [Abstract] [Full Text] [PDF]

				P. Koivunen, M. Hirsila, A. M. Remes, I. E. Hassinen, K. I. Kivirikko, and J. Myllyharju Inhibition of Hypoxia-inducible Factor (HIF) Hydroxylases by Citric Acid Cycle Intermediates: POSSIBLE LINKS BETWEEN CELL METABOLISM AND STABILIZATION OF HIF J. Biol. Chem., February 16, 2007; 282(7): 4524 - 4532. [Abstract] [Full Text] [PDF]

				T. M. Asikainen, N. S. Waleh, B. K. Schneider, R. I. Clyman, and C. W. White Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo Am J Physiol Lung Cell Mol Physiol, October 1, 2006; 291(4): L588 - L595. [Abstract] [Full Text] [PDF]

				J. C. Chavez, O. Baranova, J. Lin, and P. Pichiule The Transcriptional Activator Hypoxia Inducible Factor 2 (HIF-2/EPAS-1) Regulates the Oxygen-Dependent Expression of Erythropoietin in Cortical Astrocytes J. Neurosci., September 13, 2006; 26(37): 9471 - 9481. [Abstract] [Full Text] [PDF]

				V. H. Haase Hypoxia-inducible factors in the kidney Am J Physiol Renal Physiol, August 1, 2006; 291(2): F271 - F281. [Abstract] [Full Text] [PDF]

				W. M. Bernhardt, V. Campean, S. Kany, J.-S. Jurgensen, A. Weidemann, C. Warnecke, M. Arend, S. Klaus, V. Gunzler, K. Amann, et al. Preconditional Activation of Hypoxia-Inducible Factors Ameliorates Ischemic Acute Renal Failure J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1970 - 1978. [Abstract] [Full Text] [PDF]

				G. P. Elvidge, L. Glenny, R. J. Appelhoff, P. J. Ratcliffe, J. Ragoussis, and J. M. Gleadle Concordant Regulation of Gene Expression by Hypoxia and 2-Oxoglutarate-dependent Dioxygenase Inhibition: THE ROLE OF HIF-1{alpha}, HIF-2{alpha}, AND OTHER PATHWAYS J. Biol. Chem., June 2, 2006; 281(22): 15215 - 15226. [Abstract] [Full Text] [PDF]

				O. Aprelikova, M. Wood, S. Tackett, G. V.R. Chandramouli, and J. C. Barrett Role of ETS Transcription Factors in the Hypoxia-Inducible Factor-2 Target Gene Selection Cancer Res., June 1, 2006; 66(11): 5641 - 5647. [Abstract] [Full Text] [PDF]

				C.-J. Hu, S. Iyer, A. Sataur, K. L. Covello, L. A. Chodosh, and M. C. Simon Differential Regulation of the Transcriptional Activities of Hypoxia-Inducible Factor 1 Alpha (HIF-1{alpha}) and HIF-2{alpha} in Stem Cells. Mol. Cell. Biol., May 1, 2006; 26(9): 3514 - 3526. [Abstract] [Full Text] [PDF]

				Q. Zhang, O. W. Moe, J. A. Garcia, and C. C. W. Hsia Regulated expression of hypoxia-inducible factors during postnatal and postpneumonectomy lung growth Am J Physiol Lung Cell Mol Physiol, May 1, 2006; 290(5): L880 - L889. [Abstract] [Full Text] [PDF]

				M. J. Percy, Q. Zhao, A. Flores, C. Harrison, T. R. J. Lappin, P. H. Maxwell, M. F. McMullin, and F. S. Lee From the Cover: A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis PNAS, January 17, 2006; 103(3): 654 - 659. [Abstract] [Full Text] [PDF]

				P. H Maxwell Hypoxia-inducible factor as a physiological regulator Exp Physiol, November 1, 2005; 90(6): 791 - 797. [Abstract] [Full Text] [PDF]

				J. Yang, L. Zhang, P. J. A. Erbel, K. H. Gardner, K. Ding, J. A. Garcia, and R. K. Bruick Functions of the Per/ARNT/Sim Domains of the Hypoxia-inducible Factor J. Biol. Chem., October 28, 2005; 280(43): 36047 - 36054. [Abstract] [Full Text] [PDF]

				R. H. Wenger, D. P. Stiehl, and G. Camenisch Integration of Oxygen Signaling at the Consensus HRE Sci. Signal., October 18, 2005; 2005(306): re12 - re12. [Abstract] [Full Text] [PDF]

				E. B. Rankin, D. F. Higgins, J. A. Walisser, R. S. Johnson, C. A. Bradfield, and V. H. Haase Inactivation of the Arylhydrocarbon Receptor Nuclear Translocator (Arnt) Suppresses von Hippel-Lindau Disease-Associated Vascular Tumors in Mice Mol. Cell. Biol., April 15, 2005; 25(8): 3163 - 3172. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/12/1462 04-1640fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WARNECKE, C. Articles by ECKARDT, K.-U. Search for Related Content PubMed PubMed Citation Articles by WARNECKE, C. Articles by ECKARDT, K.-U.