FEBS Letters 583 (2009) 1314–1318

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Hypoxia-inducible factor 1a activates and is inhibited by unoccupied b

Wonchung Lim a, Jungyoon Cho a, Hyeok-yi Kwon a, Yeomyeong Park a, Mee-Ra Rhyu b, YoungJoo Lee a,* a College of Life Science, Institute of Biotechnology, Department of Bioscience and Biotechnology, Sejong University, Kwang-Jin-Gu, Seoul 143-747, Republic of Korea b Food Function Research Center, Korea Food Research Institute, Gyeonggi-Do 463-746, Republic of Korea article info abstract

Article history: Previously, we showed that hypoxia induces ligand-independent estrogen receptor (ER)a activation. Received 24 December 2008 In this study, we found that hypoxia activated the ERb-mediated transcriptional response in HEK293 Revised 9 March 2009 cells in the absence of estrogen. ERb transactivation was induced by the expression of the hypoxia- Accepted 15 March 2009 inducible factor 1a (HIF-1a) under normoxia. ERb interacted with HIF-1a, and SRC1 and CBP poten- Available online 20 March 2009 tiated the effect of HIF-1a on ERb-mediated transcription. We then examined the effect of ERb on Edited by Robert Barouki HIF1-a transactivation. Surprisingly, ERb attenuated the transcriptional activity of HIF-1a, as mea- sured by HRE-driven reporter and hypoxic induction of VEGF mRNA in HEK293 cells. Taken together, these data show that HIF-1 activates ERb-mediated transcription in the Keywords: a Estrogen receptor beta absence of a ligand, and ERb inhibits HIF-1a-mediated transcription. Hypoxia Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Hypoxia-inducible factor 1a Vascular endothelial growth factor

1. Introduction Hypoxia is defined as a state of reduced oxygen pressure in tissues and is associated with and cardiovascular disease. Estrogen and its receptors, estrogen receptor a (ERa) and b Hypoxia regulates many cellular processes, such as angiogenesis, (ERb), play an important role in many non-reproductive tissues, anaerobic metabolism, cell proliferation/survival, and promotion including the immune, cardiovascular, and central nervous sys- of metastasis, by controlling the gene expression of the major tems, as well as in normal and malignant reproductive tissues, oxygen homeostasis regulator, hypoxia inducible factor-1 (HIF-1). such as the breast, ovary, and uterus. HIF-1 is composed of a heterodimer pair with an oxygen-degraded ERb has different biological functions than ERa, as well as sim- HIF-1a subunit and a constitutively expressed HIF-1b subunit ilar activities [1,2]. Ström et al. reported that in contrast to ERa, (ARNT) [8]. Under hypoxic conditions, HIF-1a is stabilized and which is an activator of cancer cell growth, ERb inhibits cancer cell regulates transcription by binding to the hypoxia response element proliferation through the modulation of key regulators in the cell (HRE) on target genes [9]. HIF-1a-mediated activation of transcrip- cycle [3,4]. In addition, ERb can modulate the function of ERa itself. tion requires the recruitment of coactivators, such as CBP/p300, ERb regulates ERa-mediated transcriptional activation by altering and factors belonging to the p160/SRC1 family of proteins the recruitment of c-Fos and c-Jun to estrogen-responsive promot- [10–13]. Ruas et al. reported that CBP plays an important role as ers [5].ERb expression attenuates the growth-promoting activity a mediator, and SRC-1 may recruit other cofactors of the HIF- of ERa [6]. The activity of the can be 1a/Arnt/CBP/SRC1 complex [14]. modulated by the proportion of the ER subtypes [7]. Microarray The cross-talk between estrogen and hypoxic signaling path- analyses have shown that unoccupied ERb both stimulated and ways in and the role of HIF-1a in estrogen receptor suppressed many genes regulated by E2-occupied ERa [6]. Chang signaling has attracted increased interest. It has been suggested et al. also showed a unique set of genes responsive to E2-occupied that hypoxia plays a key role in the development of acquired hor- ERb, but not ERa [6]. We investigated the modulating mechanisms mone resistance in breast cancer by modulating the estrogen of ERb transactivation to better understand various estrogen-regu- receptor level [15]. Seifeddine et al. showed that 31 genes were lated biological effects on normal tissues, as well as malignant regulated by simultaneous hypoxia and E2 treatment in T47 human tumors. breast cancer cells [16]. Estrogens stabilize and activate HIF-1a by phosphorylation through the PI3K pathway in the uterus and ovar- * Corresponding author. Fax: +82 2 3408 4334. ian carcinoma cells [17,18]. Previously, we showed that hypoxia, E-mail addresses: [email protected], [email protected] (Y. Lee). via HIF-1a, regulates the expression and transcriptional activation

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.03.028 W. Lim et al. / FEBS Letters 583 (2009) 1314–1318 1315 of ERa and the physical interaction of HIF-1a and ERa [19,20]. 2.5. Reverse transcription (RT)-PCR However, the effect of hypoxia on the activation of ERb is not yet known. This study focuses on whether hypoxia regulates ERb- Total RNA was extracted using Trizol Reagent according to the mediated transcription and whether ERb is involved in the tran- manufacturer’s instruction. RNA pellets were dissolved in diethyl- scriptional response to hypoxia. We show that hypoxia induces pyrocarbonate-treated water. The yield of RNA was quantified by HIF-1a activated ERb-mediated transcription in the absence of a spectroscopy at 260 nm. Samples were aliquoted and stored at ligand, and ERb significantly decreases the transcriptional activity 80 °C until further processing. To synthesize first strand cDNA, of HIF-1a and hypoxic induction of vascular endothelial growth 3 lg total RNA was incubated at 70 °C for 5 min with 0.5 lg of ran- factor (VEGF) mRNA in HEK293 cells. dom hexamer and deionized water (up to 11 ll). The reverse tran- scription reaction was performed using 40 units of M-MLV reverse transcriptase (Promega) in 5 reaction buffer (250 mmol/l Tris–

2. Materials and methods HCl; pH 8.3, 375 mM KCl, 15 mM MgCl2, 50 mM DTT), RNase inhib- itor at 1 unit/ll, and 1 mM dNTP mixtures at 37 °C for 60 min. The 2.1. Reagents reaction was terminated by heating at 70 °C for 10 min, followed by cooling at 4 °C. The resulting cDNA was added to the PCR reac-

E2 was purchased from Sigma (St. Louis, MO, USA) and dissolved tion mixture containing 10 PCR buffer (100 mM Tris–HCl, pH 8.3, in 100% ethanol. ICI 182,780 (ICI) was obtained from ZENECA Phar- 500 mM KCl, 15 mM MgCl2), 25 units of rTaq polymerase (TakaRa, maceuticals (Tocris, UK). MG132, PD98059, or wortmannin (Sigma) Shiga, Japan), 4 ll of 2.5 mM dNTP mixtures, and 10 pmol of prim- was dissolved in dimethyl sulfoxide (DMSO). All of the compounds ers each. The final volume was 50 ll. Samples were amplified at were added to the medium such that the total solvent concentra- 94 °C for 5 min, 23 cycles of 94 °C for 45 s, 55 °C for 45 s, and tion was never higher than 0.1%. An untreated group served as a 72 °C for 45 s using Mastercycler gradient (Eppendorf, Hamburg, control. Germany). b-Actin was amplified for 20 cycles, followed by 72 °C for 5 min. The primers used were: b-actin sense primer, 50-CCTGA- 2.2. Cell culture and hypoxic conditions CCCTGAAGTACCCCA-30, b-actin antisense primer, 50-CGTCATG- CAGCTCATAGCTC-30; VEGF sense primer, 50-ATGAACTTTCTGCTCT- MCF-7 and HEK293 cells were maintained in phenol red-free CTGG-30, VEGF antisense primer, 50-TCATCTCTCCTATGTGCTGGC-30. Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (WelGENE). Cells were grown at 2.6. Statistical analysis

37 °C in a humidified atmosphere of 95% air/5% CO2 and fed every 2–3 days. Before treatment, the cells were washed with phosphate- Values shown represent the mean ± S.D. Statistical analysis was buffered saline (PBS) and cultured in DMEM/5% charcoal–dextran performed by Student’s t-test, and a P-value < 0.05 was considered stripped FBS (CD-FBS) for 2 days to eliminate any estrogenic source significant. before treatment. All treatments were done with DMEM/5% CD-

FBS. We used 10 nM E2, unless otherwise noted. For the hypoxic condition, cells were incubated at a CO2 level of 5% with 1% O2 bal- 3. Results anced with N2 using a hypoxic chamber (Forma). 3.1. Effects of hypoxia on ERb-mediated transcription in HEK293 cells 2.3. Plasmids To determine whether hypoxia activates ERb-mediated tran- The ERE-tk81-Luc plasmid, constructed by inserting the frag- scription, HEK293 cells were transfected with plasmids containing ment of the herpes simplex thymidine kinase promoter and two an ERb expression vector and an ERE-driven reporter. After trans- copies of the vitellogenin ERE into pA3-luc (ERE-Luc) [21], was a fection, cells were exposed to normoxia or hypoxia, as indicated. gift from Dr. Larry Jameson (Northwestern University, USA). The As a control, we confirmed that hypoxia significantly increases pcDNA3.1-hERa plasmid was constructed by inserting the frag- the HIF-1a-dependent transcriptional activation of the HRE-driven ment of the full-length hERa into pcDNA3.1 (Invitrogen). The hERb reporter gene in HEK293 cells (data not shown). Hypoxia activated expression vector was kindly provided by Dr. Soo-Jong Um (Sejong ERE-driven luciferase expression in cells expressing ERb (Fig. 1A), University, Korea). The hypoxia responsive element (HRE)-Luc re- and this hypoxic induction was blocked by the ER antagonist ICI porter plasmid contains four copies of the erythropoietin HRE, (Fig. 1B), indicating that hypoxia activates ERb-mediated transcrip- the SV40 promoter, and the luciferase gene.GFP-HIF-1a vector tion. However, hypoxia-activated, ERb-mediated luciferase expres- was kindly provided by Dr. Kyu-Won Kim (Seoul National sion was not further enhanced by E2 treatment (Fig. 1A). The University, Korea). The pGAL4-Luc plasmid was a gift from Dr. difference in efficiency between data reported in Fig. 1A and B Benita Katzenellenbogen (University of Illinois, USA). The pGAL4- may depend on the lot of plasmid preparation, the incubation time, HIF-1a was kindly provided by Dr. Hyun-Sung Park (Seoul Univer- and the person, etc. However, we consistently observed activation sity, Korea). of ERE-driven luciferase expression by ERb under hypoxia. To determine whether HIF-1a activates ERb-mediated tran- 2.4. Transient transfection and luciferase assay scription, we transfected HEK293 cells with an ERE-driven reporter plasmid and an expression vector for ERb with or without GFP-HIF- HEK 293 cells were transiently transfected with plasmids by 1a, which is stable and stimulates transcription of its target genes using the polyethylenimine (PEI; Polysciences) method. Lucifer- under normoxic conditions [22]. As shown in Fig. 1C, HIF-1a in- ase activity was determined 24 or 48 h after treatment with an creased ERb-mediated luciferase expression approximately five- AutoLumat LB953 luminometer using the luciferase assay system fold. However, this induction was blocked by the addition of ICI (Promega Corp., Madison, WI, USA) and expressed as relative (Fig. 1C). These results indicate that the hypoxia-induced ERb light units. The means and standard errors of three replicates transactivation is mediated by HIF-1a. are shown for the representative experiments. All transfection CBP participates in unliganded ERb coactivation [23]. SRC1 acti- experiments were repeated three or more times with similar vates ERb-mediated transcription in the absence of a ligand [23]. results. SRC1 and CBP act synergistically to enhance HIF-1a-dependent 1316 W. Lim et al. / FEBS Letters 583 (2009) 1314–1318

7 * 3 * 6 2.5 5 2 4 1.5 3 1 2 (Fold Induction) (Fold Induction) Luciferase Activity Luciferase Activity 1 0.5

0 0 con Hypoxia Hypoxia+E2 con Hypoxia Hypoxia+ICI

12 7 ‡ * ‡ 6 10

5 8 4 6 3 * 4 2 (Fold Induction) (Fold Induction) 1 2 Luciferase Activity Luciferase Activity 0 0 con HIF1- HIF-1 +ICI HIF-1 - + ++ SRC1 - - + - CBP - -- +

Fig. 1. Effects of hypoxia on ERb-mediated transcription in HEK293 cells. (A) Cells were transfected with an ERE-Luc reporter construct with the expression vector for hERb, using the PEI method. At 24 h post-transfection, cells were left untreated or treated with 1 lM ICI (B) or 10 nM E2 (A) and incubated for 24 h under normoxic or hypoxic conditions. The cells were exposed to medium conditioned by hypoxic cells (Hypoxia) or normoxic cells (con) for 24 h. After incubation, the cells were lysed and luciferase expression was determined. Values represent the mean ± S.E.M. (N = 3). *P < 0.05. (C) HEK293 cells were transfected with an ERE-Luc reporter construct and the expression vector for ERb with or without the expression vector for hHIF-1a, using the PEI method. The cells were incubated with or without 1 lM ICI for 24 h. After incubation, the cells were lysed and luciferase expression was determined. Values represent the mean ± S.D. (N = 3). *P < 0.05. All experiments were repeated at least three times. (D) HEK293 cells were transfected with ERE-Luc and hERb, along with 0.2 lg each of hHIF-1a expression plasmid in combination with SRC1 or CBP, using the PEI method. At 48 h post- transfection, luciferase activity was determined. Values represent the mean ± S.D. (N = 3). (Significance compared with control *P < 0.05; significance compared with HIF-1a àP < 0.05.) All experiments were repeated at least three times.

transcription [11]. We therefore examined the effects of these tively, these results indicate that the ERb interacts with the N-ter- coactivators on the activity of the ERE reporter. SRC1 and CBP in- minus of HIF-1a. duced a moderate increase (approximately twofold) in ERb-medi- ated luciferase expression in the absence of a ligand (data not 3.3. ERb decreases HIF-1a mediated transcription and expression of shown). Next, we analyzed the ability of CBP and SRC1 to enhance VEGF in HEK293 cells the transactivation function of HIF-1a on ERb-mediated transcrip- tion in transient transfection experiments. As shown in Fig. 1D, The effect of HIF-1a or hypoxia on ERb-mediated transcription SRC-1 and CBP significantly enhanced HIF-1a-dependent tran- and the interaction between HIF-1a and ERb raised the possibility scriptional activation of the ERE-driven reporter gene in HEK293 that ERb might directly regulate HIF-1a-mediated gene transcrip- cells. These observations indicate that SRC1 and CBP can potentiate tion. As expected, HRE-driven luciferase reporter was activated the effect of HIF-1a on ERb-mediated transcription. by GFP-HIF-1a overexpression (Fig. 3A). However, to our surprise, the mere expression of ERb in HEK293 cells significantly decreased 3.2. Interaction between ERb and HIF-1a the transcriptional activity of HIF-1a (Fig. 3A). We next examined whether overexpression of ERb was capable of suppressing endog- We previously reported that HIF-1a interacts with ERa [19,20]. enous hypoxic-responsive VEGF expression. As shown in Fig. 3B, To determine whether HIF-1a interacts with ERb as well, we car- hypoxia increased VEGF mRNA in HEK293 cells. This hypoxic ried out mammalian one-hybrid assays, using full-length HIF-1a induction of VEGF mRNA was significantly blocked by the overex- fused to the GAL4 DBD domain, in conjunction with the GAL4- pression of ERb. These results indicate that ERb inhibits hypoxic Luc construct. HEK293 cells transfected with GAL4-HIF-1a, GAL4- induction of VEGF mRNA through the modulation of HIF-1a tran- Luc, and ERb increased transactivation about fourfold compared scriptional activity in HEK293 cells. to cells transfected only with GAL4-HIF-1a and GAL4-Luc (Fig. 2A). We used a hybrid of the HIF-1a N-terminus and the VP16 transactivation domain [19] to investigate the HIF-1a-N-ter- 4. Discussion minus-mediated interaction. The transfection of cells expressing ERb with HIF-1a/VP16 increased transactivation about threefold We previously showed that hypoxia activates ERa in a ligand- over that of cells transfected with ERb and VP16 (Fig. 2B). Collec- independent manner, possibly through the interaction of HIF-1a W. Lim et al. / FEBS Letters 583 (2009) 1314–1318 1317

25 * 5

20 4 *

15 3

10 2 (Fold Induction) (Fold Induction) 5 1 Luciferase Activity Luciferase Activity

0 0 GAL4-LUC + + + VP16 + - GAL4-HIF-1 - + + H/V - + ERβ - - + ERβ + +

Fig. 2. Interaction between ERb and HIF-1a. (A) HEK293 cells were transiently transfected via the PEI method with pGAL4-Luc with or without the expression vector for GAL4-HIF-1a fusion protein, which was with or without the expression vector for hERb. At 48 h post-transfection, luciferase activity was determined. Values represent the mean ± S.D. (N = 3). *P < 0.05. All experiments were repeated at least three times. (B) HEK293 cells were transiently transfected with ERE-Luc, hERb, and VP16 or HIF-1a/VP16 (H/V), as indicated. At 48 h post-transfection, luciferase activity was determined. Values represent the mean ± S.D. (N = 3). *P < 0.05. All experiments were repeated at least three times. and ERa [19,20]. In this study, we sought to determine whether gested that, as a negative modulator of the hypoxia-dependent HIF-1a regulates ERb-mediated transcription, and ERb influences pathway, ERb inhibits angiogenesis and the growth of breast can- HIF1-a activity in reverse. We found that HIF-1a activated ERb in cer xenografts by decreasing the expression of VEGF and PDGFB a ligand-independent manner, and ERb expression decreased HIF- in T47D-ERb cells [31]. , which is known to activate 1a-mediated transcription and VEGF induction in HEK293 cells. ERb, has been shown to inhibit the growth of breast cancer xeno- Although ERb can be activated by the same stimuli activating grafts by affecting angiogenesis [32]. It has been suggested that

ERa,ERb may play a different role in regulating transcription and E2 may counteract hypoxia-induced VEGF through ERb in MDA- biological responses than does ERa. MB231 cells [33]. The selective ERb agonist diarylpropiolnitrile de- In breast cancer, hypoxia can promote tumor progression by creases pulmonary artery vasoconstriction induced by hypoxia via dedifferentiation and ERa down-regulation [24,25]. Studies have a nitric-oxide-dependent mechanism [34]. These findings indicate shown that estrogen induces VEGF expression in breast cancer that ERb and HIF-1a may have a unique connection, supporting our cells [26–29], as well as an increase in HIF-1a mRNA and protein experiments showing that ERb represses HIF-1a and is activated by expression in the uterus [30]. However, several studies have sug- HIF-1a. The hypoxic activation of ERb may augment the tumor- suppressing activity of ERb by inhibiting HIF-1a activation. How- ever, this is not the case with ERa,asERa activates HIF-1a (our unpublished data). Studies suggest that ERb plays a dominant neg- 30 -ER ative role in ERa signaling in the presence of estrogen through 25 * +ER competition with ERa for binding to a ligand, DNA, or cofactors 20 on specific promoters [35]. Our data also support the notion that the expression of ERb inhibits HIF-1a, which could be activated 15 by ERa under certain circumstances. Thus, ERb may play a domi- 10 nant negative role in the regulation of ERa actions in response to hypoxia by competing with ERa for binding to HIF-1a on specific

(Fold Induction) 5 Luciferase Activity promoters, such as the VEGF gene. 0 SRC-1 and CBP act synergistically to enhance HIF-1a-dependent con HIF-1 transcription [11]. In fact, SRC1 and CBP significantly increased the HIF-1a-dependent transcriptional activation of the HRE-driven re- con Hypoxia Hypoxia+ER porter gene in our system (data not shown). Thus, SRC-1 and CBP both significantly enhanced HIF-1a-dependent transcriptional VEGF activation of the HRE and ERb-mediated ERE-driven reporter gene,

-actin ERβ

Fig. 3. ERb decreases HIF-1a mediated transcription and expression of VEGF in HEK293 cells. (A) HEK293 cells were transfected with HRE-Luc reporter and ER HIF-1 hHIF-1a with or without the expression vector for hERb using the PEI method. At 48 h post-transfection, luciferase expression was determined. Values represent the ER HIF-1 mean ± S.D. (N = 3). *P < 0.05. All experiments were repeated at least three times. (B) HEK293 cells were transfected with hERb using the PEI method. At 24 h ERE HRE post-transfection, cells were incubated for 24 h under normoxic or hypoxic conditions. Total cellular mRNA from untransfected HEK293 cells and cells Fig. 4. Model illustrating the link between HIF-1a and ERb. HIF-1a activates ERb- transfected with hERb plasmids were analyzed using VEGF-specific primers, as mediated transcription in the absence of a ligand, and ERb inhibits HIF-1a- described in Section 2. mediated transcription. 1318 W. Lim et al. / FEBS Letters 583 (2009) 1314–1318 whether enhanced ERb transactivation by SRC1 and CBP is neces- and estrogen co-operate to regulate gene expression in T-47D human breast sary for inhibition of HIF-1a-mediated transcription is under cancer cells. J. Steroid Biochem. Mol. Biol. 104, 169–179. [17] Gao, N., Nester, R.A. and Sarkar, M.A. (2004) 4-Hydroxy estradiol but not 2- investigation. hydroxy estradiol induces expression of hypoxia-inducible factor 1alpha and In conclusion, HIF-1a activates ERb-mediated transcription in vascular endothelial growth factor A through phosphatidylinositol 3-kinase/ the absence of a ligand, and ERb inhibits HIF-1a-mediated tran- Akt/FRAP pathway in OVCAR-3 and A2780-CP70 human ovarian carcinoma cells. Toxicol. Appl. Pharmacol. 196, 124–135. scription, as summarized in Fig. 4. Further study is required to elu- [18] Kazi, A.A. and Koos, R.D. (2007) Estrogen-induced activation of hypoxia- cidate the mechanism by which HIF-1a induces ERb activation and, inducible factor-1alpha, vascular endothelial growth factor expression, and in particular, to determine the physiological significance of the edema in the uterus are mediated by the phosphatidylinositol 3-kinase/Akt pathway. Endocrinology 148, 2363–2374. interaction between ERb and HIF-1a. [19] Cho, J., Kim, D., Lee, S. and Lee, Y. (2005) Cobalt Chloride-induced estrogen receptor alpha down-regulation involves Hypoxia-Inducible Factor-1{alpha} Acknowledgment in MCF-7 human breast cancer cells. Mol. Endocrinol. 19, 1191–1199. [20] Cho, J., Bahn, J.J., Park, M., Ahn, W. and Lee, Y.J. (2006) Hypoxic activation of unoccupied estrogen-receptor-alpha is mediated by hypoxia-inducible factor- This work was supported by grants from the Korea Research 1 alpha. J. Steroid. Biochem. Mol. Biol. 100, 18–23. Foundation (E00103) to Y.J.L. 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