Cobalt Chloride-Induced Estrogen Receptor {alpha} Down-Regulation Involves Hypoxia-Inducible Factor-1{alpha} in MCF-7 Human Breast Cancer Cells

Jungyoon Cho, Dukkyung Kim, SeungKi Lee and YoungJoo Lee

College of Life Science (J.C., Y.L.), Institute of Biotechnology, Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747; Department of Medicine (D.K.), Samsung Medical Center, Sungkyunkwan University, School of Medicine, Seoul 135-710; and College of Pharmacy (S.L.), Seoul National University, Seoul 151-742, Korea

Address all correspondence and requests for reprints to: YoungJoo Lee, Ph. D., Department of Bioscience and Biotechnology, Sejong University, Kwang-Jin-Gu, Seoul 143-747, Korea. E-mail: yjlee{at}sejong.ac.kr.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) is down-regulated under hypoxia via a proteasome-dependent pathway. We studied the mechanism of ER{alpha} degradation under hypoxic mimetic conditions. Cobalt chloride-induced ER{alpha} down-regulation was dependent on the expression of newly synthesized protein(s), one possibility of which was hypoxia-inducible factor-1{alpha} (HIF-1{alpha}). To examine the role of HIF-1{alpha} expression in ER{alpha} down-regulation under hypoxic-mimetic conditions, we used a constitutively active form of HIF-1{alpha}, HIF-1{alpha}/herpes simplex viral protein 16 (VP16), constructed by replacing the transactivation domain of HIF-1{alpha} with that of VP16. Western blot analysis revealed that HIF-1{alpha}/VP16 down-regulated ER{alpha} in a dose-dependent manner via a proteasome-dependent pathway. The kinase pathway inhibitors PD98059, U0126, wortmannin, and SB203580 did not affect the down-regulation. A mammalian two-hybrid screen and immunoprecipitation assays indicated that ER{alpha} interacted with HIF-1{alpha} physically. These results suggest that ER{alpha} down-regulation under hypoxia involves protein-protein interactions between the ER{alpha} and HIF-1{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ESTROGEN RECEPTOR (ER) is a ligand-activated transcription factor. On binding of estrogen, the ER undergoes conformational changes that induce binding to the cognate estrogen-responsive element, modulating the transcription of target genes (1, 2). More than 30 regulatory proteins affect the ER-mediated transcriptional response (3). Furthermore, the differential regulation of the activity of coactivators modulates ER signal transduction pathways via direct or indirect interactions (4). Although many coregulators participate in the ER-mediated transcriptional response, the magnitude of this response depends on the initial concentration of ER (5). There is a linear relationship between the amount of glucocorticoid receptors and transcriptional gene activation (6). ER expression is a molecular predictor of tumor responsiveness to antiestrogens during breast cancer treatment (7). Estrogen causes rapid turnover of ER via the ubiquitin-proteasome pathway, with a half-life of approximately 3 h (8). However, whether estrogen-induced proteolysis is associated with receptor activation that leads to transactivation is controversial (9, 10). ER is degraded in response to various stimuli other than estrogens, such as hypoxia, dioxin, and oxytocin (11, 12, 13). Whereas the elucidation of the mechanisms that govern the regulation of ER is crucial to understanding ER function, the precise details of the regulation remain unclear.

Hypoxia is important in normal physiological processes and development as well as in tumorigenesis (14). Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates gene expression in response to changes in cellular oxygen tension, such those that occur during erythropoiesis (erythropoietin), glycolysis (glucose transporter-1, aldolase A, enolase 1, lactate dehydrogenase), vasculogenesis [VEGF (vascular endothelial growth factor)], and vasodilation (nitric oxide synthase, hemoxigenase) (15). Functional HIF-1 exists as a heterodimer that comprises an HIF-1{alpha} subunit and an aryl hydrocarbon receptor nuclear translocator (ARNT) subunit. HIF-1 activation depends on HIF-1{alpha}, which usually decays under normoxia but is stabilized rapidly under hypoxia. HIF-1 activates gene transcription via HIF-1 DNA binding sequences (15). Nevertheless, during hypoxia, some genes are regulated by HIF-1-independent mechanisms. For example, HIF-1 does not mediate the regulation of the hypoxia-induced expression of the tissue factor and {alpha}-fetoprotein genes (16, 17).

The ER is degraded under hypoxia within 6–12 h via a proteasome-dependent pathway, which has implications for clinical treatment of mammary tumors (11). HIF-1{alpha}-positive tumors express significantly lower levels of ER (18). However, the role of HIF-1{alpha} in the down-regulation of ER expression has not been examined, which is probably owing to a lack of experimental tools for overcoming the conditional regulation of HIF-1{alpha}. In this study, we examined the mechanism of the down-regulation of ER{alpha} expression under hypoxia-mimetic conditions, and the role of HIF-1{alpha} in this process. To examine the involvement of HIF-1{alpha}, we used hybrid molecules of HIF-1{alpha} and herpes simplex viral protein 16 (VP16) that were designed to be stable under normoxia. Using this system, we demonstrated that HIF-1{alpha}/VP16 hybrid was sufficient to induce the proteasome-dependent down-regulation of ER{alpha} expression and that this involved an interaction between the ER{alpha} and HIF-1{alpha} proteins.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Protein Synthesis Is Required for CoCl2-Induced Down-Regulation of ER{alpha} Protein Expression
Hypoxia induces the proteasome-dependent down-regulation of ER expression in ZR75 cells (11), but the impact of hypoxia on ER{alpha} expression in the MCF-7 human breast cancer cell line has not been examined. Therefore, we examined the effects of CoCl2, a hypoxic mimetic agent, on the expression of ER{alpha} protein in MCF-7 cells. Western blot analysis revealed that the treatment of cells with 0.5 mM CoCl2 for 24 h reduced ER{alpha} expression by approximately 60% (Fig. 1Go). As a positive control, 17ß-estradiol (E2), a well-characterized down-regulator of ER expression, was tested in parallel (8). To determine whether the CoCl2-induced degradation of ER{alpha} required the synthesis of new protein, MCF-7 cells were incubated with 0.5 mM CoCl2 in the presence or absence of the protein synthesis inhibitor cycloheximide and the protein levels were measured using Western blot analysis. Consistent with previous reports (19, 20), the E2-induced inhibition of ER{alpha} expression was partially dependent on protein synthesis in MCF-7 cells. Cycloheximide prevented the CoCl2-induced down-regulation of ER{alpha} expression, which suggested that the hypoxia-induced degradation of ER{alpha} protein required de novo protein synthesis. These results led us to investigate whether hypoxia-induced down-regulation of ER{alpha} expression depended on the synthesis of HIF-1{alpha}.



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Fig. 1. Protein Synthesis Is Required for CoCl2-Induced ER{alpha} Down-Regulation

MCF-7 cells were preincubated with (+) or without (–) 10 µM cycloheximide (CHX) for 1 h before the addition of 10 nM E2 or 0.5 mM CoCl2 for 24 h. An untreated group served as a control (CON). After the incubation, the cells were lysed and total protein extracts were resolved by SDS-PAGE and immunoblotted using an anti-ER{alpha} antibody or an anti-ß-actin antibody. The graphic representation of the amount of ER{alpha} was derived from bioimaging analyzer (ß) to calculate relative intensity of the ER{alpha} band normalized to that of ß-actin (right panel). These experiments were repeated at least three times.

 
HIF-1{alpha}/VP16 Is Sufficient for ER{alpha} Protein Degradation
HIF-1{alpha} is degraded rapidly under normoxia via ubiquitin- and proteasome-dependent pathways, whereas HIF-1{alpha} is accumulated under hypoxia (14). This conditional regulation of HIF-1{alpha} hinders studies on the effects of HIF-1{alpha} expression (21). To circumvent this problem, we used a constitutively stable form of HIF-1{alpha}, namely a hybrid of HIF-1{alpha} and VP16 (henceforth referred to as HIF-1{alpha}/VP16), which was reported initially by Vincent et al. (22). The HIF-1{alpha}/VP16 system has been used to demonstrate hypoxia-induced up-regulation by HIF-1{alpha} of the proliferator-activator-receptor {gamma} angiopoietin-related gene (23). We constructed the hybrid according to the report by Vincent et al. (22), and functional activity was probed using a hypoxia-responsive element (HRE)-dependent luciferase assay (Fig. 2AGo). To investigate whether the CoCl2-induced down-regulation of ER{alpha} expression occurred via HIF-1{alpha}, we examined ER{alpha} proteins from MCF-7 cells that had been transfected transiently with JDK-HIF-1{alpha}/VP16 for 24 h. Analyses were performed after 24 h because the expression of HIF-1{alpha}/VP16 was detected after 12–16 h of transfection (data not shown). As controls, the cells were untreated or transfected with VP16 for 24 h (Fig. 2BGo). Western blot analysis revealed that the expression of HIF-1{alpha}/VP16 hybrid protein caused ER{alpha} protein expression to be down-regulated to a level similar to the level after treatment with CoCl2 (Fig. 2BGo). Transfection with VP16 had no effect on the down-regulation of ER{alpha} expression. These results suggested that the CoCl2-induced down-regulation of ER{alpha} expression was likely mediated by HIF-1{alpha} and that the expression of HIF-1{alpha}/VP16 under normoxia can induce the degradation of ER{alpha}. For all of the experiments described hereafter, we used adenoviral HIF-1{alpha}/VP16 (Ad-HIF-1{alpha}/VP16) instead of HIF-1{alpha}/VP16 plasmid to increase the level of expression.



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Fig. 2. Expression of HIF-1{alpha}/VP16 Is Sufficient for ER{alpha} Down-Regulation

A, Schematic representation and transcriptional activation of HIF-1{alpha}/VP16 hybrid are shown. The hybrid DNA contains DNA binding/dimerization domain (amino acid 1–390) of HIF-1{alpha} and VP16 transactivation domain (TA). HeLa cells were transfected with HRE-Luc with (H/V) or without (control; CON) JDK-HIF-1{alpha}/VP16 and luciferase expression was determined. B, MCF-7 cells were transfected with JDK-HIF-1{alpha}/VP16 (H/V) or pVP16 (VP16), or incubated with or without (CON) 0.5 mM CoCl2 for 24 h. Total protein prepared from each cells was resolved by SDS-PAGE and analyzed using anti-ER{alpha} or anti-ß-actin antibody. The graphic representation of the amount of ER{alpha} was derived from bioimaging analyzer (Bio-Rad, Hercules, CA) to calculate relative intensity of the ER{alpha} band normalized to that of ß-actin (right panel). C, MCF-7 cells were incubated for 24 h with increasing concentrations [7–17%, virus/media (vol/vol)] of Ad-HIF-1{alpha}/VP16 or 12% [virus/media (vol/vol)] Ad-GFP. Total protein extracts were resolved by SDS-PAGE and immunoblotted using an anti-ER{alpha} or an anti-ß-actin antibody. The graphic representation of the amount of ER{alpha} was derived from bioimaging analyzer (Bio-Rad) to calculate relative intensity of the ER{alpha} band normalized to that of ß-actin (right panel). D, MCF-7 cells were infected with Ad-HIF-1{alpha}/VP16 (H/V), or treated with 0.5 mM CoCl2 or 10 nM E2 for 24 h. Whole cell uptake assays were performed as described in Materials and Methods. Each experiment was repeated at least three times.

 
To provide additional evidence that HIF-1{alpha}/VP16 down-regulated ER{alpha} protein expression, we treated MCF-7 cells with increasing amounts of Ad-HIF-1{alpha}/VP16 or Ad-green fluorescent protein (Ad-GFP). As shown in Fig. 2CGo, HIF-1{alpha}/VP16 promoted ER{alpha} degradation in a dose-dependent manner, whereas the Ad-GFP had no effect on ER{alpha} expression. To determine whether the decrease in ER{alpha} protein expression was associated with a reduction in the estrogen-binding ability of the cells, whole-cell estrogen uptake assays were carried out. MCF-7 cells were infected with Ad-HIF-1{alpha}/VP16 or treated with 0.5 mM CoCl2 or 10 nM E2 for 24 h. The percentage of E2 binding was measured as described in Materials and Methods. HIF-1{alpha}/VP16 reduced the specific E2 binding activity to approximately 25% that of the untreated controls (Fig. 2DGo). These data confirm that HIF-1{alpha}/VP16 decreased functional ER{alpha} expression. The E2 binding activity of Ad-HIF-1{alpha}/VP16-treated cells was similar to that of cells treated with CoCl2; this suggested that the CoCl2-induced down-regulation of ER{alpha} expression involved HIF-1{alpha}. Collectively, the aforementioned findings are consistent with the hypothesis that HIF-1{alpha}/VP16 is sufficient for the degradation of ER{alpha}.

HIF-1{alpha}/VP16-Induced Degradation of ER{alpha} Is Mediated by the Ubiquitin-Proteasome Pathway
The ubiquitin-proteasome pathway is responsible for the selective degradation of ER (8, 24). To examine the involvement of the proteasomal pathway in HIF-1{alpha}/VP16-induced down-regulation of ER{alpha} expression, MCF-7 cells were infected with Ad-HIF-1{alpha}/VP16 or treated with 10 nM E2 with or without 1 µM MG132 (a proteasomal inhibitor) for 24 h. Western blot analysis revealed that the E2-induced down-regulation of ER{alpha} expression was inhibited by MG132 (Fig. 3AGo), which is consistent with previous reports (8, 10). Similarly, the addition of MG132 completely blocked the down-regulation of ER{alpha} expression by HIF-1{alpha}/VP16. As a control, we observed no significant change in ß-actin expression under the same conditions.



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Fig. 3. Effects of Proteasome and Kinase Inhibitors on HIF-1{alpha}/VP16-Induced ER{alpha} Down-Regulation

A, MCF-7 cells were preincubated with (+) or without (–) proteasome inhibitor MG132 (1 µM) for 30 min before the addition of E2 (10 nM) or Ad-HIF-1{alpha}/VP16 (H/V) for 24 h. B, MCF-7 cells were infected with Ad-HIF-1{alpha}/VP16 (H/V) or incubated under hypoxic condition (1% O2) for 24 h. Cell lysates were immunoprecipitated (IP) with either anti-IgG or anti-ER{alpha} antibody, and precipitated proteins were immunoblotted (IB) with antiubiquitin antibody. Molecular mass markers are indicated (top). Total extracts were analyzed by immunoblots with anti-ER{alpha} antibody (bottom). The graphic representation of the amount of ER{alpha}-conjugated ubiquitin was derived from bioimaging analyzer (right panel). C-a, MCF-7 cells were preincubated with either 30 µM PD98059 (P) or 10 µM U0126 (U) for 30 min before the addition of 0.5 mM CoCl2 for 24 h. After the incubation, the cells were lysed and total protein extracts were analyzed by Western blotting using an anti-p42/p44 MAPK or phospho-p42/p44 MAPK monoclonal antibody. C-b and C-c, MCF-7 cells were preincubated with either vehicle 0.1% DMSO (D), 30 µM PD98059 (P), 150 nM wortmannin (W), 10 µM SB203580 (S), or 10 µM U0126 (U) for 30 min before the addition of 0.5 mM CoCl2 or Ad-HIF-1{alpha}/VP16 (H/V) for 24 h. Whole cell extracts were analyzed by Western blotting using an anti-ER{alpha} or anti-ß-actin antibody. All Western blots were repeated at least three times. C-d, MCF-7 cells were transfected with HRE-Luc, and then the cells were preincubated as indicated for 30 min before the addition of 0.5 mM CoCl2 (Co) for 24 h. Luciferase expression was determined after the treatment.

 
To assess the ubiquitination of ER{alpha} in response to hypoxia or HIF-1{alpha}/VP16, the cell lysates from MCF-7 cells that had been infected with Ad-HIF-1{alpha}/VP16 or had been incubated under hypoxia were immunoprecipitated with an anti-ER{alpha} antibody; these lysates were then analyzed on Western blots using antiubiquitin antibody. A predominant ubiquitinated ER{alpha} protein band was detected at approximately 85 kDa with faint multiple higher molecular mass bands (Fig. 3BGo). The ubiquitinated ER protein levels were 3- and 2-fold greater in response to hypoxia and HIF-1{alpha}/VP16, respectively. These results suggested that the HIF-1{alpha}/VP16-mediated down-regulation of ER{alpha} expression proceeded via the ubiquitin-proteasome pathway.

The activation of protein kinase pathways induces ER down-regulation even in the absence of ligand; kinase activation plays an important role in proteasome-mediated ER degradation (25, 26) and is necessary for transcription activity of HIF-1 (27). Therefore, we examined the effects of kinase inhibition on the down-regulation of ER{alpha} expression by CoCl2 and HIF-1{alpha}/VP16. MCF-7 cells were incubated for 30 min with dimethyl sulfoxide (DMSO) or with several kinase inhibitors, including inhibitors of MAPK (30 µM PD98059 or 10 µM U0126), PI3K (150 nM wortmannin), or p38 (10 µM SB203580). The concentrations of kinase inhibitors used in the present study were based on the values reported to be sufficient to inhibit the respective types of kinases without inducing nonspecific toxicity (28), and we have confirmed that PD98059 and U0126 efficiently blocked phosphorylation of p42/p44 MAPK as evaluated by Western analysis using antiphospho p42/p44 antibody (Fig. 3C-aGo). After the incubation with the kinase inhibitors, the cells were infected with Ad-HIF-1{alpha}/VP16 or treated with CoCl2 for 24 h. The effects of kinase inhibitors on CoCl2- and HIF-1{alpha}/VP16-induced down-regulation of ER{alpha} expression were analyzed using Western blots. As shown in Fig. 3CGo-b and -c, none of the protein kinase inhibitors prevented the down-regulation of ER{alpha} expression by CoCl2- or HIF-1{alpha}/VP16. These results indicated that the down-regulation of ER{alpha} expression by CoCl2 or HIF-1{alpha}/VP16 is independent of the MAPK and PI3K pathways. Similarly, aryl hydrocarbon receptor (AhR)-mediated degradation of ER was reported to occur via proteasomes in the absence of activation of kinase-dependent phosphorylation (12). In parallel, we measured the effects of kinase inhibitors on the HIF-1-induced transcriptional response. MCF-7 cells were transfected with HRE-luciferase (HRE-Luc) plasmid and incubated with PD98059 or U0126 for 30 min before being treated with CoCl2 for 24 h. HIF-1-mediated luciferase activity was inhibited by PD98059 or U0126 (Fig. 3C-dGo), which indicated that the inhibition of MAPK prevented the induction of the target genes of HIF-1 (27). These data suggest that ER{alpha} degradation under hypoxia is independent of the expression of the target genes of HIF-1.

HIF-1{alpha}/VP16 Interacts with the ER{alpha}
The basic helix-loop-helix (bHLH)-Per-ARNT-Sim (PAS) protein ARNT (an aryl hydrocarbon receptor nuclear translocator) was reported to act as a partner protein of HIF-1{alpha} and to interact with the ER (21); however, in that study, the authors did not examine the interaction between HIF-1{alpha} and ER owing to the conditional regulation of HIF-1{alpha} (21). AhR is also a bHLH-PAS protein and also reportedly interacts with ER in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin and induces ER degradation (12). In the present study, we examined whether ER{alpha} degradation occurred in the presence of an interaction between ER{alpha} and the bHLH-PAS protein HIF-1{alpha}. MCF-7 cells were infected with Ad-HIF-1{alpha}/VP16 and then either the ER{alpha} or VP16 protein was immunoprecipitated from whole-cell extracts using an anti-ER{alpha} or anti-VP16 antibody. The immunocomplexes were analyzed on Western blots using the corresponding antibody. As shown in Fig. 4AGo, an interaction between ER{alpha} and HIF-1{alpha}/VP16 was observed. It is of concern that VP16 might have influenced interaction between ER{alpha} and HIF-1{alpha}/VP16. Therefore, we have analyzed the interaction between ER{alpha} and HIF-1{alpha} using GFP-tagged HIF-1{alpha} (GFP-HIF-1{alpha}) under hypoxia. Human embryonic kidney (HEK) 293 cells were cotransfected with GFP-HIF-1{alpha} and ER{alpha} expression vectors and incubated under hypoxia. Thereafter, cell lysates were immunoprecipitated with an anti-ER{alpha} antibody and were analyzed on Western blots using an anti-HIF-1{alpha} antibody. As shown in Fig. 4BGo, interaction between ER{alpha} and GFP-HIF-1{alpha} were detected that were cotransfected with GFP-HIF-1{alpha} and ER{alpha} under hypoxia, whereas no coimmunoprecipitation was observed in the absence of GFP-HIF-1{alpha}. In addition, interaction between endogenous HIF-1{alpha} and ER{alpha} was observed under hypoxia. These data suggested that the full-length HIF-1{alpha} coimmunoprecipitated with ER{alpha}. The interaction between ER{alpha} and HIF-1{alpha} was investigated further in a mammalian two-hybrid assay in COS cells using expression plasmids containing the DNA binding domain of the yeast GAL4 protein fused to DEF domains of ER{alpha} (amino acids 263–595; GAL4-ERDEF), which is crucial for ligand binding and the interaction of coregulators. The empty pVP16 vector served as a control. The infection of cells that expressed GAL4-ERDEF with Ad-HIF-1{alpha}/VP16 increased transactivation approximately 2.5-fold compared with cells transfected with VP16 and GAL4-ERDEF (Fig. 4CGo). Collectively, these results demonstrated that the C-terminal domain of ER{alpha} interacted with the N-terminal bHLH/PAS domain of HIF-1{alpha} and was coupled to ER{alpha} degradation under hypoxia. However, how this interaction leads to the degradation of ER{alpha} remains to be investigated. Ligand-occupied AhR degrades ER via proteasomes regardless of the presence of E2 (12). It has been suggested that domains other than the DNA binding domain of the ER are responsible for the interaction between AhR and ER (12), which is consistent with the results of the present study. Our data do not preclude the possibility that HIF-1{alpha} interacts with ER{alpha} as a complex. The ubiquitin-protein isopeptide ligase activity of Mdm2 promotes the ubiquitination and degradation of p53 (29). Under hypoxia, Mdm2 interacts directly with HIF-1{alpha} by acting as a bridge between HIF-1{alpha} and p53, thereby stabilizing p53 (30). The region that is responsible for this interaction is unknown and there is apparently no direct interaction between the oxygen-dependent domain of HIF-1{alpha} and p53 (31). Saji et al. (32) showed that Mdm2 directly interacts with ER enhancing ER transactivation. It will be enlightening to identify the protein(s) that interact with both the bHLH-PAS domain of HIF-1{alpha} and the ER{alpha} and that ultimately lead to the proteasomal degradation of ER{alpha}. A few proteins, such as heat shock protein 90 and hepatitis B virus X protein, interact with the N-terminal domain of HIF-1{alpha} (33, 34). We are currently investigating the nature of the ER{alpha} interaction with HIF-1{alpha}.



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Fig. 4. Interaction between ER{alpha} and HIF-1{alpha}/VP16

A, MCF-7 cells were infected with Ad-HIF-1{alpha}/VP16 (H/V) for 24 h. Cell lysates were immunoprecipitated (IP) with either anti-IgG antibody, anti-ER{alpha} or anti-VP16 antibody, and precipitated proteins were immunoblotted (IB) with either anti-VP16 or anti-ER{alpha} antibody. B, HEK293 cells were transfected with expression vector for GFP-HIF-1{alpha} and/or hER{alpha} as indicated, and then the cells were incubated under hypoxic condition. Cell lysates were IP with anti-IgG or anti-ER{alpha} antibody and precipitated proteins (top) and total extracts (bottom) were analyzed by immunoblots with anti-HIF-1{alpha} antibody. C, COS cells were transiently transfected with pGAL4-Luc and the expression vector for GAL4-ERDEF fusion protein with either the expression vector for VP16 or infected with Ad-HIF-1{alpha}/VP16 (H/V) and luciferase expression was determined. The significance is *, P < 0.01. Each experiment was repeated at least three times.

 
Cellular hypoxia is a crucial stimulus in many pathological conditions, such as tumor growth. Decreased oxygen levels activate HIF-1 by stabilizing the HIF-1{alpha} subunit. HIF-1 expression plays a major role in tumor biology and is essential for the growth of blood vessels (14). Therefore, it is important to elucidate the effects of hypoxia on ER levels within in breast cancer cells, the growth of which depends on agonist-occupied ER (35, 36). In addition, it has been suggested that hypoxia may be a key to the development of acquired hormone resistance in breast cancer by modulating ER expression levels (11, 18, 37). VEGF, a known estrogen-responsive gene (38), has been proposed to be a candidate for causing hormonal resistance (39). Coradini et al. (39) reported that hypoxic induction of VEGF was not inhibited by antiestrogens, which provided a partial explanation of hormone resistance attributable in part to the loss of ER protein. Despite its significance, the mechanism of ER regulation under hypoxia has not been studied intensively.

In the present study, we examined the role of HIF-1{alpha} in the down-regulation of ER{alpha} expression under hypoxia. We found that the down-regulation of ER{alpha} expression under hypoxia depended on HIF-1{alpha} and that ER{alpha} was subsequently degraded via the proteasome pathway. Further study is required to elucidate the mechanism by which HIF-1{alpha} induces ER{alpha} degradation and particularly to determine the nature and significance of the interaction between ER{alpha} and HIF-1{alpha}.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
E2 and CoCl2 were purchased from Sigma (St. Louis, MO). E2 was dissolved in 100% ethanol. Cycloheximide, MG132, PD98059, SB203580, U0126, or wortmannin (Sigma) was dissolved in DMSO. All of the compounds were added to the medium such that the total solvent concentration was never higher than 0.1%. An untreated group served as a control.

Plasmids
The pMERDEF plasmid constructed by cloning DEF domain of ER{alpha} (amino acids 263–595) into the pM vector that contains the GAL4 DNA binding domain (CLONTECH Laboratories, Inc., Palo Alto, CA) and pGAL4-Luc were gift from Dr. Benita Katzenellenbogen (40). HIF-1{alpha}/VP16 DNA fragment was generated according to the methods published by Vincent et al. (22) from Genzyme Corp. (Framingham, MA). The hybrid DNA contains DNA binding/dimerization domain (amino acids 1–390) of HIF-1{alpha} and VP16 transactivation domain. The insert was cloned into either JDK (41) or pShuttle-CMV adenoviral vector (AdEasy vector system; Qbiogene, Carlsbad, CA). The HRE-Luc reporter plasmid contains four copies of the erythropoietin HRE, the simian virus 40 promoter, and the luciferase gene. GFP-HIF-1{alpha} vector was kindly provided by Dr. Kyu-Won Kim (Seoul National University, Seoul, Korea) (42).

Cell Culture and Hypoxic Condition
MCF-7, HEK 293, HeLa, and COS cells were maintained in phenol red-free DMEM containing supplemented with 10% fetal bovine serum (FBS) (HyClone Laboratories, Inc., Logan, UT) or 10% calf serum (Invitrogen Life Technologies, Grand Island, NY). Cells were grown at 37 C in a humidified atmosphere of 95% air/5% CO2 and fed every 2–3 d. Before treatment with chemicals or virus infection, the cells were washed with PBS and cultured in DMEM/5% charcoal-dextran stripped FBS (CD-FBS) or charcoal-dextran stripped calf serum for 2 d to eliminate any estrogenic source before treatment. All treatments were done with DMEM/5% CD-FBS. Ten nanomolar E2 has been used unless otherwise noted. For the hypoxic condition, cells were incubated at a CO2 level of 5% with 1% O2 balanced with N2 using a hypoxic chamber (Forma). Alternatively, cells were cultured in medium containing 500 µM CoCl2.

Transient Transfection and Luciferase Assay
Cells were transiently transfected into the cell by calcium phosphate-DNA coprecipitation method. Luciferase activity was determined 24 h after drug treatments with an AutoLumat LB9507 luminometer using the luciferase assay system (Promega, Madison, WI) and expressed as relative light units. The mean and SD of triplicate samples are shown for representative experiments. All transfection experiments were repeated three or more times with similar results.

Construction of Recombinant Adenovirus and Infection
Recombinant type 5 adenoviruses overexpressing HIF-1{alpha}/VP16 were constructed according to the manufacturer’s protocol (Qbiogene). Preconstructed pShuttle-CMV adenoviral vector containing HIF-1{alpha}/VP16 recombinant gene together with the pAdEasy-1 vector containing the adenovirus serotype 5 genome with deletions in the adenoviral E1 and E3 regions were transformed into BJ5183 (recA+) bacterial cells for homologous recombination between these two vectors. Recombinated AdEasy plasmid was transfected into HEK293A cells that contain the E1A and E1B Ad 5 viral gene necessary for viral replication. The resultant recombinant viruses were propagated in 293A cells, titered, and stored at –80 C. MCF-7 cells were infected as follows. Cells were incubated in DMEM containing 2% CD-FBS for 24 h. The medium was then removed and the cells were exposed to recombinant adenoviruses in DMEM at 37 C for 2 h and cultured for additional 24 h in the media containing the serum.

Immunoblot Analysis
Protein was isolated by using radioimmune precipitation buffer (containing 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 1% sodium dodecyl sulfate) with protease inhibitor cocktail (Sigma), dissolved in sample buffer, and boiled for 5 min before loading onto an acrylamide gel. After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane, blocked with 5% nonfat dry milk in Tris-buffered saline/0.05% Tween (TBST), and incubated with antirabbit polyclonal antibody to ER{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA), ubiquitin (Sigma), or to HIF-1{alpha} (BD Transduction Laboratories, Lexington, KY) or antimouse monoclonal antibody to ß-actin (Sigma), p42/p44 MAPK (Santa Cruz Biotechnology), phospho-p42/p44 MAPK (Santa Cruz Biotechnology), or to VP16 (Santa Cruz Biotechnology). After washing with TBST, blots were incubated with goat antirabbit or antimouse horseradish peroxidase-conjugated secondary antibody and visualized with enhanced chemiluminescence ECL kits (Amersham Bioscience, Little Chalfont, UK).

Immunoprecipitation
Two hundred micrograms (1 µg/µl) of the cell lysates were mixed with 1 µg of antibody (in 50 µl) and incubated overnight at 4 C with constant rotation. To recover immunoprecipitated complexes, 150 µl of Protein A-Sepharose, diluted 1:1 in PBS, were then added to the samples and incubated on ice for additional 2–4 h with constant rotation. The beads were pelleted by centrifugation and the bound proteins were eluted by incubation in 5x sodium dodecyl sulfate loading buffer for 5 min by boiling. The eluted proteins were analyzed by immunoblot analysis.

Whole Cell Uptake Assay
MCF-7 cells were infected with Ad-HIF-1{alpha}/VP16 or incubated with E2 (100 nM) or 0.5 mM CoCl2 for 24 h. After removal of the medium, cells were rinsed with PBS and incubated with 1 nM [2,4,6,7-3H] E2 (89 Ci/mmol; Amersham Bioscience) for 1 h at 37 C as was published (43). Aliquots of the medium were measured before and after the incubation with the cells to determine the total uptake of E2 into the cells. After removal of the medium, cells were washed with ice-cold PBS/0.1% methylcellulose twice, harvested by scraping and centrifugation, and lysed with 100% ethanol 500 µl per 60-mm dish for 10 min at room temperature. The radioactivity of extracts was measured by liquid scintillation counting.

Statistical Analysis
Values shown represent mean ± SD. Statistical analysis was performed by Student’s t test with a P value of less than 0.05 being considered statistically significant.


    ACKNOWLEDGMENTS
 
We thank Dr. Benita Katzenellenbogen for providing the pMERDEF construct. We thank Drs. Chaeok Yoon and Jonghoi Byun for useful technical advice. We are also grateful to Joonsuk Choi for technical support.


    FOOTNOTES
 
This work was supported by Korea Research Foundation Grants (KRF-2003-015-E2009; RO4-2004-000-10001-0, and BK21 to Y.J.L.).

First Published Online February 3, 2005

Abbreviations: AhR, Aryl hydrocarbon receptor; bHLH, basic helix-loop-helix; CD, charcoal-dextran; DMSO, dimethyl sulfoxide; E2, 17ß-estradiol; ER, estrogen receptor; FBS, fetal bovine serum; GFP, green fluorescent protein; HEK, human embryonic kidney; HIF-1{alpha}, hypoxia-inducible factor-1{alpha}; HRE, hypoxia-responsive element; HRE-Luc, HRE-luciferase; PAS, Per-ARNT-Sim; VEGF, vascular endothelial growth factor; VP16, herpes simplex viral protein 16.

Received for publication April 19, 2004. Accepted for publication January 26, 2005.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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