Estradiol induces type 8 17β-hydroxysteroid dehydrogenase expression: crosstalk between estrogen receptor α and C/EBPβ

in Journal of Endocrinology

Hydroxysteroid (17-beta) dehydrogenase (HSD17B) are the enzymes responsible for the reversible interconversion of 17-hydroxy and 17-keto steroids. The human and mouse type 8 17β-HSD (HSD17B8) selectively catalyze the conversion ofestradiol (E2) to estrone (E1). We previously described thatHSD17B8 is transcriptionally regulated by C/EBPβ, andthat C/EBPβ is bound to CCAAT boxes located at −5 and −46 of the transcription start site in basal conditions in HepG2 cells. Furthermore, ectopic expression of C/EBPβ transactivated the HSD17B8 promoter activity. Here, we show that HSD17B8 expression is up-regulated in response toE2 in the estrogen receptor α (ERα) positive MCF-7 cells. Results showed that this induction is mediated by ERα because i) E2 did not induce HSD17B8 expression in ERαnegative HepG2 cells, ii) ectopic expression of ERα restored E2-induced HSD17B8 expression, and iii) this induction wasblocked by the anti-ER ICI 182 780. Additional experiments showed that no estrogen response element was necessary for this regulation. However, the CCAAT boxes located at the HSD17B8 proximal promoter were required for E2-induced transcription. Furthermore, co-immunoprecipitation studies revealed tethering of ERαtoC/EBPβ inresponse to E2 in cells expressing ERα. Additionally, chromatin immunoprecipitation assays demonstrated that, in response to E2, ERα is recruited to the CCAAT boxes in which C/EBPβ is already bound. Taken together, our results reveal that ERα is involved in the transcriptional regulation of HSD17B8 gene in response to E2 through its interaction with C/EBPβ.

Abstract

Hydroxysteroid (17-beta) dehydrogenase (HSD17B) are the enzymes responsible for the reversible interconversion of 17-hydroxy and 17-keto steroids. The human and mouse type 8 17β-HSD (HSD17B8) selectively catalyze the conversion ofestradiol (E2) to estrone (E1). We previously described thatHSD17B8 is transcriptionally regulated by C/EBPβ, andthat C/EBPβ is bound to CCAAT boxes located at −5 and −46 of the transcription start site in basal conditions in HepG2 cells. Furthermore, ectopic expression of C/EBPβ transactivated the HSD17B8 promoter activity. Here, we show that HSD17B8 expression is up-regulated in response toE2 in the estrogen receptor α (ERα) positive MCF-7 cells. Results showed that this induction is mediated by ERα because i) E2 did not induce HSD17B8 expression in ERαnegative HepG2 cells, ii) ectopic expression of ERα restored E2-induced HSD17B8 expression, and iii) this induction wasblocked by the anti-ER ICI 182 780. Additional experiments showed that no estrogen response element was necessary for this regulation. However, the CCAAT boxes located at the HSD17B8 proximal promoter were required for E2-induced transcription. Furthermore, co-immunoprecipitation studies revealed tethering of ERαtoC/EBPβ inresponse to E2 in cells expressing ERα. Additionally, chromatin immunoprecipitation assays demonstrated that, in response to E2, ERα is recruited to the CCAAT boxes in which C/EBPβ is already bound. Taken together, our results reveal that ERα is involved in the transcriptional regulation of HSD17B8 gene in response to E2 through its interaction with C/EBPβ.

Keywords:

Introduction

The hydroxysteroid (17-beta) dehydrogenase (HSD17B) enzymes catalyze the oxidoreduction of hydroxyl/keto groups of androgens and estrogens and regulate intracellular availability of steroid hormones (Mindnich et al. 2004). To date, at least 14 different isozymes have been identified (Lukacik et al. 2006). The type 8 17β-HSD was initially characterized as a gene whose expression was down-regulated in recessive polycystic kidney disease (PKD) in the mouse (Aziz et al. 1993). Human and mouse HSD17B8 proteins efficiently convert estradiol (E2) into estrone (E1; Fomitcheva et al. 1998, Ohno et al. 2007). They also convert testosterone, 5α-dihydrotestosterone, and 5-androstene3β, 17β-diol into the corresponding 17-ketosteroid, but not with the same efficiency of E2. Despite its in vitro activity, structural studies suggested that the human protein could be involved in the metabolism of fatty acids (Pletnev & Duax 2005).

Expression of human HSD17B8 was found to be especially abundant in the prostate, placenta, and kidney (Ohno et al. 2007), being also detected in other tissues such as the breast, small intestine, stomach, liver, ovary, testis, and adrenals. Expression of mouse HSD17B8 gene has been detected in several somatic tissues (Fomitcheva et al. 1998, Woo et al. 2001, Pelletier et al. 2005), being particularly high in the kidney and liver.

It has been described that HSD17B8 expression is regulated by estrogens in uterus and kidney (Jelinsky et al. 2003, Khalyfa et al. 2003, Bourdeau et al. 2004). Estrogens modulate transcription in their target tissues through estrogen receptor α (ERα) or ERβ receptors using a number of signaling pathways. The ‘classical’ pathway involves direct DNA binding of the activated receptor to an estrogen response element (ERE) in the promoter region of responsive genes (Misiti et al. 2000). The alternative, ‘non-classical’ pathway involves the indirect modulation of transcription by the interaction of the ER with components of other transcription complexes like FOS, SP-1, and NFκB, via protein–protein interactions (Stein & Yang 1995, Wang et al. 1999, DeNardo et al. 2005, Dong et al. 2006, Itoh et al. 2007). In addition, a ‘non-genomic’ pathway has been described in which estrogens can signal through membrane receptors involving MAP kinase signaling (Migliaccio et al. 1996).

We have previously shown that the HSD17B8 gene is transcriptionally regulated by C/EBPβ (Villar et al. 2007b). Moreover, two CCAAT boxes located at −5 and −46 bp of the transcription start site were required for efficient transcription of the gene in HepG2 cells. Specific binding of C/EBPβ to these elements was detected and ectopic expression of C/EBPβ transactivated the HSD17B8 promoter. Here, we show that HSD17B8 expression is regulated by ERα in response to E2 in HepG2 cells. The HSD17B8 expression and promoter activity were induced upon E2 stimulation in cells transfected with ERα but not in untransfected cells, which demonstrates that this induction is ERα dependent. Additional experiments showed that no ERE was necessary for E2 response. However, the mutation of either of the two CCAAT boxes impaired the E2-induced HSD17B8 promoter activity, indicating that these motifs are essential for this regulation. Immunoprecipitation and ChIP assays demonstrated that ERα regulates HSD17B8 gene in response to E2 through its interaction with C/EBPβ. Taken together, our findings demonstrate that C/EBPβ and ERα participate in E2-induced HSD17B8 expression.

Materials and Methods

Reagents

HepG2 and MCF-7 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. ERα (pHEO) expression vector was provided by Dr Pierre Chambon (Institut de Genitique et de Biologie Moleculaire et Cellulaire, Illkirch-Strasbourg, France; Green et al. 1986). C/EBPβ (pMSV-C/EBPβ) expression vector was a gift from Dr Steven L McKnight (UT-Southwestern Medical Center, Dallas, TX, USA; Cao et al. 1991). Luciferase reporter plasmids were previously described (Villar et al. 2007b). E2 and 25OH-cholesterol were supplied by Sigma–Aldrich. C/EBPβ (C-19) and ERα (HC-20) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). ICI 182 780 (Fulvestrant) was purchased from Sigma–Aldrich.

Quantitative RT-PCR

Total RNA was isolated from HepG2 cells using the RNeasy Mini Kit (Qiagen). RNA was reverse transcribed using the Superscript II kit (Invitrogen). Real-time PCR was performed with Brilliant SYBR Green Master Mix (Stratagene, La Jolla, CA, USA) and a Chromo-4 thermocycler (MJ Research, Bio-Rad). Primers used for detecting HSD17B8 mRNA were: sense strand 5′-ACATCAGTAGCATCGTAGG-3′ and antisense strand 5′-GGAGGACAGAGTTACAGC-3′. Primers used for detecting GAPDH were: sense strand 5′-GGAGTCCACTGGCGTCTTC-3′ and antisense strand 5′-ATCTTGAGGCTGTTGTCATACTTC-3′. Results from five independent experiments, each one performed in triplicate were normalized to the level of GAPDH mRNA expressed as fold change from controls.

Transfection

Transient transfections were carried out as previously described (Villar et al. 2007b). Briefly, 5×105 cells were seeded in six-well plates, in triplicate, and transfected with 2 μg HSD17B8 promoter construct plus 3 ng pRL-SV40 vector as a control for transfection efficiency. Additionally, 2 μg ERα expression vector were co-transfected when indicated. Cells were harvested 24 h later, and reporter gene activity was measured in cells extracts. Luciferase activity was determined using a Berthold Lumat LB 9501 luminometer (Berthold Technologies, Oak Ridge, TN, USA) and the dual-luciferase reporter system (Promega Corp). Data were normalized to Renilla luciferase activity and total protein concentration. pGL3 basic and pGL3 control vectors (Promega) were used as transfection controls.

Immunoprecipitation and immunoblotting

After treatments, HepG2 cells were washed with PBS 1×, trypsinized and lysed with Modified-RIPA buffer (Complete Mini-EDTA protease inhibitor tablets (Roche), 0.5 M Tris–HCl pH 8.0, 10% NP-40, 1.5 M NaCl and H2O). Five hundred micrograms of protein mixtures were then precleared with 50 μl TrueBlot anti-Rabbit IP Beads (eBioscience, San Diego, CA, USA) and incubated on ice for 1 h. Supernatants were incubated with anti-ERα, anti-C/EBPβ, or with a non-specific IgG control overnight at 4 °C, and were further incubated for another 4 h after the addition of 50 μl protein beads. After centrifugation, beads were washed at least five times with 500 μl lysis buffer, and eluted with 30 μl Laemmli Sample Buffer (Bio-Rad) with 2% β-mercaptoethanol.

Methods for SDS-PAGE electrophoresis of cellular proteins, and transfer onto polyvinylidene difluoride membranes were as previously described (Alonso et al. 2003, Villar et al. 2007a). Membranes were incubated with anti-ERα or anti- C/EBPβ; washed with Tween 20 in PBS; incubated with peroxidase-conjugated secondary antibody; and the signal was then detected with a chemiluminescence-based system (Pierce, Rockford, IL, USA).

Chromatin immunoprecipitation (ChIP) assay

ChIPs were performed using the ChIP Kit and Shearing Kit (Active Motif, Carlsbad, CA, USA). HepG2 cells were fixed with 37% formaldehyde and lysed according to the manufacturer's instructions. Chromatin was sheared by sonication on ice, and agarose gel electrophoresis was used to select chromatin fragments with an average size of ≤500 bp. Primers 5′-CCTAAGCAGCAGTGTCGG-3′ (upper strand) and 5′-TGTGGGTGGGTGGGAATC-3′ (lower strand) were used for PCR amplification of input DNA and DNA precipitated with anti-C/EBPβ, anti-ERα, or with a non-specific control IgG. Primers were designed to amplify a 167-bp fragment of the HSD17B8 proximal promoter (positions −142 to +25) that encompasses the CCAAT boxes required for promoter activity.

Statistical analysis

Data were analyzed using one-factor ANOVAs. Following significance in the ANOVAs, data were subjected to analysis with Tanhane posttests in which the value necessary for significance (P<0.05) is lowered by dividing the number of comparisons that are made. Analysis was done using SPSS 12.0 for Windows (Chicago, IL, USA). Results are expressed as mean±s.e.m.

Results

E2 increases the expression of HSD17B8 in an ERα-dependent manner

Microarray studies in uterus and kidney have shown that HSD17B8 could be regulated by estrogens in those tissues (Jelinsky et al. 2003, Khalyfa et al. 2003). To confirm whether or not this gene is regulated by estrogens, we treated the breast cancer cell line MCF-7 with E2 10−8 M for 24 h. Real time RT-PCR showed that E2 treatment induced HSD17B8 expression by about 2.5-fold (Fig. 1A). Importantly, the anti-ER ICI 182 780 suppressed this induction, which suggested that this process is ER-dependent. To confirm this, HepG2 cells, a hepatocarcinoma cell line that does not express ERα, were treated with E2. As expected, E2 treatment does not increase HSD17B8 expression (Fig. 1B). However, when HepG2 cells were transfected with an ERα expression vector, E2 induced HSD17B8 expression by about 2.5-fold. More importantly, the anti-ER ICI 182 780 suppressed this induction which confirms that E2-induced HSD17B8 expression is dependent on ERα. Taken together, these results clearly demonstrate that HSD17B8 expression is induced by E2 by a mechanism mediated by ERα.

Figure 1
Figure 1

Estrogens activate HSD17B8 expression. (A) Effect of E2 on HSD17B8 expression in MCF-7 cells. Cells were treated with 10−8 M E2 and 10−7 M ICI 182 780 alone or in combination for 24 h. (B) Effect of E2 on HSD17B8 expression in HepG2 cells. Cells were treated with 10−8 M E2 and 10−7 M ICI alone or in combination for 24 h, in the presence or absence of ERα. Afterwards, RNA was extracted and HSD17B8 expression measured by qPCR. GAPDH was used as loading control. Columns, mean of five independent experiments each done in triplicate; bars, s.e.m., *P<0.05, in all cases.

Citation: Journal of Endocrinology 200, 1; 10.1677/JOE-08-0134

E2 increases the expression of HSD17B8 in an ERE-independent mechanism

To further investigate the E2 activation of HSD17B8 transcription, we tested whether the promoter region of the HSD17B8 gene responded to E2. HepG2 cells were transfected with pJV260, a construct that contained the most active part of the HSD17B8 promoter, and treated with E2 10−8 M for 24 h. As shown in Fig. 2, E2 treatment increased the HSD17B8 promoter activity more than 2–fold. Once again, this induction was only observed in the cells expressing ERα, which confirmed that E2-induced HSD17B8 expression is mediated by ERα. pGL3-promoter that was used as negative control and pERE, a plasmid carrying three copies of the vitellogenin ERE cloned upstream of the luciferase gene into pGL3-promoter, was used as positive control. Taken together, these data confirm that HSD17B8 expression is regulated by E2 and this is mediated by ERα.

Figure 2
Figure 2

E2 increases the HSD17B8 promoter activity in an ERE-independent manner. HepG2 cells were transfected with various human HSD17B8 promoter deletion constructs (schematic on the left) in the presence or absence of E2 10−8 M and ERα. Promoter activity (right) was determined as described in Materials and Methods and expressed as -fold over control (cells treated with vehicle). Columns, mean of five independent experiments each done in triplicate; bars, s.e.m., *P<0.05, in all cases.

Citation: Journal of Endocrinology 200, 1; 10.1677/JOE-08-0134

Computational analysis on the human HSD17B8 promoter revealed the presence of a putative ERE half-site located at −82 (Villar et al. 2007b). ERα is able to activate transcription through ERE half-sites either binding directly to DNA (Kato et al. 1992, Ediger et al. 2002) or binding to other transcription factors previously bound to this element (Stossi et al. 2006). To evaluate whether or not this ERE half-site participates in E2-induced HSD17B8 expression, HepG2 cells were co-transfected with ERα and pJV260 pJV150, or pJV75. pJV260 and pJV150 constructs contained the ERE half-site. However, pJV75 is a deletion construct lacking this element. As shown in Fig. 2, treatment with E2 induced the promoter activity of all of them by more than 2–fold, which demonstrates that this element is not implicated in the regulation of HSD17B8 expression and suggests that HSD17B8 gene is transcriptionally regulated by E2 in an ERE-independent mechanism.

E2 action on HSD17B8 is through the CCAAT boxes presented at the promoter of the gene

Previous studies demonstrated that two CCAAT boxes located at −5 and −46 are essential for HSD17B8 promoter activity (Villar et al. 2007b). Furthermore, C/EBPβ bound to these boxes in vitro and transactivated the HSD17B8 promoter activity. After showing that E2 induces the transcriptional activity of the minimal promoter, it became important to explore whether or not these CCAAT boxes participate in E2-induced HSD17B8 promoter activity. Therefore, HepG2 cells were transfected with pJV260 or with constructs in which these CCAAT boxes are mutated. E2 treatment increased the promoter activity of the wild-type pJV260 by about 2.5-fold. However, mutation of either of the two CCAAT boxes prevented the E2-induced HSD17B8 promoter activity (Fig. 3). These results demonstrate that CCAAT boxes located at −5 and −46 relative to the start site are necessary for E2-induced HSD17B8 transcription.

Figure 3
Figure 3

CCAAT boxes are essential for the E2-induced HSD17B8 expression. CCAAT boxes located at −5 (pMC1) and −46 (pMC2) were mutated. HepG2 cells were co-transfected with the different mutants and the ERα expression vector, and treated with 10−8 M E2. Promoter activity was determined as previously described and expressed as -fold over control (cells treated with vehicle). Columns, mean of five independent experiments each done in triplicate; bars, s.e.m., *P<0.05, in all cases.

Citation: Journal of Endocrinology 200, 1; 10.1677/JOE-08-0134

Estrogens do not regulate C/EBPβ expression in HepG2 cells

Estrogens are known to regulate expression of some transcription factors like C-MYC (Dubik et al. 1987), FOS, Sp1, and NF-YA (Dong et al. 2007). Recently, it has been described that dehydroepiandrosterone modulates the transcription of HSD11B2 by increasing C/EBPβ expression (Balazs et al. 2007). Since C/EBPβ transactivates HSD17B8, upregulation of C/EBPβ by E2 would explain the induction of HSD17B8 expression by E2. To check if E2 increases C/EBPβ expression, MCF-7 and HepG2 cells expressing ERα were treated with E2 10−8 M. Real time RT-PCR showed that E2 does not induce HSD17B8 expression in either of the cell lines (Fig. 4), which clearly indicates that E2-induced HSD17B8 expression is not mediated by the upregulation of C/EBPβ.

Figure 4
Figure 4

E2 does not regulate C/EBPβ expression. (A) MCF-7 cells were treated with 10−8 M E2 for 24 h. (B) HepG2 cells were treated with 10−8 M E2 for 24 h in the presence or absence of ERα. Then, RNA was extracted and C/EBPβ expression was measured by qPCR. GAPDH was used as loading control. Columns, mean of five independent experiments each done in triplicate; bars, s.e.m., *P<0.05, in all cases.

Citation: Journal of Endocrinology 200, 1; 10.1677/JOE-08-0134

C/EBPβ and ERα interact in HepG2 cells expressing ERα

A direct interaction between ERα and C/EBPβ bound to DNA has been described in MCF-7 cells (Dong et al. 2006). Since C/EBPβ transactivates HSD17B8 (Villar et al. 2007b) and E2-induced HSD17B8 expression is ERE-independent, we then decided to test whether or not ERα interacts with C/EBPβ to promote HSD17B8 expression in response to E2. Therefore, HepG2 cells expressing ERα were treated with E2 and co-immunoprecipitation assays with C/EBPβ (Fig. 5A) or ERα (Fig. 5B) specific antibodies were performed. Immunoprecipitated complexes were detected with either ERα (Fig. 5A) or C/EBPβ (Fig. 5B). IgG was included as negative control. In the absence of E2, ERα showed minimal interaction with C/EBPβ (Fig. 5A and B, middle panels). However, upon E2 treatment there was an increase in the amount of the complex formed between ERα and C/EBPβ (Fig. 5A and B, bottom panels). No interaction was observed in cells not expressing ERα (Fig. 5A and B, top panels). Taken together these results indicate that E2 promotes the formation of a complex between ERα and C/EBPβ.

Figure 5
Figure 5

E2 induces interaction between C/EBPβ and ERα. HepG2 cells were treated with 10−8 M E2 or vehicle for 24 h in the presence or absence of ERα. After treatments, cells were harvested and protein complexes immunoprecipitated (IP) using specific antibodies against C/EBPβ, ERα, or control IgG. Western blots (WB) with (A) C/EBPβ or (B) ERα specific antibodies were performed as described in Materials and Methods.

Citation: Journal of Endocrinology 200, 1; 10.1677/JOE-08-0134

Recruitment of ERα to HSD17B8 promoter in response to E2

After showing that E2 induces the formation of the complex ERα-C/EBPβ, we next investigated the recruitment of ERα and C/EBPβ to the HSD17B8 promoter in response to E2. Therefore, ChIP assays were performed in HepG2 cells expressing ERα treated with 10−8 M E2 or vehicle. HepG2 cells not expressing ERα exposed to vehicle were used as a control. Chromatin was immunoprecipitated with ERα, C/EBPβ, or IgG specific antibodies. For PCR analysis a set of primers that amplified a fragment that encompassed the CCAAT boxes located within the proximal promoter of HSD17B8 were used (Fig. 6A). As expected, no ERα recruitment was observed in cells not expressing ERα (Fig. 6B, top panel). Interestingly, in the absence of hormone, ERα was slightly recruited to the HSD17B8 promoter (6B, middle panel). However, after E2 treatment, the recruitment of ERα to the HSD17B8 promoter was highly induced (6B, lower panel). On the other side, recruitment of C/EBPβ to the HSD17B8 promoter was not affected by E2 treatment nor ERα expression, which suggests that C/EBPβ is not affected by E2 treatment. Based on these findings, we conclude that in response to E2, ERα binds to C/EBPβ already bound to HSD17B8 5′ untranslated region promoting its expression.

Figure 6
Figure 6

Recruitment of ERα to the HSD17B8 promoter in response to E2. (A) Panel illustrating the position of the HSD17B8 promoter primers used for the amplification of the ERα, C/EBPβ, or IgG immunoprecipitated DNA fragments. (B) Chromatin immunoprecipitation (ChIP) assay showing that ERα bound to the 17HSD8 promoter upon E2 (10−8 M) stimulation for 24 h. ChIP was performed with chromatin prepared from untransfected HepG2 cells treated with vehicle (top panel), transfected with ERα HepG2 cells treated with vehicle (middle panel) and transfected with ERα HepG2 cells treated with 10−8 M E2. Results of amplification of soluble chromatin before precipitation are shown as control (input).

Citation: Journal of Endocrinology 200, 1; 10.1677/JOE-08-0134

Discussion

HSD17B8 protein efficiently converts E2 into E1 in vitro, which suggests that it might be involved in the metabolism of estrogens. Here, we show that HSD17B8 is regulated by estrogens (Figs 1 and 2), and that this regulation is ERα-mediated. Results from MCF-7 cells (Fig. 1A), a breast cancer cell line ERα positive, and from HepG2 cells (Fig. 1B), a hepatocarcinoma cell line with no ERα expression, revealed that in the presence of ERα E2-induced HSD17B8 expression by about 2.5-fold. This induction was blocked by the addition of the anti-estrogens ICI 182 780, and no induction was observed in the absence of ERα in HepG2 cells, which clearly demonstrates that E2-induced HSD17B8 expression is through ERα.

Our results confirm previous results that showed that the HSD17B8 gene is regulated by estrogens in uterus and kidney (Jelinsky et al. 2003, Khalyfa et al. 2003, Bourdeau et al. 2004). The biological effects of estrogens are mediated by ERα and ERβ, which are members of the superfamily of nuclear receptors (Bjornstrom & Sjoberg 2005). The classical mechanism of ER action involves estrogens binding to receptors in the nucleus, after which the receptors dimerize and bind to specific response elements (ERE) in the promoter of target genes. Although, Bourdeau et al. identified a putative ERE about 3 kb upstream of the transcription start site of the human HSD17B8, we demonstrate that transcriptional regulation of HSD17B8 by ERα does not require direct binding of the agonist-activated ERα to this element (Fig. 2). The human HSD17B8 proximal promoter contains an ERE half-site located at −82. It has been described that ERE half-sites bind ERα but not ERβ (Vanacker et al. 1999). Our results clearly show that not the ERE half-site but CCAAT boxes present in the human HSD17B8 proximal promoter are required for the induction of HSD17B8 expression by E2 (Fig. 3).

In addition to the classical mechanism, two other different mechanisms called non-genomic and non-classical have been described for ER action. The non-classical mechanism, often referred as transcriptional crosstalk, involves tethering of the receptor with other transcription factor complexes that contact the DNA. In previous work, we demonstrated that CCAAT boxes located at −5 and −46 contribute dramatically to the basal promoter activity of HSD17B8. We also detected specific binding of C/EBPβ to those motives, and we showed that ectopic expression of C/EBPβ transactivated the HSD17B8 promoter. Here, we describe that C/EBPβ and ERα crosstalk to promote HSD17B8 transcription in response to E2 (Figs 5 and 6). Interestingly, previous studies have shown C/EBPβ and ERα transcriptional crosstalk, to repress the human IL6 promoter in human osteoblast cell lines (Stein & Yang 1995) and to activate the human prolactin receptor gene in human breast cancer MCF-7 cells (Dong et al. 2006). In the latter, E2 induced the formation of a complex between C/EBPβ, Sp1, and ERα. In the present study, using co-IP, we provide strong evidence of an interaction between ERα and C/EBPβ in response to E2 in human hepatoma HepG2 cells (Fig. 5). Our results also show that E2 induces recruitment of ERα but not C/EBPβ to the HSD17B8 promoter (Fig. 6). This suggests that E2-activated ERα tethers to C/EBPβ already bound to the HSD17B8 promoter, thus, enhancing its expression.

C/EBPβ belongs to a family of transcription factors that contain a highly conserved, basic leucine zipper domain at the C-terminus that is involved in dimerization and DNA binding (Ramji & Foka 2002). C/EBPβ is a critical regulator of proliferation and/or differentiation in multiple tissues, including the liver, adipose tissue, immune system, and mammary gland (Wedel & Ziegler-Heitbrock 1995, Greenbaum et al. 1998, Seagroves et al. 1998, Tang et al. 2003). Importantly, C/EBPβ is essential for female reproduction because of a critical role in ovarian follicle development (Sterneck et al. 1997). C/EBPβ is known to be a critical mediator of steroid hormone responsiveness in the uterus (Mantena et al. 2006), where it mediates E2-induced epithelium proliferation and, additionally, is rapidly induced by E2. In the present study, we clearly show that the induction of HSD17B8 expression by E2 in HepG2 cells is not due to an increase in C/EBPβ expression after E2 treatment (Fig. 4).

C/EBPβs transcription factors have been involved in the regulation of HSD11B1 and 11β-HSD2, enzymes involved in glucocorticoid metabolism (Williams et al. 2000, Gout et al. 2006). Impaired intracellular metabolism of steroids has been suggested to contribute to the development of various pathologic conditions including PKD. Interestingly, HSD17B8 and HSD11B1 expression are downregulated in PKD (Aziz et al. 1994), which suggests that C/EBPβs transcription factor might play an important role in PKD. A significant decrease in HSD17B8 expression has also been described in tumor tissue of oral cavity patients without lymph node metastasis compared with its surrounding healthy tissue (Reinders et al. 2007). Whether or not ERα and C/EBPβ play a role in this downregulation remains to be investigated.

In conclusion, the fact that HSD17B8 converts E2 into E1 suggests that HSD17B8 plays an important role in the control of the intracellular levels of active estrogens. Here, we show that estrogens regulate the expression of HSD17B8, a gene implicated in their own regulation. We also provide strong evidence of C/EBPβ role in E2/ERα mediated HSD17B8 expression. However, the molecular mechanisms underlying the effects of estrogens are likely to be specific for the cell type, and thus, the gene responses are likely to be diverse. Therefore, additional experiments are required to demonstrate if HSD17B8 is regulated by E2 in tissues where estrogens play an important role in the initiation and progression of cancer.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by a grant from the Departmento de Salud, Gobierno de Navarra, Spain.

Acknowledgements

M R and J C are fellows from the Departamento de Educación y Cultura, Gobierno de Navarra, Spain.

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  • EdigerTRParkSEKatzenellenbogenBS2002Estrogen receptor inducibility of the human Na+/H+ exchanger regulatory factor/ezrin-radixin-moesin binding protein 50 (NHE-RF/EBP50) gene involving multiple half-estrogen response elements. Molecular Endocrinology1618281839.

    • Search Google Scholar
    • Export Citation
  • FomitchevaJBakerMEAndersonELeeGYAzizN1998Characterization of Ke 6, a new 17beta-hydroxysteroid dehydrogenase, and its expression in gonadal tissues. Journal of Biological Chemistry2732266422671.

    • Search Google Scholar
    • Export Citation
  • GoutJTirardJThevenonCRiouJPBegeotMNavilleD2006CCAAT/enhancer-binding proteins (C/EBPs) regulate the basal and cAMP-induced transcription of the human 11beta-hydroxysteroid dehydrogenase encoding gene in adipose cells. Biochimie8811151124.

    • Search Google Scholar
    • Export Citation
  • GreenSWalterPKumarVKrustABornertJMArgosPChambonP1986Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature320134139.

    • Search Google Scholar
    • Export Citation
  • GreenbaumLELiWCressmanDEPengYCilibertoGPoliVTaubR1998CCAAT enhancer- binding protein beta is required for normal hepatocyte proliferation in mice after partial hepatectomy. Journal of Clinical Investigation1029961007.

    • Search Google Scholar
    • Export Citation
  • ItohYHayashiHMiyazawaKKojimaSAkahoshiTOnozakiK200717β-Estradiol induces IL-1alpha gene expression in rheumatoid fibroblast-like synovial cells through estrogen receptor alpha (ERalpha) and augmentation of transcriptional activity of Sp1 by dissociating histone deacetylase 2 from ERalpha. Journal of Immunology17830593066.

    • Search Google Scholar
    • Export Citation
  • JelinskySAHarrisHABrownELFlanaganKZhangXTunkeyCLaiKLaneMVSimcoeDKEvansMJ2003Global transcription profiling of estrogen activity: estrogen receptor alpha regulates gene expression in the kidney. Endocrinology144701710.

    • Search Google Scholar
    • Export Citation
  • KatoSToraLYamauchiJMasushigeSBellardMChambonP1992A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5′-TGACC-3′ motifs acting synergistically. Cell68731742.

    • Search Google Scholar
    • Export Citation
  • KhalyfaAKlingeCMHallWCZhaoXMillerMMWangE2003Transcription profiling of estrogen target genes in young and old mouse uterus. Experimental Gerontology3810871099.

    • Search Google Scholar
    • Export Citation
  • LukacikPKavanaghKLOppermannU2006Structure and function of human 17β-hydroxysteroid dehydrogenases. Molecular and Cellular Endocrinology2486171.

    • Search Google Scholar
    • Export Citation
  • MantenaSRKannanACheonYPLiQJohnsonPFBagchiICBagchiMK2006C/EBPbeta is a critical mediator of steroid hormone-regulated cell proliferation and differentiation in the uterine epithelium and stroma. PNAS10318701875.

    • Search Google Scholar
    • Export Citation
  • MigliaccioADi DomenicoMCastoriaGde FalcoABontempoPNolaEAuricchioF1996Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO Journal1512921300.

    • Search Google Scholar
    • Export Citation
  • MindnichRMollerGAdamskiJ2004The role of 17 beta-hydroxysteroid dehydrogenases. Molecular and Cellular Endocrinology218720.

  • MisitiSNanniSFontemaggiGCongYSWenJHirteHWPiaggioGSacchiAPontecorviABacchettiS2000Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells. Molecular and Cellular Biology2037643771.

    • Search Google Scholar
    • Export Citation
  • OhnoSNishikawaKHondaYNakajinS2007Expression in E. coli and tissue distribution of the human homologue of the mouse Ke 6 gene, 17beta-hydroxysteroid dehydrogenase type 8. Molecular and Cellular Biochemistry309209215.

    • Search Google Scholar
    • Export Citation
  • PelletierGLuu-TheVLiSLabrieF2005Localization of type 8 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization. Journal of Histochemistry and Cytochemistry5312571271.

    • Search Google Scholar
    • Export Citation
  • PletnevVZDuaxWL2005Rational proteomics IV: modeling the primary function of the mammalian 17beta-hydroxysteroid dehydrogenase type 8. Journal of Steroid Biochemistry and Molecular Biology94327335.

    • Search Google Scholar
    • Export Citation
  • RamjiDPFokaP2002CCAAT/enhancer-binding proteins: structure, function and regulation. Biochemical Journal365561575.

  • ReindersJRozemullerEHvan der WeidePOkaASlootwegPJInokoHTilanusMG2007Genes in the HLA region indicative for head and neck squamous cell carcinoma. Molecular Immunology44848855.

    • Search Google Scholar
    • Export Citation
  • SeagrovesTNKrnacikSRaughtBGayJBurgess-BeusseBDarlingtonGJRosenJM1998C/EBPbeta, but not C/EBPalpha, is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland. Genes and Development1219171928.

    • Search Google Scholar
    • Export Citation
  • SteinBYangMX1995Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta. Molecular and Cellular Biology1549714979.

    • Search Google Scholar
    • Export Citation
  • SterneckETessarolloLJohnsonPF1997An essential role for C/EBPbeta in female reproduction. Genes and Development1121532162.

  • StossiFLikhiteVSKatzenellenbogenJAKatzenellenbogenBS2006Estrogen-occupied estrogen receptor represses cyclin G2 gene expression and recruits a repressor complex at the cyclin G2 promoter. Journal of Biological Chemistry2811627216278.

    • Search Google Scholar
    • Export Citation
  • TangQQOttoTCLaneMD2003CCAAT/enhancer-binding protein beta is required for mitotic clonal expansion during adipogenesis. PNAS100850855.

    • Search Google Scholar
    • Export Citation
  • VanackerJMPetterssonKGustafssonJALaudetV1999Transcriptional targets shared by estrogen receptor- related receptors (ERRs) and estrogen receptor (ER) alpha, but not by ERbeta. EMBO Journal1842704279.

    • Search Google Scholar
    • Export Citation
  • VillarJArenasMIMacCarthyCMBlanquezMJTiradoOMNotarioV2007aPCPH/ENTPD5 expression enhances the invasiveness of human prostate cancer cells by a protein kinase C delta-dependent mechanism. Cancer Research671085910868.

    • Search Google Scholar
    • Export Citation
  • VillarJCelayJAlonsoMMRotinenMde MiguelCMigliaccioMEncioI2007bTranscriptional regulation of the human type 8 17beta-hydroxysteroid dehydrogenase gene by C/EBPbeta. Journal of Steroid Biochemistry and Molecular Biology105131139.

    • Search Google Scholar
    • Export Citation
  • WangWDongLSavilleBSafeS1999Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Molecular Endocrinology1313731387.

    • Search Google Scholar
    • Export Citation
  • WedelAZiegler-HeitbrockHW1995The C/EBP family of transcription factors. Immunobiology193171185.

  • WilliamsLJLyonsVMacLeodIRajanVDarlingtonGJPoliVSecklJRChapmanKE2000C/EBP regulates hepatic transcription of 11beta -hydroxysteroid dehydrogenase type 1. A novel mechanism for cross-talk between the C/EBP and glucocorticoid signaling pathways. Journal of Biological Chemistry2753023230239.

    • Search Google Scholar
    • Export Citation
  • WooDLeeGYAndersonEAzizN2001Immature ovaries and polycystic kidneys in the congenital polycystic kidney mouse may be due to abnormal sex steroid metabolism. Molecular and Cellular Endocrinology176155162.

    • Search Google Scholar
    • Export Citation
*

(M M Alonso is now at the Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.)

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    Estrogens activate HSD17B8 expression. (A) Effect of E2 on HSD17B8 expression in MCF-7 cells. Cells were treated with 10−8 M E2 and 10−7 M ICI 182 780 alone or in combination for 24 h. (B) Effect of E2 on HSD17B8 expression in HepG2 cells. Cells were treated with 10−8 M E2 and 10−7 M ICI alone or in combination for 24 h, in the presence or absence of ERα. Afterwards, RNA was extracted and HSD17B8 expression measured by qPCR. GAPDH was used as loading control. Columns, mean of five independent experiments each done in triplicate; bars, s.e.m., *P<0.05, in all cases.

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    E2 increases the HSD17B8 promoter activity in an ERE-independent manner. HepG2 cells were transfected with various human HSD17B8 promoter deletion constructs (schematic on the left) in the presence or absence of E2 10−8 M and ERα. Promoter activity (right) was determined as described in Materials and Methods and expressed as -fold over control (cells treated with vehicle). Columns, mean of five independent experiments each done in triplicate; bars, s.e.m., *P<0.05, in all cases.

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    CCAAT boxes are essential for the E2-induced HSD17B8 expression. CCAAT boxes located at −5 (pMC1) and −46 (pMC2) were mutated. HepG2 cells were co-transfected with the different mutants and the ERα expression vector, and treated with 10−8 M E2. Promoter activity was determined as previously described and expressed as -fold over control (cells treated with vehicle). Columns, mean of five independent experiments each done in triplicate; bars, s.e.m., *P<0.05, in all cases.

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    E2 does not regulate C/EBPβ expression. (A) MCF-7 cells were treated with 10−8 M E2 for 24 h. (B) HepG2 cells were treated with 10−8 M E2 for 24 h in the presence or absence of ERα. Then, RNA was extracted and C/EBPβ expression was measured by qPCR. GAPDH was used as loading control. Columns, mean of five independent experiments each done in triplicate; bars, s.e.m., *P<0.05, in all cases.

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    E2 induces interaction between C/EBPβ and ERα. HepG2 cells were treated with 10−8 M E2 or vehicle for 24 h in the presence or absence of ERα. After treatments, cells were harvested and protein complexes immunoprecipitated (IP) using specific antibodies against C/EBPβ, ERα, or control IgG. Western blots (WB) with (A) C/EBPβ or (B) ERα specific antibodies were performed as described in Materials and Methods.

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    Recruitment of ERα to the HSD17B8 promoter in response to E2. (A) Panel illustrating the position of the HSD17B8 promoter primers used for the amplification of the ERα, C/EBPβ, or IgG immunoprecipitated DNA fragments. (B) Chromatin immunoprecipitation (ChIP) assay showing that ERα bound to the 17HSD8 promoter upon E2 (10−8 M) stimulation for 24 h. ChIP was performed with chromatin prepared from untransfected HepG2 cells treated with vehicle (top panel), transfected with ERα HepG2 cells treated with vehicle (middle panel) and transfected with ERα HepG2 cells treated with 10−8 M E2. Results of amplification of soluble chromatin before precipitation are shown as control (input).

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  • DeNardoDGKimHTHilsenbeckSCubaVTsimelzonABrownPH2005Global gene expression analysis of estrogen receptor transcription factor cross talk in breast cancer: identification of estrogen-induced/activator protein-1-dependent genes. Molecular Endocrinology19362378.

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  • DongSZhangZTakaharaH2007Estrogen-enhanced peptidylarginine deiminase type IV gene (PADI4) expression in MCF-7 cells is mediated by estrogen receptor-alpha-promoted transfactors activator protein-1, nuclear factor-Y, and Sp1. Molecular Endocrinology2116171629.

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  • DubikDDembinskiTCShiuRP1987Stimulation of c-myc oncogene expression associated with estrogen-induced proliferation of human breast cancer cells. Cancer Research4765176521.

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    • Export Citation
  • EdigerTRParkSEKatzenellenbogenBS2002Estrogen receptor inducibility of the human Na+/H+ exchanger regulatory factor/ezrin-radixin-moesin binding protein 50 (NHE-RF/EBP50) gene involving multiple half-estrogen response elements. Molecular Endocrinology1618281839.

    • Search Google Scholar
    • Export Citation
  • FomitchevaJBakerMEAndersonELeeGYAzizN1998Characterization of Ke 6, a new 17beta-hydroxysteroid dehydrogenase, and its expression in gonadal tissues. Journal of Biological Chemistry2732266422671.

    • Search Google Scholar
    • Export Citation
  • GoutJTirardJThevenonCRiouJPBegeotMNavilleD2006CCAAT/enhancer-binding proteins (C/EBPs) regulate the basal and cAMP-induced transcription of the human 11beta-hydroxysteroid dehydrogenase encoding gene in adipose cells. Biochimie8811151124.

    • Search Google Scholar
    • Export Citation
  • GreenSWalterPKumarVKrustABornertJMArgosPChambonP1986Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature320134139.

    • Search Google Scholar
    • Export Citation
  • GreenbaumLELiWCressmanDEPengYCilibertoGPoliVTaubR1998CCAAT enhancer- binding protein beta is required for normal hepatocyte proliferation in mice after partial hepatectomy. Journal of Clinical Investigation1029961007.

    • Search Google Scholar
    • Export Citation
  • ItohYHayashiHMiyazawaKKojimaSAkahoshiTOnozakiK200717β-Estradiol induces IL-1alpha gene expression in rheumatoid fibroblast-like synovial cells through estrogen receptor alpha (ERalpha) and augmentation of transcriptional activity of Sp1 by dissociating histone deacetylase 2 from ERalpha. Journal of Immunology17830593066.

    • Search Google Scholar
    • Export Citation
  • JelinskySAHarrisHABrownELFlanaganKZhangXTunkeyCLaiKLaneMVSimcoeDKEvansMJ2003Global transcription profiling of estrogen activity: estrogen receptor alpha regulates gene expression in the kidney. Endocrinology144701710.

    • Search Google Scholar
    • Export Citation
  • KatoSToraLYamauchiJMasushigeSBellardMChambonP1992A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5′-TGACC-3′ motifs acting synergistically. Cell68731742.

    • Search Google Scholar
    • Export Citation
  • KhalyfaAKlingeCMHallWCZhaoXMillerMMWangE2003Transcription profiling of estrogen target genes in young and old mouse uterus. Experimental Gerontology3810871099.

    • Search Google Scholar
    • Export Citation
  • LukacikPKavanaghKLOppermannU2006Structure and function of human 17β-hydroxysteroid dehydrogenases. Molecular and Cellular Endocrinology2486171.

    • Search Google Scholar
    • Export Citation
  • MantenaSRKannanACheonYPLiQJohnsonPFBagchiICBagchiMK2006C/EBPbeta is a critical mediator of steroid hormone-regulated cell proliferation and differentiation in the uterine epithelium and stroma. PNAS10318701875.

    • Search Google Scholar
    • Export Citation
  • MigliaccioADi DomenicoMCastoriaGde FalcoABontempoPNolaEAuricchioF1996Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells. EMBO Journal1512921300.

    • Search Google Scholar
    • Export Citation
  • MindnichRMollerGAdamskiJ2004The role of 17 beta-hydroxysteroid dehydrogenases. Molecular and Cellular Endocrinology218720.

  • MisitiSNanniSFontemaggiGCongYSWenJHirteHWPiaggioGSacchiAPontecorviABacchettiS2000Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells. Molecular and Cellular Biology2037643771.

    • Search Google Scholar
    • Export Citation
  • OhnoSNishikawaKHondaYNakajinS2007Expression in E. coli and tissue distribution of the human homologue of the mouse Ke 6 gene, 17beta-hydroxysteroid dehydrogenase type 8. Molecular and Cellular Biochemistry309209215.

    • Search Google Scholar
    • Export Citation
  • PelletierGLuu-TheVLiSLabrieF2005Localization of type 8 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization. Journal of Histochemistry and Cytochemistry5312571271.

    • Search Google Scholar
    • Export Citation
  • PletnevVZDuaxWL2005Rational proteomics IV: modeling the primary function of the mammalian 17beta-hydroxysteroid dehydrogenase type 8. Journal of Steroid Biochemistry and Molecular Biology94327335.

    • Search Google Scholar
    • Export Citation
  • RamjiDPFokaP2002CCAAT/enhancer-binding proteins: structure, function and regulation. Biochemical Journal365561575.

  • ReindersJRozemullerEHvan der WeidePOkaASlootwegPJInokoHTilanusMG2007Genes in the HLA region indicative for head and neck squamous cell carcinoma. Molecular Immunology44848855.

    • Search Google Scholar
    • Export Citation
  • SeagrovesTNKrnacikSRaughtBGayJBurgess-BeusseBDarlingtonGJRosenJM1998C/EBPbeta, but not C/EBPalpha, is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland. Genes and Development1219171928.

    • Search Google Scholar
    • Export Citation
  • SteinBYangMX1995Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta. Molecular and Cellular Biology1549714979.

    • Search Google Scholar
    • Export Citation
  • SterneckETessarolloLJohnsonPF1997An essential role for C/EBPbeta in female reproduction. Genes and Development1121532162.

  • StossiFLikhiteVSKatzenellenbogenJAKatzenellenbogenBS2006Estrogen-occupied estrogen receptor represses cyclin G2 gene expression and recruits a repressor complex at the cyclin G2 promoter. Journal of Biological Chemistry2811627216278.

    • Search Google Scholar
    • Export Citation
  • TangQQOttoTCLaneMD2003CCAAT/enhancer-binding protein beta is required for mitotic clonal expansion during adipogenesis. PNAS100850855.

    • Search Google Scholar
    • Export Citation
  • VanackerJMPetterssonKGustafssonJALaudetV1999Transcriptional targets shared by estrogen receptor- related receptors (ERRs) and estrogen receptor (ER) alpha, but not by ERbeta. EMBO Journal1842704279.

    • Search Google Scholar
    • Export Citation
  • VillarJArenasMIMacCarthyCMBlanquezMJTiradoOMNotarioV2007aPCPH/ENTPD5 expression enhances the invasiveness of human prostate cancer cells by a protein kinase C delta-dependent mechanism. Cancer Research671085910868.

    • Search Google Scholar
    • Export Citation
  • VillarJCelayJAlonsoMMRotinenMde MiguelCMigliaccioMEncioI2007bTranscriptional regulation of the human type 8 17beta-hydroxysteroid dehydrogenase gene by C/EBPbeta. Journal of Steroid Biochemistry and Molecular Biology105131139.

    • Search Google Scholar
    • Export Citation
  • WangWDongLSavilleBSafeS1999Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions. Molecular Endocrinology1313731387.

    • Search Google Scholar
    • Export Citation
  • WedelAZiegler-HeitbrockHW1995The C/EBP family of transcription factors. Immunobiology193171185.

  • WilliamsLJLyonsVMacLeodIRajanVDarlingtonGJPoliVSecklJRChapmanKE2000C/EBP regulates hepatic transcription of 11beta -hydroxysteroid dehydrogenase type 1. A novel mechanism for cross-talk between the C/EBP and glucocorticoid signaling pathways. Journal of Biological Chemistry2753023230239.

    • Search Google Scholar
    • Export Citation
  • WooDLeeGYAndersonEAzizN2001Immature ovaries and polycystic kidneys in the congenital polycystic kidney mouse may be due to abnormal sex steroid metabolism. Molecular and Cellular Endocrinology176155162.

    • Search Google Scholar
    • Export Citation