Liver Receptor Homologue-1 (LRH-1) Regulates Expression of Aromatase in Preadipocytes
2002; Elsevier BV; Volume: 277; Issue: 23 Linguagem: Inglês
10.1074/jbc.m201117200
ISSN1083-351X
AutoresColin D. Clyne, Caroline J. Speed, Jiong Zhou, Evan R. Simpson,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoEstrogen biosynthesis from C19steroids is catalyzed by aromatase cytochrome P450. Aromatase is expressed in breast adipose tissue through the use of a distal, cytokine-responsive promoter (promoter I.4). Breast tumors, however, secrete soluble factors that stimulate aromatase expression through an alternative proximal promoter, promoter II. In other estrogenic tissues such as ovaries, transcription from promoter II requires the presence of the Ftz-F1 homologue steroidogenic factor-1 (SF-1); adipose tissue, however, does not express SF-1. We have explored the hypothesis that in adipose tissue, an alternative Ftz-F1 family member, liver receptor homologue-1 (LRH-1), substitutes for SF-1 in driving transcription from promoter II. In transient transfection assays using 3T3-L1 preadipocytes, promoter II reporter constructs were modestly (2–3-fold) stimulated by either treatment with activators of protein kinases A or C (PKA/C) or by cotransfection with LRH-1. In combination, these treatments synergistically activated promoter II (>30-fold). Induction by LRH-1 (but not by PKA/C) required an AGGTCA motif at −130 base pairs, to which LRH-1 bound in gel shift assays. Activity of GAL4-LRH-1 fusion proteins was not altered by activators of PKA or PKC. Quantitative real-time PCR revealed that LRH-1 (but not SF-1) is expressed in the preadipocyte fraction of human adipose tissue at levels comparable with that of liver. Differentiation of cultured human preadipocytes into mature adipocytes was associated with a time-dependent induction of peroxisome proliferator-activated receptor-γ (PPARγ), and rapid loss of LRH-1 and aromatase expression. We conclude that LRH-1 is a preadipocyte-specific nuclear receptor that regulates expression of aromatase in adipose tissue. Alterations in LRH-1 expression and/or activity in adipose tissue could therefore have considerable effects on local estrogen production and breast cancer development. Estrogen biosynthesis from C19steroids is catalyzed by aromatase cytochrome P450. Aromatase is expressed in breast adipose tissue through the use of a distal, cytokine-responsive promoter (promoter I.4). Breast tumors, however, secrete soluble factors that stimulate aromatase expression through an alternative proximal promoter, promoter II. In other estrogenic tissues such as ovaries, transcription from promoter II requires the presence of the Ftz-F1 homologue steroidogenic factor-1 (SF-1); adipose tissue, however, does not express SF-1. We have explored the hypothesis that in adipose tissue, an alternative Ftz-F1 family member, liver receptor homologue-1 (LRH-1), substitutes for SF-1 in driving transcription from promoter II. In transient transfection assays using 3T3-L1 preadipocytes, promoter II reporter constructs were modestly (2–3-fold) stimulated by either treatment with activators of protein kinases A or C (PKA/C) or by cotransfection with LRH-1. In combination, these treatments synergistically activated promoter II (>30-fold). Induction by LRH-1 (but not by PKA/C) required an AGGTCA motif at −130 base pairs, to which LRH-1 bound in gel shift assays. Activity of GAL4-LRH-1 fusion proteins was not altered by activators of PKA or PKC. Quantitative real-time PCR revealed that LRH-1 (but not SF-1) is expressed in the preadipocyte fraction of human adipose tissue at levels comparable with that of liver. Differentiation of cultured human preadipocytes into mature adipocytes was associated with a time-dependent induction of peroxisome proliferator-activated receptor-γ (PPARγ), and rapid loss of LRH-1 and aromatase expression. We conclude that LRH-1 is a preadipocyte-specific nuclear receptor that regulates expression of aromatase in adipose tissue. Alterations in LRH-1 expression and/or activity in adipose tissue could therefore have considerable effects on local estrogen production and breast cancer development. Estrogen biosynthesis from C19 steroids is catalyzed by the enzyme aromatase cytochrome P450 (1Simpson E.R. Mahendroo M.S. Means G.D. Kilgore M.W. Hinshelwood M.M. Graham-Lorence S. Amarneh B. Ito Y. Fisher C.R. Michael M.D. Mendelson C.R. Bulun S.E. Endocr. Rev. 1994; 15: 342-355Crossref PubMed Scopus (1060) Google Scholar). In humans, aromatase is expressed in both the granulosa and luteal cells of the ovary and also in various extra-glandular sites, including the placenta, brain, bone, testis, and adipose tissue (2Bulun S.E. Simpson E.R. Breast Cancer Res. Treat. 1994; 30: 19-29Crossref PubMed Scopus (88) Google Scholar). Aromatase is encoded by the CYP19 gene, which maps to chromosome 15q21.2 in humans (3Chen S.A. Besman M.J. Sparkes R.S. Zollman S. Klisak I. Mohandas T. Hall P.F. Shively J.E. DNA (N. Y.). 1988; 7: 27-38Crossref PubMed Scopus (211) Google Scholar,4Sebastian S. Bulun S.E. J. Clin. Endocrinol. Metab. 2001; 86: 4600-4602Crossref PubMed Scopus (179) Google Scholar). The structure and hormonal regulation of CYP19 are complex: the gene spans 123 kb, with a coding region of 30 kb comprising nine translated exons (4Sebastian S. Bulun S.E. J. Clin. Endocrinol. Metab. 2001; 86: 4600-4602Crossref PubMed Scopus (179) Google Scholar, 5Means G.D. Mahendroo M.S. Corbin C.J. Mathis J.M. Powell F.E. Mendelson C.R. Simpson E.R. J. Biol. Chem. 1989; 264: 19385-19391Abstract Full Text PDF PubMed Google Scholar, 6Toda K. Terashima M. Kawamoto T. Sumimoto H. Yokoyama Y. Kuribayashi I. Mitsuuchi Y. Maeda T. Yamamoto Y. Sagara Y. Eur. J. Biochem. 1990; 193: 559-565Crossref PubMed Scopus (126) Google Scholar, 7Harada N. Yamada K. Saito K. Kibe N. Dohmae S. Takagi Y. Biochem. Biophys. Res. Commun. 1990; 166: 365-372Crossref PubMed Scopus (136) Google Scholar). A number of untranslated exons I, each driven by a unique promoter, exist upstream of exon II (8Means G.D. Kilgore M.W. Mahendroo M.S. Mendelson C.R. Simpson E.R. Mol. Endocrinol. 1991; 5: 2005-2013Crossref PubMed Scopus (235) Google Scholar, 9Mahendroo M.S. Means G.D. Mendelson C.R. Simpson E.R. J. Biol. Chem. 1991; 266: 11276-11281Abstract Full Text PDF PubMed Google Scholar, 10Toda K. Shizuta Y. Eur. J. Biochem. 1993; 213: 383-389Crossref PubMed Scopus (63) Google Scholar). These are spliced to a common site in the 5′-untranslated region. Tissue-specific regulation of CYP19 expression is achieved through the use of these distinct promoters, each of which is regulated by distinct hormonal factors. Thus in the ovary, CYP19 expression is regulated by FSH, which acts (through cAMP) via promoter II (11Steinkampf M.P. Mendelson C.R. Simpson E.R. Mol. Endocrinol. 1987; 1: 465-471Crossref PubMed Scopus (122) Google Scholar, 12Michael M.D. Kilgore M.W. Morohashi K. Simpson E.R. J. Biol. Chem. 1995; 270: 13561-13566Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar), whereas in placenta, promoter I.I regulates CYP19 expression in response to retinoids (13Sun T. Zhao Y. Mangelsdorf D.J. Simpson E.R. Endocrinology. 1998; 139: 1684-1691Crossref PubMed Google Scholar). In bone and adipose tissue, by contrast, a distal promoter (promoter I.4) drives CYP19 expression under the control of glucocorticoids, class 1 cytokines, or TNFα (14Shozu M. Simpson E.R. Mol. Cell. Endocrinol. 1998; 139: 117-129Crossref PubMed Scopus (115) Google Scholar, 15Zhao Y. Mendelson C.R. Simpson E.R. Mol. Endocrinol. 1995; 9: 340-349Crossref PubMed Google Scholar, 16Zhao Y. Nichols J.E. Bulun S.E. Mendelson C.R. Simpson E.R. J. Biol. Chem. 1995; 270: 16449-16457Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 17Zhao Y. Nichols J.E. Valdez R. Mendelson C.R. Simpson E.R. Mol. Endocrinol. 1996; 10: 1350-1357Crossref PubMed Google Scholar). In postmenopausal women, aromatase activity in adipose tissue is the major source of circulating estrogens (18Siiteri P.K. Macdonald P.C. Green R.O. Astwoon E.B. Handbook of Physiology. American Physiological Society, Bethesda, MD1973: 619-629Google Scholar, 19Simpson E.R. Zhao Y. Agarwal V.R. Michael M.D. Bulun S.E. Hinshelwood M.M. Graham-Lorence S. Sun T. Fisher C.R. Qin K. Mendelson C.R. Recent Prog. Horm. Res. 1997; 52: 185-213PubMed Google Scholar). In normal breast adipose tissue aromatase activity and CYP19 expression are low. However, in adipose tissue of breast cancer patients, estrogen levels, aromatase activity, and CYP19 expression are elevated (20Thorsen T. Tangen M. Stoa K.F. Eur. J. Cancer Clin. Oncol. 1982; 18: 333-337Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 21van Landeghem A.A. Poortman J. Nabuurs M. Thijssen J.H. Cancer Res. 1985; 45: 2900-2906PubMed Google Scholar, 22Bulun S.E. Price T.M. Aitken J. Mahendroo M.S. Simpson E.R. J. Clin. Endocrinol. Metab. 1993; 77: 1622-1628Crossref PubMed Scopus (246) Google Scholar, 23Harada N. J. Steroid Biochem. Mol. Biol. 1997; 61: 175-184Crossref PubMed Google Scholar). This occurs in response to tumor-derived factors (such as prostaglandin E2) produced by breast tumor fibroblasts and epithelium as well as infiltrating macrophages (24Zhao Y. Agarwal V.R. Mendelson C.R. Simpson E.R. J. Steroid Biochem. Mol. Biol. 1997; 61: 203-210Crossref PubMed Google Scholar). It is this local source of estrogen that provides the drive for growth of estrogen receptor-positive tumors and which is the target of anti-estrogen adjuvant therapies in postmenopausal women. However, current strategies of anti-estrogen therapy such as pure estrogen receptor antagonists or aromatase enzyme inhibitors act in a global fashion and inhibit estrogen action or synthesis in all sites of production. This has the potential to result in bone loss and other sequelae of estrogen insufficiency such as cognitive dysfunction and hepatic steatosis with prolonged treatment (25Paganini-Hill A. Clark L.J. Breast Cancer Res. Treat. 2000; 64: 165-176Crossref PubMed Scopus (198) Google Scholar, 26Murata Y. Ogawa Y. Saibara T. Nishioka A. Fujiwara Y. Fukumoto M. Inomata T. Enzan H. Onishi S. Yoshida S. Oncol. Rep. 2000; 7: 1299-1304PubMed Google Scholar, 27Pinto H.C. Baptista A. Camilo M.E. de Costa E.B. Valente A. de Moura M.C. J. Hepatol. 1995; 23: 95-97Abstract Full Text PDF PubMed Scopus (91) Google Scholar). Thus there is a clear need for more specific, tissue-selective anti-estrogens. The increased CYP19 expression in response to breast tumor-derived factors is associated with a switch in promoter usage from the normal adipose-specific promoter I.4 to the cAMP-responsive promoter II (28Agarwal V.R. Bulun S.E. Leitch M. Rohrich R. Simpson E.R. J. Clin. Endocrinol. Metab. 1996; 81: 3843-3849Crossref PubMed Scopus (252) Google Scholar, 29Harada N. Utsumi T. Takagi Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11312-11316Crossref PubMed Scopus (349) Google Scholar, 30Zhou D. Zhou C. Chen S. J. Steroid Biochem. Mol. Biol. 1997; 61: 273-280Crossref PubMed Google Scholar). Since these two promoters are regulated by different cohorts of transcription factors and coactivators, it follows that the differential regulation of CYP19 expression via alternative promoters in disease-free and cancerous breast adipose tissue may permit the development of selective aromatase modulators, which target the aberrant overexpression in cancerous breast, while sparing estrogen action in other sites of synthesis such as normal adipose tissue, bone, and brain (31Simpson E.R. Davis S.R. Endocrinology. 2001; 142: 4589-4594Crossref PubMed Scopus (310) Google Scholar). A more complete understanding of the mechanisms regulating CYP19 transcription from promoter II in breast adipose tissue is a prerequisite for the development of such selective aromatase modulators. In classic steroidogenic tissue such as ovary and testis, promoter II is regulated by steroidogenic factor-1 (SF-1 1The abbreviations used are: SF-1steroidogenic factor-1LRH-1liver receptor homologue-1ERestrogen receptorERRαestrogen receptor-related receptor-αPPARγperoxisome proliferator-activated receptor-γNREnuclear receptor half-siteDBDDNA binding domainPKAprotein kinase APKCprotein kinase CFSKforskolinPMAphorbol 12-myristate 13-acetateFtz-F1fushi tarazu F1 /Ad4BP/NR5A1) (32(1999) Cell 97, 161–163Google Scholar), which binds to a nuclear receptor half-site (NRE) within the promoter to mediate basal transcription and, in part, cAMP-induced transcription (12Michael M.D. Kilgore M.W. Morohashi K. Simpson E.R. J. Biol. Chem. 1995; 270: 13561-13566Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Although adipose tissue does not express SF-1 (Fig. 2herein), 2C. D. Clyne, C. J. Speed, J. Zhou, and E. R. Simpson, unpublished observations. the NRE within promoter II has been shown to bind other negative and positive transcription factors in adipose stromal cells and breast cancer cell lines including chicken ovalbumin upstream promoter transcription factor (COUP-TF) and ERRα (33Yang C. Zhou D. Chen S. Cancer Res. 1998; 58: 5695-5700PubMed Google Scholar). Thus the activity of promoter II in adipose tissue is controlled, at least in part, by the balance of stimulatory and inhibitory transcription factors binding to this site. steroidogenic factor-1 liver receptor homologue-1 estrogen receptor estrogen receptor-related receptor-α peroxisome proliferator-activated receptor-γ nuclear receptor half-site DNA binding domain protein kinase A protein kinase C forskolin phorbol 12-myristate 13-acetate fushi tarazu F1 In seeking to identify transcription factors that could potentially activate CYP19 transcription in adipose tissue through this promoter II NRE, we have focused the current study on liver receptor homologue-1 (LRH-1, also known as CYP7A promoter binding factor), α-fetoprotein transcription factor, human B1-binding factor, and NR5A2 (32(1999) Cell 97, 161–163Google Scholar, 34Nitta M., Ku, S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar, 35Galarneau L. Pare J.F. Allard D. Hamel D. Levesque L. Tugwood J.D. Green S. Belanger L. Mol. Cell. Biol. 1996; 16: 3853-3865Crossref PubMed Google Scholar, 36Li M. Xie Y.H. Kong Y.Y., Wu, X. Zhu L. Wang Y. J. Biol. Chem. 1998; 273: 29022-29031Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). LRH-1 and SF-1 are the two human homologues of the Drosophila nuclear receptor Ftz-F1 (37Lavorgna G. Ueda H. Clos J. Wu C. Science. 1991; 252: 848-851Crossref PubMed Scopus (247) Google Scholar) and share common DNA binding and transactivation properties. Whereas SF-1 expression is mainly restricted to steroidogenic tissues of the reproductive axis (38Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar), LRH-1 is expressed at high levels in liver where it regulates expression of genes involved in cholesterol metabolism and bile acid synthesis including cholesterol 7α-hydroxylase (CYP7A) (34Nitta M., Ku, S. Brown C. Okamoto A.Y. Shan B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6660-6665Crossref PubMed Scopus (249) Google Scholar, 39Goodwin B. Jones S.A. Price R.R. Watson M.A. McKee D.D. Moore L.B. Galardi C. Wilson J.G. Lewis M.C. Roth M.E. Maloney P.R. Willson T.M. Kliewer S.A. Mol. Cell. 2000; 6: 517-526Abstract Full Text Full Text PDF PubMed Scopus (1515) Google Scholar, 40Lu T.T. Makishima M. Repa J.J. Schoonjans K. Kerr T.A. Auwerx J. Mangelsdorf D.J. Mol. Cell. 2000; 6: 507-515Abstract Full Text Full Text PDF PubMed Scopus (1229) Google Scholar), sterol 12α-hydroxylase (CYP8B1) (41Castillo-Olivares A. Gil G. J. Biol. Chem. 2000; 275: 17793-17799Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and the cholesteryl ester transfer protein (42Luo Y. Liang C.P. Tall A.R. J. Biol. Chem. 2001; 276: 24767-24773Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). LRH-1 is also expressed in the pancreas, ovary, intestine, and colon (43Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar) and has been reported to regulate adrenal expression of 11β-hydroxylase (44Wang Z.N. Bassett M. Rainey W.E. J. Mol. Endocrinol. 2001; 27: 255-258Crossref PubMed Scopus (56) Google Scholar). It has, however, been reported to be absent from adipose tissue (43Lu T.T. Repa J.J. Mangelsdorf D.J. J. Biol. Chem. 2001; 276: 37735-37738Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). In the current study we show that LRH-1 can bind to and strongly activate promoter II of the CYP19 gene. Importantly, LRH-1 is expressed at high levels in the undifferentiated stromal compartment of human adipose tissue (the site of CYP19 expression), but not in differentiated adipocytes, and therefore represents a physiologically relevant regulator of estrogen biosynthesis in breast. PII-516 is a CYP19 promoter II/luciferase construct containing −516/−17 nucleotides of human CYP19 promoter II and has been described previously (12Michael M.D. Kilgore M.W. Morohashi K. Simpson E.R. J. Biol. Chem. 1995; 270: 13561-13566Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar,45Michael M.D. Michael L.F. Simpson E.R. Mol. Cell. Endocrinol. 1997; 134: 147-156Crossref PubMed Scopus (139) Google Scholar). The nuclear receptor half-site at position −130 within this construct was mutated by PCR-directed mutagenesis (AGGTCA → AaaTCA) to produce pII-516mNRE. An expression construct encoding mouse LRH-1 (pCMX-LRH-1) was a generous gift from David Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). The cDNA insert of pCMX-LRH-1 was subcloned into pcDNA3.1+ (Invitrogen) for use in in vitro transcription/translation reactions. pBIND (Promega) encodes amino acids 1–147 of yeast GAL4. To produce a GAL4/LRH-1 fusion construct in which the DBD and Ftz-F1 box of LRH-1 was replaced by the GAL4 DBD (pBIND-LRHΔ), the appropriate LRH-1 cDNA sequence was amplified by PCR from pCMX-LRH-1 and cloned into pBIND. PCR fidelity and correct reading frame of the resultant plasmid were confirmed by sequencing. The pSV-β vector (Promega) encodes full-length β-galactosidase and was used to correct for transfection efficiency. An expression construct encoding SF-1 was generously provided by Ken-ichirou Morohashi, (Kyushu University, Fukuoka, Japan). Human adipose stromal cells were isolated and cultured as described previously (46Ackerman G.E. Smith M.E. Mendelson C.R. MacDonald P.C. Simpson E.R. J. Clin. Endocrinol. Metab. 1981; 53: 412-417Crossref PubMed Scopus (357) Google Scholar). 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at a density of 40,000 cells/ml. Cells were transfected for 22 h with 2.2 μg of total DNA comprising 1.0 μg of luciferase reporter, 1.0 μg of expression construct (or empty vector), and 0.2 μg of pSV-β, using FuGENE 6 reagent (Roche Molecular Biochemicals). Cells were serum-starved for 24 h prior to experimental treatment, after which luciferase and β-galactosidase activities of soluble cell extracts were measured using the Luciferase Assay System (Promega) and Galacto-light system (Tropix), respectively. Differentiation of cultured human preadipocytes was induced by a 3-day incubation in adipogenic medium (Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (3%), Troglitazone (1 μm), insulin (10 nm), dexamethasone (1 μm), triiodothyronine (200 pm), and 1-methyl-3-isobutylxanthine (0.5 mm)). Cells were then incubated in adipogenic medium lacking 1-methyl-3-isobutylxanthine and Troglitazone for a further 9 days. Medium was changed every 3 days. Nuclear extracts were prepared from confluent adipose stromal cells by the method of Schreiber et al. (47Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3917) Google Scholar). Recombinant proteins were transcribed/translated in vitro using the TnT Quick-coupled transcription/translation system (Promega). 5 μg of nuclear extract or 0.5 μl of translation product were incubated with 20,000 cpm of 32P-labeled probe for 15 min at room temperature in 20 μl of binding buffer (20 mm HEPES pH 8.0, 1 mm EDTA, 10% glycerol, 50 mm KCl, 50 μg/ml poly(dI·dC/dI·dC), 1 mg/ml bovine serum albumin, 10 mm dithiothreitol) before electrophoresis using a 5.4% polyacrylamide gel and 0.5 × TBE (final concentrations 44.5 mm Tris, 44.5 mm boric acid, 1 mmEDTA, pH 8.0) as running buffer for 3 h at 200 V. Gels were dried and radioactive complexes visualized by phosphorimaging. Where antibodies were included in the reaction (directed against LRH-1 or p65, SantaCruz), protein extract and antibody were preincubated on ice for 10 min before addition of probe. In some experiments protein extracts were heated to 37 °C for 3 min either before or after addition of probe. Protein lysates of human mammary fat and adipose stromal cells were generated using a lysis buffer (137 mm NaCl, 0.5% Nonidet P-40, 10 mm Tris-HCl, pH 7.5, and a Protease Inhibitor Cocktail tablet (Roche Molecular Biochemicals)), separated on a 10% SDS-polyacrylamide gel (15 μg/lane), and transferred to nitrocellulose filter. Filters were probed with a CYP7A promoter binding factor (LRH-1) antibody (Santa Cruz), then stripped and re-probed with a β-tubulin antibody to confirm equal protein loading. Detection was by a ECL Plus Western Blotting Detection System (Amersham Biosciences). Total RNA was prepared from freshly isolated mouse tissues or various cell lines using the QiaAMP RNA Blood Mini kit (Qiagen). First strand cDNA synthesis from 250 ng of total RNA was performed using avian myeloblastosis virus reverse transcriptase (Roche) primed by random hexamers. PCR reactions were carried out using the following primer sets (all 5′ → 3′): LRH-1 (sense, CTG ATA CTG GAA CTT TTG AA; antisense, CTT CAT TTG GTC ATC AAC CTT); SF-1 (sense, TGC AGA ATG GCC GAC CAG; antisense, TGG CGG TAG ATG TGG TC); PPARγ (sense, ATT CTG GCC CAC CAA CTT TGG G; antisense, ATT GCC ATG AGC GAG TTG GAA GGC); CYP19 (sense, TTG GAA ATG GTC AAC CCG AT; antisense, CAG GAA TCT GCC GTG GGA GA); 18S (sense, CGG CTA CCA CAT CCA AGG AA; antisense, GCT GGA ATT ACC GCG GCT). Real-time PCR amplification of LRH-1 and 18S was performed on the LightCycler (Roche Molecular Biochemicals) using SYBR Green reaction mix (Roche Molecular Biochemicals) and the primers described above. cDNA samples were diluted 1:20 in water immediately before use. Experimental samples were quantified by comparison with standards of known concentration (0.01–100 fg/μl). To identify receptors that could bind and activate promoter II through the NRE, 3T3L1 mouse preadipocytes were cotransfected with a luciferase reporter construct harboring 516 nucleotides of CYP19 promoter II (12Michael M.D. Kilgore M.W. Morohashi K. Simpson E.R. J. Biol. Chem. 1995; 270: 13561-13566Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 45Michael M.D. Michael L.F. Simpson E.R. Mol. Cell. Endocrinol. 1997; 134: 147-156Crossref PubMed Scopus (139) Google Scholar) and expression vectors encoding various nuclear receptors (Fig 1). Luciferase activity was not significantly altered by cotransfection with ERRα, NGFIB, Nurr1, Nor1 (Fig 1), or either ERα or ERβ in the presence or absence of ligand (not shown). In contrast, both SF-1 and LRH-1 stimulated promoter II activity 9- and 6-fold, respectively. Although the stimulatory effect of SF-1 on CYP19 promoter II in gonadal cells has been well characterized (38Parker K.L. Schimmer B.P. Endocr. Rev. 1997; 18: 361-377Crossref PubMed Scopus (556) Google Scholar), the role of LRH-1 in CYP19 transcription has not previously been investigated. SF-1 and LRH-1 are expressed at high levels in steroidogenic tissues and liver, respectively. To address the possible roles of these receptors in the regulation of CYP19 in adipose tissue, we determined the expression profiles of each receptor in various cell lines and adipose tissue specimens (Fig. 2A). LRH-1 mRNA expression was detected by RT-PCR in mouse adrenal and liver, human adrenal and liver cell lines (H295R and HepG2), the 3T3L1 mouse preadipocyte cell line, and the MCF-7 human breast cancer cell line. LRH-1 was also expressed in four out of four human adipose tissue specimens, and in four of four primary cultures of adipose stromal cells derived from different individuals. In contrast, expression of SF-1 mRNA was restricted to mouse adrenal and human adrenocortical H295R cells. We also investigated SF-1 and LRH-1 expression in human primary breast cancer tissue (Fig. 2B). All seven breast cancer specimens examined expressed LRH-1, whereas SF-1 was undetectable. Thus LRH-1, but not SF-1, is expressed in breast adipose and cancer tissues. To quantify LRH-1 mRNA expression in these tissues, we performed real-time PCR (Fig. 3A). Expressed as the ratio of LRH-1 molecules:18S molecules per μg of total RNA; LRH-1 expression in adipose tissue was ∼20% that of liver. However, the relative expression in primary cultured adipose stromal cells was much higher, ∼11-fold higher than in whole adipose tissue and 2.5-fold higher than in liver. LRH-1 protein was also readily detectable by Western blotting in isolated preadipocytes, but not in whole adipose tissue (Fig. 3B). Thus although LRH-1 mRNA levels in adipose tissue are relatively low compared with liver, LRH-1 expression is enriched in the adipose stromal cell compartment. This suggests that LRH-1, like CYP19, may be a marker of the undifferentiated preadipocyte phenotype. To test this hypothesis, primary cultured human preadipocytes were induced to differentiate into adipocytes by a 12-day incubation in adipogenic medium (Fig. 3C). Under such conditions lipid droplets became visible after 6 days, and by day 12 ∼50% of the cells exhibited abundant lipid accumulation (Fig. 3C, lower panel). The mature adipocyte phenotype was confirmed by a rapid and sustained expression of PPARγ (Fig. 3D, upper panel). LRH-1 mRNA was readily detectable in untreated preadipocytes but was dramatically reduced following 3 days of culture in adipogenic medium and undetectable at days 9 and 12.CYP19 mRNA expression also displayed a time-dependent decrease with progression of differentiation. Therefore, LRH-1 is expressed at high levels in human preadipocytes, but not mature adipocytes. Since this expression profile mirrors that of CYP19, LRH-1 is a potential physiological regulator of CYP19 expression in breast adipose stromal cells. Aromatase activity and CYP19 mRNA expression are strongly induced by prostaglandin E2 derived from breast cancer cells and/or macrophages infiltrating the tumor site (24Zhao Y. Agarwal V.R. Mendelson C.R. Simpson E.R. J. Steroid Biochem. Mol. Biol. 1997; 61: 203-210Crossref PubMed Google Scholar). PGE2 binds to EP1 and EP2 receptors linked to PKC and PKA signaling pathways, activation of which together maximally stimulates CYP19 expression via promoter II (24Zhao Y. Agarwal V.R. Mendelson C.R. Simpson E.R. J. Steroid Biochem. Mol. Biol. 1997; 61: 203-210Crossref PubMed Google Scholar). To assess the effect of these pathways on LRH-1-induced CYP19 transcription, 3T3L1 cells were cotransfected with the CYP19 promoter II reporter construct and increasing concentrations of LRH-1 expression construct. Cells were then incubated in the presence or absence of the adenylyl cyclase activator forskolin and the PKC activator PMA for 8 h (Fig. 4). In the absence of stimulation, LRH-1 dose-dependently increased promoter II activity reaching a maximum of 3-fold over basal at 1.0 μg of LRH-1 plasmid. Treatment with forskolin and PMA increased basal promoter II activity 4-fold; however, in the presence of these agents LRH-1 strongly induced promoter II activity reaching a maximum of 30-fold at 1.0 μg of LRH-1. The synergistic effects of LRH-1 and FSK + PMA raised the possibility that LRH-1 contributes to PKA and/or PKC induction of promoter II. The primary amino acid sequence of LRH-1 contains several potential consensus PKA and PKC phosphorylation sites (PKA: Ser-32, Thr-142, Ser-382; PKC: Ser-32, Ser-126, Thr-154, Ser-350, Thr-512). To determine whether transactivation by LRH-1 can be directly modified by phosphorylation, we constructed a fusion construct in which the DNA binding domain of LRH-1 is replaced by the DBD of the yeast transcription factor GAL4. This fusion construct was transfected into 3T3-L1 cells along with a GAL4-responsive luciferase reporter gene, and cells treated with FSK and PMA, alone or in combination, for 16 h. As a control, we also treated 3T3-L1 cells transfected with pII-516 with these agents (Fig. 5A). Treatment with FSK increased activity of pII-516 ∼5-fold. PMA, while ineffective on its own, increased FSK-induced activity to 8.5-fold. These changes in activity of pII-516 mirror the effects of FSK and PMA on endogenous aromatase activity in adipose stromal cells (24Zhao Y. Agarwal V.R. Mendelson C.R. Simpson E.R. J. Steroid Biochem. Mol. Biol. 1997; 61: 203-210Crossref PubMed Google Scholar). 3T3-L1 cells transfected with the GAL4 DBD and a GAL4-responsive luciferase reporter had low levels of luciferase activity that were not altered following treatment with FSK or PMA (Fig. 5B, upper panel). Luciferase activity in cells transfected with the GAL4 DBD/LRH-1 fusion construct were ∼15-fold higher; however, treatment with FSK or PMA, alone or in combination, did not further affect luciferase activity. Therefore, activity of LRH-1 is not regulated by PKA or PKC signaling pathways. Induction of expression from promoter II by PKA and PKC likely occurs through use of other hormone-sensitive cis-elements, for example the CRE-like element upstream from the NRE (45Michael M.D. Michael L.F. Simpson E.R. Mol. Cell. Endocrinol. 1997; 134: 147-156Crossref PubMed Scopus (139) Google Scholar) in the case of PKA, with LRH-1 functioning as a basal transcription or competence factor. This would be consistent with, and analogous to, the role of SF-1 in cAMP stimulation of promoter II
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