Identification of NR5A1 (SF-1/AD4BP) Gene Expression Modulators By

489 Identification of NR5A1 (SF-1/AD4BP ) gene expression modulators by large-scale gain and loss of function studies Noriko Sakai*, Hiromi Terami*, Shinobu Suzuki, Megumi Haga, Ken Nomoto, Nobuko Tsuchida, Ken-ichirou Morohashi1, Naoaki Saito2, Maki Asada3, Megumi Hashimoto3, Daisuke Harada3, Hiroshi Asahara3, Tetsuya Ishikawa, Fumiki Shimada and Kazuhiro Sakurada Department of Stem Cell Based Drug Discovery, Research Center Kobe, Bayer Yakuhin Ltd, Kobe, Hyogo 650-0047, Japan 1Division of Sex Differentiation, National Institute for Basic Biology, Myodaiji-cho, Okazaki, Nagoya 444-8787, Japan 2Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Hyogo 657-8501, Japan 3Department of Systems Biomedicine, National Research Institute for Child Health and Development, 157-8535 Tokyo, Japan (Correspondence should be addressed to K Sakurada; Email: [email protected]) *(N Sakai and H Terami contributed equally to this work) Abstract Nuclear receptor subfamily 5, group A, member 1 (NR5A1 involved in the NR5A1 gene expression. Among these, we previously known as SF-1/AD4BP) is a transcription factor identified crosstalk between erb-B3 and PKC-d signaling involved in the development of adrenal/gonadal tissues and cascades. In addition, the gain of function studies indicated steroidogenic linage cell differentiation in adult somatic stem that sex-determining region Y (SRY)-box 15 (SOX15), cells. To understand the cellular signaling network that TEA domain family member 4, KIAA1257 (a gene of regulates NR5A1 gene expression, loss of function screening unknown function), ADAM metallopeptidase with throm- with an siRNA kinome library, and gain of function bospondin type 1 motif 6, Josephin domain containing 1, screening with an addressable full-length cDNA library centromere protein, TATA box-binding protein-associated representing one quarter of the human genome was carried factor 5-like RNA polymerase, and inducible T-cell out. The NR5A1 gene expression was activated in co-stimulator activate NR5A1 gene expression. These mesenchymal stem cells by siRNA directed against protein results provide new insights into the molecular mechanisms kinase C (PKC)-d, erb-B3, RhoGAP (ARHGAP26), and of NR5A1 gene expression. hexokinase 2, none of which were previously known to be Journal of Endocrinology (2008) 198, 489–497 Introduction FDA (Food and Drug Administration) recommends using the lowest dose of hormone replacement therapy (HRT) for Ovarian folliculogenesis has an essential role in oocyte the shortest duration (Cirigliano 2007). One of the major maturation and steroid production (Palter et al.2001). The wall problems of estrogen replacement therapy is the optimization of of a mature preovulatory follicle consists of granulosa, theca the dose of estrogen in each individual patient (Cirigliano 2007). interna, and theca externa cells that produce three classes of Production of steroids in the ovary is tightly regulated by the steroid hormones, progestins, androgens, and estrogens hypothalamus–anterior pituitary axis. Androgen production (Vanderhyden 2002). Theca interna cells produce androgens, from theca interna cells is stimulated by luteinizing hormone whereas granulosa cells convert androgens to estrogens. (LH; Magoffin 2005). Follicle-stimulating hormone (FSH) Progesterone is the major product of granulosa cells stimulates the conversion of androgen to estrogen in granulosa (Vanderhyden 2002). The human follicle pool decreases cells by inducing aromatase, which catalyzes estrogen significantly with age and the number of follicles is reduced to biosynthesis from C19 steroids (Simpson & Davis 2001). On almost zero at menopause (Broekmans et al.2007). The decline the other hand, estrogen negatively regulates the production of in estrogen at menopause may cause symptoms such as dizziness LH and FSH in the anterior pituitary (de Ziegler et al.2007). and depression, and post-menopausal women are at risk of Thus, a treatment that can regenerate this hormone loop might developing osteoporosis (Broekmans et al.2007). Hormone have advantages compared with standard HRT, as it should replacement is effective for menopause disorders; however, result in tight control of hormone levels in blood by the body’s large-scale clinical studies demonstrated that estrogen treatment natural feedback loops. can be a risk factor for myocardial infarction, stroke, breast One possible therapeutic strategy to achieve this is to cancer, and uterine cancer (Rossouw et al.2002). At present, the develop transplantable estrogen-producing cells in vitro. Journal of Endocrinology (2008) 198, 489–497 DOI: 10.1677/JOE-08-0027 0022–0795/08/0198–489 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/01/2021 09:14:11PM via free access 490 N SAKAI, H TERAMI and others . NF5A1 gene expression modulators To achieve this, nuclear receptor subfamily 5, group A, Materials and Methods member 1 (NF5A1 that was previously known as SF-1 or AD4BP) is a key molecule. NF5A1 is the steroidogenic Cell culture tissue-specific transcription factor that controls the expression Two lines of HEK293, HEK293 FT cells and HEK293 low of the steroidogenic cytochrome P450 genes in endocrine cells, were obtained from Invitrogen and Microbix (Ontario, tissues, such as the adrenal cortex, testis, and ovary (Morohashi et al. 1992). Previously, Crawford et al. (1997) Canada) respectively. HEK293 FT cells were established from using stable ectopic expression of NF5A1 in mouse a fast-growing variant of the 293 cell line that stably expresses embryonic stem (ES) cells, showed that NF5A1 regulates the SV40 large T antigen and these cells were mainly used in differentiation and promotes steroidogenesis in ES cells, our study. HEK293 low cells were used when lower demonstrating that NF5A1 is a dominant regulator of the background of reporter gene expression is required in the steroidogenic cell phenotype. Also, it has been reported that confirmation study, due to its lower transfection efficiency primary long-term-cultured mesenchymal stem cells (MSCs) compared with HEK293 FT cells. HEK293 were maintained infected with an adenovirus-encoding bovine SF-1 (bSF-1) in Iscove’s modified Dulbecco’s medium (IMDM; supple- synthesized de novo multiple steroid hormones (Gondo et al. mented with 10% fetal bovine serum (FBS); Sigma–Aldrich). 2004, Tanaka et al. 2007). This has been confirmed by Yazawa Human bone marrow-derived MSCs (lot number 4F1560; et al. (2006) who established steroidogenic cells by NF5A1 Lonza, Basel, Switzerland) were cultured in MSC medium (MSCGM; Lonza). When the cultures reached subcon- forced expression. Another therapeutic strategy is to develop pharmacological fluence, the cells were harvested with 0 05% trypsin and ! 3 2 interventions to activate endogenous steroidogenesis in the 1 mM EDTA and were replated at 5 10 cells per cm . ovary. Primordial follicles appear to contain only granulosa cells. It is well known that theca cells differentiate from a siRNAs, cDNAs, and reagents population of unspecialized MSCs in the ovarian stroma (Magoffin 2005). Consistent with these findings, theca stem Both siRNA kinome library and validated siRNAs for hit cells, which appear similar to fibroblasts and MSCs, were molecules were purchased from Qiagen. The human cDNA isolated recently (Honda et al.2007). The presence of theca stem library Mammalian gene collection (MGC), consisting of cells/ovarian MSCs in post-menopausal women has not been 6344 clones, was purchased from ATCC. IMAGE clones for shown. However, it is likely that ovarian MSCs share common protein kinase C (PKC)-d, sex-determining region Y (SRY)- features with MSCs in other tissues. Thus, the therapeutic box 15 (SOX15), TEA domain family member 4 (TEAD-4), strategy for pharmacological interventions is to develop drugs KIAA1257, ADAM metallopeptidase, Josephin domain that can stimulate the differentiation of endogenous theca/ containing 1 ( JOSD1), centromere protein (CENP), TATA ovarian MSCs into estrogen-producing cells. box-binding protein-associated factor 5-like RNA poly- Adipocytes, which differentiate from MSCs, are known to merase (TAF5)-like RNA polymerase, and inducible T-cell produce estrogen in an LH/FSH-independent manner. co-stimulator (ICOS) were all purchased from Open Although the expression of estrogen biosynthesis enzymes is BioSystems. Rottelerin (R5648) was purchased from Sigma. regulated by NF5A1 in the ovary, a different transcription factor (NF5A2 previously known as LRH-1) regulates the Plasmids expression of aromatase in adipocytes (Clyne et al.2002). NF5A2 also regulates aromatase expression in the ovary bNF5A1 (bSF-1) cDNA was cloned into both pMXs (kindly (Saxena et al.2007); however, NF5A1 is not expressed in donated by Dr Kitamura, University of Tokyo, Japan) and adipocytes. Thus, we have focused our studies on the pcDNA3.1 vector. The promoter region of human NF5A1 identification of NF5A1 gene expression modulators in MSCs. sequence (5 kbp) was digested (BamHI and NotI) from To date, only limited factors have been identified that human BAC clone 10J18 (Invitrogen) and was ligated with 0 regulate NF5A1 gene expression (Scherrer et al. 2002). 77mer double stranded oligo DNAs (5 -GGC GGC CGC However, as an siRNA kinome library has been shown to be GGG GAC CCC AGG CTG CCG GTC TCC GCC GGC useful to identify new regulators of the target of the interest CCT CCC TGA CCC GCT GTC CCT CCG CAG

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