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Stem cell differentiation into steroidogenic cell lineages by NR5A family
Molecular and Cellular Endocrinology 336 (2011) 123–126
Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Review
Stem cell differentiation into steroidogenic cell lineages by NR5A family Kaoru Miyamoto a,∗ , Takashi Yazawa a , Tetsuya Mizutani a , Yoshitaka Imamichi a , Shin-ya Kawabe a , Masafumi Kanno a , Takahiro Matsumura a , Yunfeng Ju a , Akihiko Umezawa b a b
Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Shimoaizuki 23, Matsuoka, Eiheiji-chou, Fukui 910-1193, Japan National Research Institute for Child Health and Development, Setagaya 2-10-1, Tokyo 157-8535, Japan
a r t i c l e
i n f o
Article history: Received 1 September 2010 Received in revised form 26 November 2010 Accepted 26 November 2010 Keywords: SF-1 Differentiation ES cells LRH-1 Chromatin
a b s t r a c t Transformants of mesenchymal stem cells (MSCs) stably expressing steroidogenic factor-1 (SF-1) undergo differentiation into steroidogenic cell-lineages by stimulation with cyclic-adenosine mono-phosphate (cAMP). Another member of NR5A nuclear orphan receptors, Liver-specific receptor homologue-1 (LRH1), was also able to differentiate MSCs. On the other hand, we found that embryonic stem (ES) cells were hardly induced to differentiate into steroidogenic cell-lineage by the similar treatment. In this study, we developed a novel method to differentiate ES cells into steroidogenic cells. We introduced SF-1 into mouse ES cells at ROSA26 locus under regulation of Tetracycline-off (Tet-off) in order to express SF-1 in the cells at desired period. When SF-1 was induced to express after the ES cells had been differentiated into mesenchymal cell-lineage, steroid hormones were produced from the SF-1 expressing cells. This provides a safer method for supplying sufficient amount of differentiated cells toward future regenerative medicine. © 2010 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of NR5A family on steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differentiation of ES cells into steroidogenic cell lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Steroid hormones are mainly synthesized in the adrenal cortex and the gonads. All steroid hormones are converted from cholesterol via sequential reactions catalyzed by cytochrome P450 steroid hydroxylases and hydroxysteroid dehydrogenases, most of which are under control of NR5A family orphan nuclear receptors (Lala et al., 1992; Morohashi et al., 1992; Saxena et al., 2007). Steroidogenic factor-1 (NR5A1/SF-1/Ad4BP) and liver-specific receptor homolog-1 (NR5A2/LRH-1) belong to the subfamily of nuclear receptors. Since NR5A1 and NR5A2 have quite similar conformational structures (Fayard et al., 2004; Krylova et al., 2005), it is conceivable that they have some overlapping roles on development
Abbreviations: SF-1, steroidogenic factor-1; LRH-1, liver-specific receptor homologue-1; MSCs, mesenchymal stem cells; ES cells, embryonic stem cells; Tetoff, tetracycline-off. ∗ Corresponding author. Tel.: +81 776 61 8315; fax: +81 776 61 8102. E-mail address: [email protected] (K. Miyamoto). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.11.031
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and functions in steroidogenic organs. We recently have shown that SF-1 can induce the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as testicular Leydig cells and adrenocortical cells with the aid of cAMP stimulation (Yazawa et al., 2009, 2006). In this review we describe the similar potential of another member of NR5A family, LRH-1, on the differentiation of MSCs into steroidogenic cells. Upon treatment of MSCs with NR5A family genes we found that all MSCs were converted into steroidogenic cells (Yazawa et al., 2009). On the other hand, it became evident that ES cells were hardly induced to differentiate into steroidogenic cells by the similar treatment, in spite of the original report by Crawford et al. (1997). In this review, we also describe successful differentiation of ES cells into steroidogenic cell lineage by a novel method. 2. Roles of NR5A family on steroidogenesis Steroidogenic factor-1 (SF-1; also known as Ad4BP) and liverspecific receptor homologue-1 (LRH-1) belong to the NR5A
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K. Miyamoto et al. / Molecular and Cellular Endocrinology 336 (2011) 123–126
Fig. 1. Induced differentiation of mesenchymal stem cells into steroidogenic cells. Various MSCs from different sources could be induced into steroidogenic cells, although steroidogenic profiles were different from each other. Both NR5A family genes, SF-1 and LRH-1, gave similar steroidogenic profiles in each MSCs.
human StAR gene by electrophoretic mobility sift (EMSA) and chromatin immunoprecipitation (ChIP) assays. A luciferase reporter assay revealed that the region worked as a strong enhancer to exert the maximal transcription of StAR gene. Chromosome conformation capture analysis revealed that upstream of StAR gene formed a chromatin loop both in the differentiated MSCs and KGN cells which express SF-1 endogenously. In addition, SF-1 knockdown resulted in disruption of the chromatin loop formation in KGN cells. These results indicate that the novel enhancer participates in the human StAR gene activation through SF-1 dependent alterations of chromatin structure including histone eviction and chromatin loop formation (Mizutani et al., 2010; Owen-Hughes and Workman, 1996; Vicent et al., 2004) (Fig. 2). As for CYP17 gene, promoter analysis, EMSA, and chromatin immunoprecipitation assay using SF-1 or LRH-1-transduced hMSCs indicated that three NR5A binding sites were responsible for CYP17 transactivation. The CYP17 promoter region was highly methylated in hMSCs, whereas it was demethylated by the introduction of LRH1 and cAMP treatment (Yazawa et al., 2009). These results indicate that both SF-1 and LRH-1 represent key regulators of the differentiation of MSCs into steroidogenic cell lineage through altering chromosomal structures.
3. Differentiation of ES cells into steroidogenic cell lineage subfamily of nuclear receptors (Fayard et al., 2004). They function as monomers to regulate genes by binding to similar response elements. SF-1 is expressed in the adrenal cortex, testicular Leydig and Sertoli cells, ovarian theca and granulosa cells, pituitary gonadotropes, hypothalamus, and spleen (Parker and Schimmer, 1997). It regulates the cell specific expression of a variety of different genes involved in steroidogenesis, including a number of steroid hydroxylases (Lala et al., 1992; Morohashi et al., 1992). LRH-1, however, is highly expressed in tissues of endodermal origin, such as liver and intestine (Fayard et al., 2004; Lee and Moore, 2008). It is essential for Oct4 (an essential gene for the maintenance of inner cell mass and pluripotency of embryonic stem cells) expression at the epiblast stage, and its disruption causes early embryonic death (Gu et al., 2005). In the adult, LRH-1 functions in the control of cholesterol and bile acid homeostasis by regulating the transcription of a number of genes including CYP7A1 and CYP8A1 (Fayard et al., 2004). LRH-1 has also been reported to be involved in steroidogenesis in the gonad and intestine (Volle et al., 2007). LRH-1 and SF-1 have similar actions on rat granulose cell steroidogenesis (Boerboom et al., 2000; Duggavathi et al., 2008; Volle et al., 2007), and impaired progesterone production in LRH-1 hetero-knock-out mice leads to a reduction of female reproductive functions (Labelle-Dumais et al., 2007). MSCs have been shown to be differentiated into various cell types such as adipocytes, chontrocytes and muscle cells. With the aid of cAMP, SF-1 can induce the differentiation of bone marrowderived MSCs into steroidogenic cells such as testicular Leydig cells and adrenocortical cells (Yazawa et al., 2006). Similarly introduction of LRH-1 and cAMP treatment resulted in the differentiation of human MSCs (hMSCs) into steroidogenic cells (Yazawa et al., 2009) (Fig. 1). The MSCs differentiation seems to involve chromatin alterations (Adams and Workman, 1993; Barrera and Ren, 2006; Owen-Hughes and Workman, 1996); induced by the nuclear receptor family members (Li et al., 2007; Owen-Hughes and Workman, 1996). Introduction of SF-1 into MSCs induced expression of genes related to steroidogenesis including steroidogenic acute regulatory protein (StAR) (Mizutani et al., 2010; Yazawa et al., 2009, 2006). We found that transcriptional regulation of StAR gene was dependent on chromosomal conformation in the genome. We identified novel SF-1 binding sites between 3,000 and 3,400 bp upstream of
Originally Crawford et al. (1997) reported that stable expression of SF-1 has been shown to direct embryonic stem cells toward the steroidogenic cell lineage. However, the steroidogenic capacity of the cells was very limited since a membrane-permeable substrate, 20˛-hydroxycholesterol, was necessary to produce progesterone, which was the only steroid produced from the cells. Following the report, MSCs have been shown to be successfully converted into steroidogenic cell lineage by introduction of NR5A family, while it became evident that ES cells were hardly converted into steroidogenic cell lineage by similar treatment. It is quite interesting that Guo et al. identified NR5a1 and NR5A2 as potent inducers of ground state pluripotency (Guo and Smith, 2010). In addition, Oct-3/4 can be replaced by LRH-1 for the reprogramming of murine somatic cells into induced pluripotent stem (iPS) cells (Heng et al., 2010). SF-1 and LRH-1 have similar potential for the regulation of Oct-3/4 expression (Gu et al., 2005), and therefore the same potential for induction of somatic cells into iPS cells (Heng et al., 2010). Niwa et al. (2000) demonstrated that quantitative expression of Oct-3/4 defines the fate of ES cells. A less than twofold increase in Oct-3/4 expression causes differentiation of ES cells into primitive endoderm and mesoderm, whereas repression of Oct-3/4 expression induces loss of pluripotency and causes dedifferentiation of cells into the trophectoderm. It has been shown that DAX-1, a common transcriptional inhibitor of Oct-3/4, SF-1 and LRH-1 are also important for the pluripotency and survival of ES cells (Khalfallah et al., 2009; Sun et al., 2009; Yu et al., 1998). DAX1 expression is detectable in ES cells and its expression is reduced upon differentiation of the cells into each germ layer. DAX-1 knockdown induces loss of pluripotency even under culture conditions for maintaining the undifferentiated state (Khalfallah et al., 2009; Sun et al., 2009), whereas complete deletion of DAX-1 by gene targeting results in cell death (Yu et al., 1998). These facts strongly suggest that regulated and coordinated expression of NR5A genes is essential for the pluripotency and survival of ES cells. These properties of the NR5A family are likely to cause difficulties in the induction of steroidogenic cells by NR5A members directly from ES cells. Considering these possibility, we tried to express SF-1 after differentiation of ES cells into mesenchymal stem cells. Takashima et al. (2007) showed that the earliest wave of MSC in the embryonic trunk is generated from Sox1 positive neuroep-
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Fig. 2. Chromosomal conformational changes are required for the activation of steroidogenesis related genes during differentiation of MSCs by expression of SF-1. DNA loop was formed upon SF-1 binding to the upstream of human StAR gene, resulting in the activation of the gene during the differentiation.
ithelium but not from mesoderm. In order to specify the culture condition for ES cell differentiation to multipotent MSCs, they compared two methods that have been used for inducing mesenchymal cell lineages from ES cells. The first is culture on collagen IV-coated dish under serum-containing medium, which supports generation of mesodermal cells that give rise to osteocytes, chondrocytes and myocytes (Sakurai et al., 2006), and the other involves the treatment of ES cells with pulse exposures to retinoic acid (RA) from day 2 to day 5 of culture on collagen IV-coated dish. Only the latter could give rise to MSCs, and the observation in ES cell culture was confirmed in E9.5 embryos. According to the above observation, inducible expression of SF1 by ROSA-TET system was applied to establish pathway for ES cell differentiation into steroidogenic cells (Yazawa et al., in this issue). For the purpose, ES cell line, EBRTcH3, carrying a tetracy-
cline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus (Masui et al., 2005) was used. SF-1 cDNA along with Venus fluorescent gene was integrated to the ROSA-TET locus by a knock-in method. Withdrawal of tetracycline (Tc) from the culture medium resulted in the induction of Venus fluorescence in virtually all cells within 48 h, whereas no fluorescence was detected in cells cultured in the presence of Tc. SF-1 mRNA and proteins were also induced in the absence of Tc, although ES cells kept the undifferentiated states and never expressed any steroidogenic marker genes including Cyp11a1 in the presence of LIF. In addition, ES cells stopped proliferation and died after several days in the absence LIF and Tc. These results were in agreement with previous observations that steroidogenic cells could not be induced directly from ES cells (Yazawa et al., 2006). To avoid such troubles, expression of SF-1 was induced after differentiation of the ES cells into
Fig. 3. Differentiation of ES cells into steroidogenic cells. Direct expression of SF-1 in ES cells failed to differentiate the cells into steroidogenic cells. After differentiation of ES cells into mesenchymal stem cells, expression of SF-1 led to the cells into steroidogenic cell-lineage mainly expressing glucocorticoids.
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MSCs. For differentiation of ES cells into multipotent MSCs, they were cultured on collagen IV-coated dish and treated with pulse exposures to retinoic acid (RA). In contrast to undifferentiated ES cells, the induced MSCs were able to survive after SF-1 expression in the absence of LIF. As expected, expression of SF-1 in the induced MSCs resulted in the expression of various steroidogenesis related genes, such as Cyp11a1, Hsd3b1, Cyp17, Cyp21 Cyp11b1 and Acthr. The gene expression pattern was quite similar to that of adrenocortical cells, especially fasciculata cells. Consistent with the gene expression profile, corticosterone was the main secreted steroid hormone from these cells, since Cyp17 expression is barely detectable in adult murine adrenal gland. These results indicate that ES cells could also be differentiated into steroidogenic cells by SF-1 via multipotent MSCs (Fig. 3). This approach might also provide the opportunity, as the usage of MSCs, leading to the development of the cell and gene therapy for the deficiency of steroidogenesis. 4. Concluding remarks In this review, roles of NR5A family proteins for stem cell differentiation into steroidogenic cells are described. In addition to SF-1, LRH-1 is also effective in differentiation of stem cells into sterodogenic cells. In contrast to SF-1, expression of LHR-1 is widely observed in tissues and cells. It suggests that induction of endogenous LRH-1 gene expression, which will lead stem cells into steroidogenic cells, may be easier than that of SF-1. This may provide a novel and milder way for generating steroidogenic cells from stem cells without integrating exogenous genes. This review also describes differentiation of ES cells into steroidogenic cells by a novel method. Targeted knock-in of NR5A family gene to ES cells and induced expression of the gene will provide a safer procedure to generate sufficient amount of steroidogenic cells for future regenerative therapy on the deficiency of steroidogenesis. References Adams, C.C., Workman, J.L., 1993. Nucleosome displacement in transcription. Cell 72, 305–308. Barrera, L.O., Ren, B., 2006. The transcriptional regulatory code of eukaryotic cells – insights from genome-wide analysis of chromatin organization and transcription factor binding. Curr. Opin. Cell Biol. 18, 291–298. Boerboom, D., Pilon, N., Behdjani, R., Silversides, D.W., Sirois, J., 2000. Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process. Endocrinology 141, 4647–4656. Crawford, P.A., Sadovsky, Y., Milbrandt, J., 1997. Nuclear receptor steroidogenic factor 1 directs embryonic stem cells toward the steroidogenic lineage. Mol. Cell. Biol. 17, 3997–4006. Duggavathi, R., Volle, D.H., Mataki, C., Antal, M.C., Messaddeq, N., Auwerx, J., Murphy, B.D., Schoonjans, K., 2008. Liver receptor homolog 1 is essential for ovulation. Genes Dev. 22, 1871–1876. Fayard, E., Auwerx, J., Schoonjans, K., 2004. LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends Cell Biol. 14, 250–260. Gu, P., Goodwin, B., Chung, A.C.K., Xu, X., Wheeler, D.A., Price, R.R., Galardi, C., Peng, L., Latour, A.M., Koller, B.H., et al., 2005. Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development. Mol. Cell. Biol. 25, 3492–3505.
Guo, G., Smith, A., 2010. A genome-wide screen in EpiSCs identifies NR5A nuclear receptors as potent inducers of ground state pluripotency. Development 137, 3185–3192. Heng, J.C., Feng, B., Han, J., Jiang, J., Kraus, P., Ng, J.H., Orlov, Y.L., Huss, M., Yang, L., Lufkin, T., et al., 2010. The nuclear receptor NR5A2 can replace Oct4 in the reprogramming of murine somatic cells to pluripotent cells. Cell Stem Cell 6, 167–174. Khalfallah, O., Rouleau, M., Barbry, P., Bardoni, B., Lalli, E., 2009. DAX-1 knockdown in mouse embryonic stem cells induces loss of pluripotency and multilineage differentiation. Stem Cells 27, 1529–1537. Krylova, I.N., Sablin, E.P., Moore, J., Xu, R.X., Waitt, G.M., MacKay, J.A., Juzumiene, D., Bynum, J.M., Madauss, K., Montana, V., et al., 2005. Structural analyses reveal phosphatidyl inositols as ligands for the NR5 orphan receptors SF-1 and LRH-1. Cell 120, 343–355. Labelle-Dumais, C., Pare, J.-F., Belanger, L., Farookhi, R., Dufort, D., 2007. Impaired progesterone production in NR5A2+/− mice leads to a reduction in female reproductive function. Biol. Reprod. 77, 217–225. Lala, D.S., Rice, D.A., Parker, K.L., 1992. Steroidogenic factor I, a key regulator of steroidogenic enzyme expression, is the mouse homolog of fushi tarazu-factor I. Mol. Endocrinol. 6, 1249–1258. Lee, Y.K., Moore, D.D., 2008. Liver receptor homolog-1, an emerging metabolic modulator. Front. Biosci. 13, 5950–5958. Li, B., Carey, M., Workman, J.L., 2007. The role of chromatin during transcription. Cell 128, 707–719. Masui, S., Shimosato, D., Toyooka, Y., Yagi, R., Takahashi, K., Niwa, H., 2005. An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit. Nucleic Acids Res. 33, e43. Mizutani, T., Yazawa, T., Ju, Y., Imamichi, Y., Uesaka, M., Inaoka, Y., Matsuura, K., Kamiki, Y., Oki, M., Umezawa, A., et al., 2010. Identification of a novel distal control region upstream of the human steroidogenic acute regulatory protein (StAR) gene that participates in SF-1-dependent chromatin architecture. J. Biol. Chem. 285, 28240–28251. Morohashi, K., Honda, S., Inomata, Y., Handa, H., Omura, T., 1992. A common transacting factor, Ad4-binding protein, to the promoters of steroidogenic P-450S. J. Biol. Chem. 267, 17913–17919. Niwa, H., Miyazaki, J., Smith, A.G., 2000. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376. Owen-Hughes, T., Workman, J.L., 1996. Remodeling the chromatin structure of a nucleosome array by transcription factor-targeted trans-displacement of histones. EMBO J. 15, 4702–4712. Parker, K.L., Schimmer, B.P., 1997. Steroidogenic factor 1: a key determinant of endocrine development and function. Endocr. Rev. 18, 361–377. Sakurai, H., Era, T., Jakt, L.M., Okada, M., Nakai, S., Nishikawa, S., Nishikawa, S.-I., 2006. In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility. Stem Cells 24, 575–586. Saxena, D., Escamilla-Hernandez, R., Little-Ihrig, L., Zeleznik, A.J., 2007. Liver receptor homolog-1 and steroidogenic factor-1 have similar actions on rat granulosa cell steroidogenesis. Endocrinology 148, 726–734. Sun, C., Nakatake, Y., Akagi, T., Ura, H., Matsuda, T., Nishiyama, A., Koide, H., Ko, M.S., Niwa, H., Yokota, T., 2009. Dax1 binds to Oct3/4 and inhibits its transcriptional activity in embryonic stem cells. Mol. Cell. Biol. 29, 4574–4583. Takashima, Y., Era, T., Nakao, K., Kondo, S., Kasuga, M., Smith, A.G., Nishikawa, S.-I., 2007. Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 129, 1377–1388. Vicent, G.P., Nacht, A.S., Smith, C.L., Peterson, C.L., Dimitrov, S., Beato, M., 2004. DNA instructed displacement of histones H2A and H2B at an inducible promoter. Mol. Cell 16, 439–452. Volle, D.H., Duggavathi, R., Magnier, B.C., Houten, S.M., Cummins, C.L., Lobaccaro J-MA, Verhoeven, G., Schoonjans, K., Auwerx, J., 2007. The small heterodimer partner is a gonadal gatekeeper of sexual maturation in male mice. Genes Dev. 21, 303–315. Yazawa, T., Inanoka, Y., Mizutani, T., Kuribayashi, M., Umezawa, A., Miyamoto, K., 2009. Liver receptor homolog-1 regulates the transcription of steroidogenic enzymes and induces the differentiation of mesenchymal stem cells into steroidogenic cells. Endocrinology 150, 3885–3893. Yazawa, T., Mizutani, T., Yamada, K., Kawata, H., Sekiguchi, T., Yoshino, M., Kajitani, T., Shou, Z., Umezawa, A., Miyamoto, K., 2006. Differentiation of adult stem cells derived from bone marrow stroma into Leydig or adrenocortical cells. Endocrinology 147, 4104–4111. Yu, R.N., Achermann, J.C., Ito, M., Jameson, J.L., 1998. The role of DAX-1 in reproduction. Trends Endocrinol. Metab. 9, 169–175.
Contents lists available at ScienceDirect
Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce
Review
Stem cell differentiation into steroidogenic cell lineages by NR5A family Kaoru Miyamoto a,∗ , Takashi Yazawa a , Tetsuya Mizutani a , Yoshitaka Imamichi a , Shin-ya Kawabe a , Masafumi Kanno a , Takahiro Matsumura a , Yunfeng Ju a , Akihiko Umezawa b a b
Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Shimoaizuki 23, Matsuoka, Eiheiji-chou, Fukui 910-1193, Japan National Research Institute for Child Health and Development, Setagaya 2-10-1, Tokyo 157-8535, Japan
a r t i c l e
i n f o
Article history: Received 1 September 2010 Received in revised form 26 November 2010 Accepted 26 November 2010 Keywords: SF-1 Differentiation ES cells LRH-1 Chromatin
a b s t r a c t Transformants of mesenchymal stem cells (MSCs) stably expressing steroidogenic factor-1 (SF-1) undergo differentiation into steroidogenic cell-lineages by stimulation with cyclic-adenosine mono-phosphate (cAMP). Another member of NR5A nuclear orphan receptors, Liver-specific receptor homologue-1 (LRH1), was also able to differentiate MSCs. On the other hand, we found that embryonic stem (ES) cells were hardly induced to differentiate into steroidogenic cell-lineage by the similar treatment. In this study, we developed a novel method to differentiate ES cells into steroidogenic cells. We introduced SF-1 into mouse ES cells at ROSA26 locus under regulation of Tetracycline-off (Tet-off) in order to express SF-1 in the cells at desired period. When SF-1 was induced to express after the ES cells had been differentiated into mesenchymal cell-lineage, steroid hormones were produced from the SF-1 expressing cells. This provides a safer method for supplying sufficient amount of differentiated cells toward future regenerative medicine. © 2010 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3. 4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roles of NR5A family on steroidogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Differentiation of ES cells into steroidogenic cell lineage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction Steroid hormones are mainly synthesized in the adrenal cortex and the gonads. All steroid hormones are converted from cholesterol via sequential reactions catalyzed by cytochrome P450 steroid hydroxylases and hydroxysteroid dehydrogenases, most of which are under control of NR5A family orphan nuclear receptors (Lala et al., 1992; Morohashi et al., 1992; Saxena et al., 2007). Steroidogenic factor-1 (NR5A1/SF-1/Ad4BP) and liver-specific receptor homolog-1 (NR5A2/LRH-1) belong to the subfamily of nuclear receptors. Since NR5A1 and NR5A2 have quite similar conformational structures (Fayard et al., 2004; Krylova et al., 2005), it is conceivable that they have some overlapping roles on development
Abbreviations: SF-1, steroidogenic factor-1; LRH-1, liver-specific receptor homologue-1; MSCs, mesenchymal stem cells; ES cells, embryonic stem cells; Tetoff, tetracycline-off. ∗ Corresponding author. Tel.: +81 776 61 8315; fax: +81 776 61 8102. E-mail address: [email protected] (K. Miyamoto). 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.11.031
123 123 124 126 126
and functions in steroidogenic organs. We recently have shown that SF-1 can induce the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as testicular Leydig cells and adrenocortical cells with the aid of cAMP stimulation (Yazawa et al., 2009, 2006). In this review we describe the similar potential of another member of NR5A family, LRH-1, on the differentiation of MSCs into steroidogenic cells. Upon treatment of MSCs with NR5A family genes we found that all MSCs were converted into steroidogenic cells (Yazawa et al., 2009). On the other hand, it became evident that ES cells were hardly induced to differentiate into steroidogenic cells by the similar treatment, in spite of the original report by Crawford et al. (1997). In this review, we also describe successful differentiation of ES cells into steroidogenic cell lineage by a novel method. 2. Roles of NR5A family on steroidogenesis Steroidogenic factor-1 (SF-1; also known as Ad4BP) and liverspecific receptor homologue-1 (LRH-1) belong to the NR5A
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Fig. 1. Induced differentiation of mesenchymal stem cells into steroidogenic cells. Various MSCs from different sources could be induced into steroidogenic cells, although steroidogenic profiles were different from each other. Both NR5A family genes, SF-1 and LRH-1, gave similar steroidogenic profiles in each MSCs.
human StAR gene by electrophoretic mobility sift (EMSA) and chromatin immunoprecipitation (ChIP) assays. A luciferase reporter assay revealed that the region worked as a strong enhancer to exert the maximal transcription of StAR gene. Chromosome conformation capture analysis revealed that upstream of StAR gene formed a chromatin loop both in the differentiated MSCs and KGN cells which express SF-1 endogenously. In addition, SF-1 knockdown resulted in disruption of the chromatin loop formation in KGN cells. These results indicate that the novel enhancer participates in the human StAR gene activation through SF-1 dependent alterations of chromatin structure including histone eviction and chromatin loop formation (Mizutani et al., 2010; Owen-Hughes and Workman, 1996; Vicent et al., 2004) (Fig. 2). As for CYP17 gene, promoter analysis, EMSA, and chromatin immunoprecipitation assay using SF-1 or LRH-1-transduced hMSCs indicated that three NR5A binding sites were responsible for CYP17 transactivation. The CYP17 promoter region was highly methylated in hMSCs, whereas it was demethylated by the introduction of LRH1 and cAMP treatment (Yazawa et al., 2009). These results indicate that both SF-1 and LRH-1 represent key regulators of the differentiation of MSCs into steroidogenic cell lineage through altering chromosomal structures.
3. Differentiation of ES cells into steroidogenic cell lineage subfamily of nuclear receptors (Fayard et al., 2004). They function as monomers to regulate genes by binding to similar response elements. SF-1 is expressed in the adrenal cortex, testicular Leydig and Sertoli cells, ovarian theca and granulosa cells, pituitary gonadotropes, hypothalamus, and spleen (Parker and Schimmer, 1997). It regulates the cell specific expression of a variety of different genes involved in steroidogenesis, including a number of steroid hydroxylases (Lala et al., 1992; Morohashi et al., 1992). LRH-1, however, is highly expressed in tissues of endodermal origin, such as liver and intestine (Fayard et al., 2004; Lee and Moore, 2008). It is essential for Oct4 (an essential gene for the maintenance of inner cell mass and pluripotency of embryonic stem cells) expression at the epiblast stage, and its disruption causes early embryonic death (Gu et al., 2005). In the adult, LRH-1 functions in the control of cholesterol and bile acid homeostasis by regulating the transcription of a number of genes including CYP7A1 and CYP8A1 (Fayard et al., 2004). LRH-1 has also been reported to be involved in steroidogenesis in the gonad and intestine (Volle et al., 2007). LRH-1 and SF-1 have similar actions on rat granulose cell steroidogenesis (Boerboom et al., 2000; Duggavathi et al., 2008; Volle et al., 2007), and impaired progesterone production in LRH-1 hetero-knock-out mice leads to a reduction of female reproductive functions (Labelle-Dumais et al., 2007). MSCs have been shown to be differentiated into various cell types such as adipocytes, chontrocytes and muscle cells. With the aid of cAMP, SF-1 can induce the differentiation of bone marrowderived MSCs into steroidogenic cells such as testicular Leydig cells and adrenocortical cells (Yazawa et al., 2006). Similarly introduction of LRH-1 and cAMP treatment resulted in the differentiation of human MSCs (hMSCs) into steroidogenic cells (Yazawa et al., 2009) (Fig. 1). The MSCs differentiation seems to involve chromatin alterations (Adams and Workman, 1993; Barrera and Ren, 2006; Owen-Hughes and Workman, 1996); induced by the nuclear receptor family members (Li et al., 2007; Owen-Hughes and Workman, 1996). Introduction of SF-1 into MSCs induced expression of genes related to steroidogenesis including steroidogenic acute regulatory protein (StAR) (Mizutani et al., 2010; Yazawa et al., 2009, 2006). We found that transcriptional regulation of StAR gene was dependent on chromosomal conformation in the genome. We identified novel SF-1 binding sites between 3,000 and 3,400 bp upstream of
Originally Crawford et al. (1997) reported that stable expression of SF-1 has been shown to direct embryonic stem cells toward the steroidogenic cell lineage. However, the steroidogenic capacity of the cells was very limited since a membrane-permeable substrate, 20˛-hydroxycholesterol, was necessary to produce progesterone, which was the only steroid produced from the cells. Following the report, MSCs have been shown to be successfully converted into steroidogenic cell lineage by introduction of NR5A family, while it became evident that ES cells were hardly converted into steroidogenic cell lineage by similar treatment. It is quite interesting that Guo et al. identified NR5a1 and NR5A2 as potent inducers of ground state pluripotency (Guo and Smith, 2010). In addition, Oct-3/4 can be replaced by LRH-1 for the reprogramming of murine somatic cells into induced pluripotent stem (iPS) cells (Heng et al., 2010). SF-1 and LRH-1 have similar potential for the regulation of Oct-3/4 expression (Gu et al., 2005), and therefore the same potential for induction of somatic cells into iPS cells (Heng et al., 2010). Niwa et al. (2000) demonstrated that quantitative expression of Oct-3/4 defines the fate of ES cells. A less than twofold increase in Oct-3/4 expression causes differentiation of ES cells into primitive endoderm and mesoderm, whereas repression of Oct-3/4 expression induces loss of pluripotency and causes dedifferentiation of cells into the trophectoderm. It has been shown that DAX-1, a common transcriptional inhibitor of Oct-3/4, SF-1 and LRH-1 are also important for the pluripotency and survival of ES cells (Khalfallah et al., 2009; Sun et al., 2009; Yu et al., 1998). DAX1 expression is detectable in ES cells and its expression is reduced upon differentiation of the cells into each germ layer. DAX-1 knockdown induces loss of pluripotency even under culture conditions for maintaining the undifferentiated state (Khalfallah et al., 2009; Sun et al., 2009), whereas complete deletion of DAX-1 by gene targeting results in cell death (Yu et al., 1998). These facts strongly suggest that regulated and coordinated expression of NR5A genes is essential for the pluripotency and survival of ES cells. These properties of the NR5A family are likely to cause difficulties in the induction of steroidogenic cells by NR5A members directly from ES cells. Considering these possibility, we tried to express SF-1 after differentiation of ES cells into mesenchymal stem cells. Takashima et al. (2007) showed that the earliest wave of MSC in the embryonic trunk is generated from Sox1 positive neuroep-
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Fig. 2. Chromosomal conformational changes are required for the activation of steroidogenesis related genes during differentiation of MSCs by expression of SF-1. DNA loop was formed upon SF-1 binding to the upstream of human StAR gene, resulting in the activation of the gene during the differentiation.
ithelium but not from mesoderm. In order to specify the culture condition for ES cell differentiation to multipotent MSCs, they compared two methods that have been used for inducing mesenchymal cell lineages from ES cells. The first is culture on collagen IV-coated dish under serum-containing medium, which supports generation of mesodermal cells that give rise to osteocytes, chondrocytes and myocytes (Sakurai et al., 2006), and the other involves the treatment of ES cells with pulse exposures to retinoic acid (RA) from day 2 to day 5 of culture on collagen IV-coated dish. Only the latter could give rise to MSCs, and the observation in ES cell culture was confirmed in E9.5 embryos. According to the above observation, inducible expression of SF1 by ROSA-TET system was applied to establish pathway for ES cell differentiation into steroidogenic cells (Yazawa et al., in this issue). For the purpose, ES cell line, EBRTcH3, carrying a tetracy-
cline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus (Masui et al., 2005) was used. SF-1 cDNA along with Venus fluorescent gene was integrated to the ROSA-TET locus by a knock-in method. Withdrawal of tetracycline (Tc) from the culture medium resulted in the induction of Venus fluorescence in virtually all cells within 48 h, whereas no fluorescence was detected in cells cultured in the presence of Tc. SF-1 mRNA and proteins were also induced in the absence of Tc, although ES cells kept the undifferentiated states and never expressed any steroidogenic marker genes including Cyp11a1 in the presence of LIF. In addition, ES cells stopped proliferation and died after several days in the absence LIF and Tc. These results were in agreement with previous observations that steroidogenic cells could not be induced directly from ES cells (Yazawa et al., 2006). To avoid such troubles, expression of SF-1 was induced after differentiation of the ES cells into
Fig. 3. Differentiation of ES cells into steroidogenic cells. Direct expression of SF-1 in ES cells failed to differentiate the cells into steroidogenic cells. After differentiation of ES cells into mesenchymal stem cells, expression of SF-1 led to the cells into steroidogenic cell-lineage mainly expressing glucocorticoids.
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MSCs. For differentiation of ES cells into multipotent MSCs, they were cultured on collagen IV-coated dish and treated with pulse exposures to retinoic acid (RA). In contrast to undifferentiated ES cells, the induced MSCs were able to survive after SF-1 expression in the absence of LIF. As expected, expression of SF-1 in the induced MSCs resulted in the expression of various steroidogenesis related genes, such as Cyp11a1, Hsd3b1, Cyp17, Cyp21 Cyp11b1 and Acthr. The gene expression pattern was quite similar to that of adrenocortical cells, especially fasciculata cells. Consistent with the gene expression profile, corticosterone was the main secreted steroid hormone from these cells, since Cyp17 expression is barely detectable in adult murine adrenal gland. These results indicate that ES cells could also be differentiated into steroidogenic cells by SF-1 via multipotent MSCs (Fig. 3). This approach might also provide the opportunity, as the usage of MSCs, leading to the development of the cell and gene therapy for the deficiency of steroidogenesis. 4. Concluding remarks In this review, roles of NR5A family proteins for stem cell differentiation into steroidogenic cells are described. In addition to SF-1, LRH-1 is also effective in differentiation of stem cells into sterodogenic cells. In contrast to SF-1, expression of LHR-1 is widely observed in tissues and cells. It suggests that induction of endogenous LRH-1 gene expression, which will lead stem cells into steroidogenic cells, may be easier than that of SF-1. This may provide a novel and milder way for generating steroidogenic cells from stem cells without integrating exogenous genes. This review also describes differentiation of ES cells into steroidogenic cells by a novel method. Targeted knock-in of NR5A family gene to ES cells and induced expression of the gene will provide a safer procedure to generate sufficient amount of steroidogenic cells for future regenerative therapy on the deficiency of steroidogenesis. References Adams, C.C., Workman, J.L., 1993. Nucleosome displacement in transcription. Cell 72, 305–308. Barrera, L.O., Ren, B., 2006. The transcriptional regulatory code of eukaryotic cells – insights from genome-wide analysis of chromatin organization and transcription factor binding. Curr. Opin. Cell Biol. 18, 291–298. Boerboom, D., Pilon, N., Behdjani, R., Silversides, D.W., Sirois, J., 2000. Expression and regulation of transcripts encoding two members of the NR5A nuclear receptor subfamily of orphan nuclear receptors, steroidogenic factor-1 and NR5A2, in equine ovarian cells during the ovulatory process. Endocrinology 141, 4647–4656. Crawford, P.A., Sadovsky, Y., Milbrandt, J., 1997. Nuclear receptor steroidogenic factor 1 directs embryonic stem cells toward the steroidogenic lineage. Mol. Cell. Biol. 17, 3997–4006. Duggavathi, R., Volle, D.H., Mataki, C., Antal, M.C., Messaddeq, N., Auwerx, J., Murphy, B.D., Schoonjans, K., 2008. Liver receptor homolog 1 is essential for ovulation. Genes Dev. 22, 1871–1876. Fayard, E., Auwerx, J., Schoonjans, K., 2004. LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends Cell Biol. 14, 250–260. Gu, P., Goodwin, B., Chung, A.C.K., Xu, X., Wheeler, D.A., Price, R.R., Galardi, C., Peng, L., Latour, A.M., Koller, B.H., et al., 2005. Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development. Mol. Cell. Biol. 25, 3492–3505.
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