Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Takashi Baba Last modified date:2023.11.29

Associate Professor / Sex differences (Molecular Biology) / Department of Molecular Biology / Faculty of Medical Sciences


Papers
1. Fumiya Takahashi, Takashi Baba, Antonius Christianto, Shogo Yanai, Keisuke Ishiwata, Kazuhiko Nakabayashi, Kenichiro Hata, Tomohiro Ishii, Tomonobu Hasegawa, Man Ho Choi, Ken-ichirou Morohashi, Development of Sexual Dimorphism Through the Adrenal Cortex, Caused by Androgen-Induced Global Gene Suppression, Sneak Peek, 10.2139/ssrn.4425878, 2023.04.
2. Thomas Fleming, Yukiko Kikuchi, Mikoto Nakajo, Masaya Tachizawa, Tomoaki Inazumi, Soken Tsuchiya, Yukihiko Sugimoto, Daisuke Saito, Mikita Suyama, Yasuyuki Ohkawa, Takashi Baba, Ken-ichirou Morohashi, Kataaki Okubo, Prostaglandin E2 receptor Ptger4b regulates female-specific peptidergic neurons and female sexual receptivity in medaka, Communications Biology, 10.1038/s42003-022-04195-x, 5, 1, 1215, 2022.11, Abstract

In vertebrates, female receptivity to male courtship is highly dependent on ovarian secretion of estrogens and prostaglandins. We recently identified female-specific neurons in the medaka (Oryzias latipes) preoptic area that express Npba, a neuropeptide mediating female sexual receptivity, in response to ovarian estrogens. Here we show by transcriptomic analysis that these neurons express a multitude of neuropeptides, in addition to Npba, in an ovarian-dependent manner, and we thus termed them female-specific, sex steroid-responsive peptidergic (FeSP) neurons. Our results further revealed that FeSP neurons express a prostaglandin E2 receptor gene, ptger4b, in an ovarian estrogen-dependent manner. Behavioral and physiological examination of ptger4b-deficient female medaka found that they exhibit increased sexual receptivity while retaining normal ovarian function and that their FeSP neurons have reduced firing activity and impaired neuropeptide release. Collectively, this work provides evidence that prostaglandin E2/Ptger4b signaling mediates the estrogenic regulation of FeSP neuron activity and female sexual receptivity..
3. Yanai S, Baba T, Inui K, Miyabayashi K, Han S, Inoue M, Takahashi F, Kanai Y, Ohkawa Y, Choi MH, Morohashi KI., Gene expression and functional abnormalities in XX/Sry Leydig cells, SCIENTIFIC REPORTS, 10.1038/s41598-020-80741-z, 11, 1, 2021.01.
4. Soyun Han, Takashi Baba, Shogo Yanai, Dong Jun Byun, Ken-ichirou Morohashi, Jae-Hong Kim, Man Ho Choi, GC-MS-based metabolic signatures reveal comparative steroidogenic pathways between fetal and adult mouse testes, ANDROLOGY, 10.1111/andr.12893, 9, 1, 400-406, 2021.01, Background Previous studies on gonadal steroidogenesis have not compared metabolic pathways between fetal and adult mouse testes to date. Objectives To evaluate comparative metabolic signatures of testicular steroids between fetus and adult mice using gas chromatography-mass spectrometry (GC-MS)-based steroid profiling. Materials and methods GC-MS with molecular-specific scan modes was optimized for selective and sensitive detection of 23 androgens, 7 estrogens, 14 progestogens, and 13 corticoids from mouse testes with a quantification limit of 0.1-5.0 ng/mL and reproducibility (coefficient of variation: 0.3%-19.9%). Based on 26 steroids quantitatively detected in testes, comparative steroid signatures were analyzed for mouse testes of 8 fetuses on embryonic day 16.5 and 8 adults on postnatal days 56-60. Results In contrast to large amounts of steroids in adult testes (P
5. Ken-ichirou Morohashi, Miki Inoue, Takashi Baba, Coordination of Multiple Cellular Processes by NR5A1/Nr5a1, ENDOCRINOLOGY AND METABOLISM, 10.3803/EnM.2020.402, 35, 4, 756-764, 2020.12, The agenesis of the gonads and adrenal gland in revealed by knockout mouse studies strongly suggested a crucial role for Nr5a1 (SF-1 or Ad4BP) in organ development. In relation to these striking phenotypes, NR5A1/Nr5a1 has the potential to reprogram cells to steroidogenic cells, endow pluripotency, and regulate cell proliferation. However, due to limited knowledge regarding NR5A1 target genes, the mechanism by which NR5A1/Nr5a1 regulates these fundamental processes has remained unknown. Recently, newly-established technologies have enabled the identification of NR5A1 target genes related to multiple metabolic processes, as well as the aforementioned biological processes. Considering that active cellular processes are expected to be accompanied by active metabolism, NR5A1 may act as a key factor for processes such as cell differentiation, proliferation, and survival by coordinating these processes with cellular metabolism. A complete and definite picture of the cellular processes coordinated by NR5A1/Nr5a1 could be depicted by accumulating evidence of the potential target genes through whole genome studies..
6. Chikako Yokoyama, Yuta Chigi, Takashi Baba, Atsushi Ohshitanai, Yumi Harada, Fumiya Takahashi, Ken-ichirou Morohashi, Three populations of adult Leydig cells in mouse testes revealed by a novel mouse HSD3B1-specific rat monoclonal antibody, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2019.02.100, 511, 4, 916-920, 2019.04, Leydig cells play a pivotal function in the synthesis of a male sex steroid, testosterone. The ability of the steroid production is dependent on the expression of the steroidogenic genes, such as HSD3B (3 beta-hydroxysteroid dehydrogenase/Delta 5- Delta 4 isomerase). It has been established that two different types of Leydig cells, fetal Leydig cells (FLCs) and adult Leydig cells (ALC5), are developed in mammalian testes. FLCs and ALC5 are characterized by different sets of marker gene expression. In the case of mouse Leydig cells, Hsd3b1 (Hsd3b type 1) is expressed both in FLCs and ALCs whereas Hsd3b6 (Hsd3b type 6) is expressed in ALC5 but not in FLCs. However, because the antibodies established so far for HSD3B were unable to distinguish between the HSD3B1 and HSD3B6 isoforms, it remained unclear whether both of them are expressed in every ALC. Therefore, in the present study, we generated a rat monoclonal antibody specific for mouse HSD3B1. Intriguingly, this monoclonal antibody together with an antibody specific for HSD3B6 identified three populations of ALCs based on the expression levels of these HSD3Bs. (C) 2019 Elsevier Inc. All rights reserved..
7. Yuko Katoh-Fukui, Takashi Baba, Tetsuya Sato, Hiroyuki Otake, Yuko Nagakui-Noguchi, Miyuki Shindo, Mikita Suyama, Yasuyuki Ohkawa, Hideki Tsumura, Ken-ichirou Morohashi, Maki Fukami, Mouse polycomb group gene Cbx2 promotes osteoblastic but suppresses adipogenic differentiation in postnatal long bones, BONE, 10.1016/j.bone.2018.10.021, 120, 219-231, 2019.03, A set of key developmental genes is essential for skeletal growth from multipotent progenitor cells at weaning. Polycomb group proteins, which regulate such genes contributes to the cell lineage commitment and subsequent differentiation via epigenetic chromatin modification and remodeling. However, it is unclear which cell lineage and gene sets are targeted by polycomb proteins during skeletal growth. We now report that mice deficient in a polycomb group gene cbx2(cterm/cterm) exhibited skeletal hypoplasia in the tibia, femur, and cranium. Long bone cavities in these mice contained fewer multipotent mesenchymal stromal cells. RNA-sequencing of bone marrow cells showed downregulation and upregulation of osteoblastic and adipogenic genes, respectively. Furthermore, the expression levels of genes specifically expressed in B-cell precursors were decreased. Forced expression of Cbx2 in cbx2(cterm/cterm) bone marrow stromal cell recovered fibroblastic colony formation and suppressed adipogenic differentiation. Collectively, our results suggest that Cbx2 controls the maintenance and adipogenic differentiation of mesenchymal stromal cells in the bone marrow..
8. Miki Inoue, Takashi Baba, Ken ichirou Morohashi, Recent progress in understanding the mechanisms of Leydig cell differentiation, Molecular and Cellular Endocrinology, 10.1016/j.mce.2017.12.013, 468, 39-46, 2018.06, Leydig cells in fetal and adult testes play pivotal roles in eliciting male characteristics by producing androgen. Although numerous studies of Leydig cells have been performed, the mechanisms for differentiation of the two cell types (fetal Leydig and adult Leydig cells), their developmental and functional relationship, and their differential characteristics remain largely unclear. Based on recent technical progress in genome-wide analysis and in vitro investigation, novel and fascinating observations concerning the issues above have been obtained. Focusing on fetal and adult Leydig cells, this review summarizes the recent progress that has advanced our understanding of the cells..
9. Ken Ichirou Morohashi, Miki Inoue, Bing Li, Takashi Baba, Regulation of metabolic pathways in steroidogenic cells by Ad4BP/SF-1, Cell Biology of the Ovary Stem Cells, Development, Cancer, and Clinical Aspects, 10.1007/978-981-10-7941-2_3, 35-43, 2018.06, Ad4BP/SF-1 (NR5A1), a member of the nuclear receptor superfamily, is known to play crucial roles in the regulation of steroidogenesis in the gonads and adrenal cortex, and many studies have demonstrated that all steroidogenic genes are direct targets of Ad4BP/SF-1. In addition, in vivo KO studies demonstrated that no steroidogenic organs formed in gene-disrupted mice, strongly suggesting that Ad4BP/SF-1 is essential for the development of the gonads and adrenal gland. However, it remains unclear how Ad4BP/SF-1 regulates the development of the steroidogenic organs and which additional non-steroidogenic genes are targeted. We surveyed the target genes of Ad4BP/SF-1 in steroidogenic cells by mRNA deep sequencing and ChIP-sequence analyses using an Ad4BP/SF-1 antibody. Nearly all genes in energy-producing glycolytic pathways were found to be regulated by Ad4BP/SF-1. We also showed that the key genes implicated in NADPH production are the direct targets. Since sufficient supplies of ATP and NADPH are necessary for cellular survival and production of steroids, Ad4BP/SF-1 possibly orchestrates tissue-specific steroidogenic pathway and these housekeeping metabolic pathways..
10. Baba T, Otake H, Inoue M, Sato T, Ishihara Y, Moon JY, Tsuchiya M, Miyabayashi K, Ogawa H, Shima Y, Wang L, Sato R, Yamazaki T, Suyama M, Nomura M, Choi MH, Ohkawa Y, Morohashi KI., Ad4BP/SF-1 regulates cholesterol synthesis to boost the production of steroids, Communications Biology, 10.1038/s42003-018-0020-z, 18, 2018.02.
11. Takashi Baba, The role of Ad4BP/SF-1 in the development of adrenal gland and the synthesis of adrenal steroid, Japanese Journal of Clinical Urology, 72, 6, 424-429, 2018.01.
12. Kanako Miyabayashi, Yuichi Shima, Miki Inoue, Tetsuya Sato, Takashi Baba, Yasuyuki Ohkawa, Mikita Suyama, Ken-ichirou Morohashi, Alterations in fetal Leydig cell gene expression during fetal and adult development, Sexual Development, 10.1159/000453323, 11, 2, 53-63, 2017.05, [URL], Fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) develop in the mammalian prenatal and postnatal testes, respectively. In mice, FLCs emerge in the interstitial space of the testis as early as embryonic day 12.5 and thereafter increase in number during the fetal stage. We previously established a transgenic mouse line in which FLCs are labeled with EGFP and demonstrated that the EGFP-labeled FLCs were present even in adult testes. However, the characteristics of FLCs during postnatal stages remained unclear. In the present study, a comparison of the transcriptomes of FLCs from prenatal and postnatal testes and of ALCs from adult testes revealed that FLCs gradually alter their characteristics across developmental stages and come to roughly resemble ALCs. Many cholesterogenic genes simultaneously expressed a unique alternation pattern, while many oxidative phosphorylation and β-oxidation (both ….
13. Jhih-Siang Syu, Takashi Baba, Jyun-Yuan Huang, Hidesato Ogawa, Chi-Han Hsieh, Jin-Xian Hu, Ting-Yu Chen, Tzu-Chien Lin, Megumi Tsuchiya, Ken-Ichirou Morohashi, Bu-Miin Huang, Fu-I. Lu, Chia-Yih Wang, Lysosomal activity maintains glycolysis and cyclin E1 expression by mediating Ad4BP/SF-1 stability for proper steroidogenic cell growth, SCIENTIFIC REPORTS, 10.1038/s41598-017-00393-4, 7, 1, 240-240, 2017.03, The development and differentiation of steroidogenic organs are controlled by Ad4BP/SF-1 (adrenal 4 binding protein/steroidogenic factor 1). Besides, lysosomal activity is required for steroidogenesis and also enables adrenocortical cell to survive during stress. However, the role of lysosomal activity on steroidogenic cell growth is as yet unknown. Here, we showed that lysosomal activity maintained Ad4BP/SF-1 protein stability for proper steroidogenic cell growth. Treatment of cells with lysosomal inhibitors reduced steroidogenic cell growth in vitro. Suppression of autophagy did not affect cell growth indicating that autophagy was dispensable for steroidogenic cell growth. When lysosomal activity was inhibited, the protein stability of Ad4BP/SF-1 was reduced leading to reduced S phase entry. Interestingly, treatment of cells with lysosomal inhibitors reduced glycolytic gene expression and supplying the cells with pyruvate alleviated the growth defect. ChIP-sequence/ChIP studies indicated that Ad4BP/SF-1 binds to the upstream region of Ccne1 (cyclin E1) gene during G1/S phase. In addition, treatment of zebrafish embryo with lysosomal inhibitor reduced the levels of the interrenal (adrenal) gland markers. Thus lysosomal activity maintains steroidogenic cell growth via stabilizing Ad4BP/SF-1 protein..
14. Yurina Shishido, Takashi Baba, Tetsuya Sato, Yuichi Shima, Kanako Miyabayashi, Miki Inoue, Haruhiko Akiyama, Hiroshi Kimura, Yoshiakira Kanai, Yasuhiro Ishihara, Shogo Haraguchi, Akira Miyazaki, Damjana Rozman, Takeshi Yamazaki, Man-Ho Choi, Yasuyuki Ohkawa, Mikita Suyama, Ken-ichirou Morohashi, Differential lactate and cholesterol synthetic activities in XY and XX Sertoli cells, SCIENTIFIC REPORTS, 10.1038/srep41912, 7, 41912-41912, 2017.02, SRY, a sex-determining gene, induces testis development in chromosomally female (XX) individuals. However, mouse XX Sertoli cells carrying Sry (XX/Sry Sertoli cells) are incapable of fully supporting germ cell development, even when the karyotype of the germ cells is XY. While it has therefore been assumed that XX/Sry Sertoli cells are not functionally equivalent to XY Sertoli cells, it has remained unclear which specific functions are affected. To elucidate the functional difference, we compared the gene expression of XY and XX/Sry Sertoli cells. Lactate and cholesterol metabolisms, essential for nursing the developing germ cells, were down-regulated in XX/Sry cells, which appears to be caused at least in part by the differential expression of histone modification enzymes SMCX/SMCY (H3K4me3 demethylase) and UTX/UTY (H3K27me3 demethylase) encoded by the sex chromosomes. We suggest that down-regulation of lactate and cholesterol metabolism that may be due to altered epigenetic modification affects the nursing functions of XX/Sry Sertoli cells..
15. Bing Li, Takashi Baba, Kanako Miyabayashi, Tetsuya Sato, Yuichi Shima, Tomomi Ichinose, Daisuke Miura, Yasuyuki Ohkawa, Mikita Suyama, Ken Ichirou Morohashi, Role of Ad4-binding protein/steroidogenic factor 1 in regulating NADPH production in adrenocortical Y-1 cells, Endocrine Journal, 10.1507/endocrj.EJ16-0467, 64, 3, 315-324, 2017.01, Ad4-binding protein/steroidogenic factor 1 (Ad4BP/SF-1), a member of the nuclear receptor superfamily, is expressed in steroidogenic cells and regulates all steroidogenic gene expression. We recently employed mRNA and chromatin immunoprecipitation sequence (ChIP-seq) to demonstrate that Ad4BP/SF-1 directly regulates the expression of nearly all glycolytic genes. The pentose phosphate pathway (PPP) contributes to the production of nicotinamide adenine dinucleotide phosphate (NADPH). Although the expression of PPP genes and intracellular NADPH were decreased by Ad4BP/SF-1 knockdown, these genes were not the direct targets of Ad4BP/SF-1. This study therefore investigates whether Ad4BP/SF-1 directly regulates genes implicated in NADPH production. Examination of previously published data sets of mRNA sequence (mRNA-seq) and ChIP-seq strongly suggested a possibility that other NADPH-producing genes, such as malic enzyme 1 (Me1) and methylenetetrahydrofolate dehydrogenase 2 (Mthfd2), are the direct targets of Ad4BP/SF-1. Reporter gene assays and determination of intracellular NADPH concentration supported the notion that Ad4BP/SF-1 regulates NADPH production by regulating these genes. NADPH is required for macromolecule synthesis of compounds such as steroids, and for detoxification of reactive oxygen species. When synthesizing steroid hormones, steroidogenic cells consume NADPH through enzymatic reactions mediated by steroidogenic P450s. NADPH is also consumed through elimination of reactive oxygen species produced as the byproducts of the P450 reactions. Overall, Ad4BP/SF-1 potentially maintains the intracellular NADPH level through cooperative regulation of genes involved in the biological processes for consumption and supply..
16. Maki Igarashi, Kei Takasawa, Akiko Hakoda, Junko Kanno, Shuji Takada, Mami Miyado, Takashi Baba, Ken-ichirou Morohashi, Toshihiro Tajima, Kenichiro Hata, Kazuhiko Nakabayashi, Yoichi Matsubara, Ryohei Sekido, Tsutomu Ogata, Kenichi Kashimada, Maki Fukami, Identical NR5A1 Missense Mutations in Two Unrelated 46,XX Individuals with Testicular Tissues, HUMAN MUTATION, 10.1002/humu.23116, 38, 1, 39-42, 2017.01, The role of monogenic mutations in the development of 46,XX testicular/ovotesticular disorders of sex development (DSD) remains speculative. Although mutations in NR5A1 are known to cause 46,XY gonadal dysgenesis and 46,XX ovarian insufficiency, such mutations have not been implicated in testicular development of 46,XX gonads. Here, we identified identical NR5A1 mutations in two unrelated Japanese patients with 46,XX testicular/ovotesticular DSD. The p.Arg92Trp mutation was absent from the clinically normal mothers and from 200 unaffected Japanese individuals. In silico analyses scored p.Arg92Trp as probably pathogenic. In vitro assays demonstrated that compared with wild-type NR5A1, the mutant protein was less sensitive to NR0B1-induced suppression on the SOX9 enhancer element. Other sequence variants found in the patients were unlikely to be associated with the phenotype. The results raise the possibility that specific mutations inNR5A1 underlie testicular development in genetic females..
17. Miki Inoue, Yuichi Shima, Kanako Miyabayashi, Kaori Tokunaga, Tetsuya Sato, Takashi Baba, Yasuyuki Ohkawa, Haruhiko Akiyama, Mikita Suyama, Ken-ichirou Morohashi, Isolation and Characterization of Fetal Leydig Progenitor Cells of Male Mice, ENDOCRINOLOGY, 10.1210/en.2015-1773, 157, 3, 1222-1233, 2016.03, Fetal and adult Leydig cells develop in mammalian prenatal and postnatal testes, respectively. In mice, fetal Leydig cells (FLCs) emerge in the interstitial space of the testis at embryonic day 12.5 and thereafter increase in number, possibly through differentiation from progenitor cells. However, the progenitor cells have not yet been identified. Previously, we established transgenic mice in which FLCs are labeled strongly with enhanced green fluorescent protein (EGFP). Interestingly, fluorescence-activated cell sorting provided us with weakly EGFP-labeled cells as well as strongly EGFP-labeled FLCs. In vitro reconstruction of fetal testes demonstrated that weakly EGFP-labeled cells contain FLC progenitors. Transcriptome from the 2 cell populations revealed, as expected, marked differences in the expression of genes required for growth factor/receptor signaling and steroidogenesis. In addition, genes for energy metabolisms such as glycolytic pathways and the citrate cycle were activated in strongly EGFP-labeled cells, suggesting that metabolism is activated during FLC differentiation..
18. Yuichi Shima, Sawako Matsuzaki, Kanako Miyabayashi, Hiroyuki Otake, Takashi Baba, Shigeaki Kato, Ilpo Huhtaniemi, Ken-ichirou Morohashi, Fetal Leydig Cells Persist as an Androgen-Independent Subpopulation in the Postnatal Testis, MOLECULAR ENDOCRINOLOGY, 10.1210/me.2015-1200, 29, 11, 1581-1593, 2015.11, Two distinct types of Leydig cells emerge during the development of eutherian mammals. Fetal Leydig cells (FLCs) appear shortly after gonadal sex differentiation, and play a crucial role in masculinization of male fetuses. Meanwhile, adult Leydig cells (ALCs) emerge after birth and induce the secondary male-specific sexual maturation by producing testosterone. Previous histological studies suggested that FLCs regress completely soon after birth. Furthermore, gene disruption studies indicated that androgen signaling is dispensable for FLC differentiation but indispensable for postnatal ALC differentiation. Here, we performed lineage tracing of FLCs using a FLC enhancer of the Ad4BP/SF-1 (Nr5a1) gene and found that FLCs persist in the adult testis. Given that postnatal FLCs expressed androgen receptor (AR) as well as LH receptor (LuR), the effects of AR disruption on FLCs and ALCs were analyzed by crossing AR knockout (KO) mice with FLC-specific enhanced green fluorescent protein (EGFP) mice. Moreover, to eliminate the influence of elevated LH levels in ARKO mice, LuRKO mice and AR/LuR double-KO mice were analyzed. The proportion of ALCs to postnatal FLCs was decreased in ARKO mice, and the effect was augmented in the double-KO mice, suggesting that androgen signaling plays important roles in ALCs, but not in FLCs. Finally, ARKO was achieved in an FLC-specific manner (FLCARKO mice), but the FLC number and gene expression pattern appeared unaffected. These findings support the conclusion that FLCs persist as an androgen-independent Leydig subpopulation in the postnatal testis..
19. Yuko Katoh-Fukui, Maki Igarashi, Keisuke Nagasaki, Reiko Horikawa, Toshiro Nagai, Takayoshi Tsuchiya, Erina Suzuki, Mami Miyado, Kenichiro Hata, Kazuhiko Nakabayashi, Keiko Hayashi, Yoichi Matsubara, Takashi Baba, Ken-ichirou Morohashi, Arisa Igarashi, Tsutomu Ogata, Shuji Takada, Maki Fukami, Testicular dysgenesis/regression without campomelic dysplasia in patients carrying missense mutations and upstream deletion of SOX9, MOLECULAR GENETICS & GENOMIC MEDICINE, 10.1002/mgg3.165, 3, 6, 550-557, 2015.11, SOX9 haploinsufficiency underlies campomelic dysplasia (CD) with or without testicular dysgenesis. Current understanding of the phenotypic variability and mutation spectrum of SOX9 abnormalities remains fragmentary. Here, we report three patients with hitherto unreported SOX9 abnormalities. These patients were identified through molecular analysis of 33 patients with 46,XY disorders of sex development (DSD). Patients 1-3 manifested testicular dysgenesis or regression without CD. Patients 1 and 2 carried probable damaging mutations p.Arg394Gly and p.Arg437Cys, respectively, in the SOX9 C-terminal domain but not in other known 46, XY DSD causative genes. These substitutions were absent from similar to 120,000 alleles in the exome database. These mutations retained normal transactivating activity for the Col2a1 enhancer, but showed impaired activity for the Amh promoter. Patient 3 harbored a maternally inherited similar to 491 kb SOX9 upstream deletion that encompassed the known 32.5 kb XY sex reversal region. Breakpoints of the deletion resided within nonrepeat sequences and were accompanied by a short-nucleotide insertion. The results imply that testicular dysgenesis and regression without skeletal dysplasia may be rare manifestations of SOX9 abnormalities. Furthermore, our data broaden pathogenic SOX9 abnormalities to include C-terminal missense substitutions which lead to target-gene-specific protein dysfunction, and enhancer-containing upstream microdeletions mediated by nonhomologous end-joining..
20. Kanako Miyabayashi, Kaori Tokunaga, Hiroyuki Otake, Takashi Baba, Yuichi Shima, Ken-ichirou Morohashi, Heterogeneity of Ovarian Theca and Interstitial Gland Cells in Mice, PLOS ONE, 10.1371/journal.pone.0128352, 10, 6, e0128352, 2015.06, It has been established that two developmentally and functionally distinct cell types emerge within the mammalian testis and adrenal gland throughout life. Fetal and adult types of steroidogenic cells (i.e., testicular Leydig cells and adrenocortical cells) develop in the prenatal and postnatal period, respectively. Although the ovary synthesizes steroids postnatally, the presence of fetal-type steroidogenic cells has not been described. We had previously established transgenic mouse lines in which fetal Leydig cells were labeled with an EGFP reporter gene by the FLE (fetal Leydig enhancer) of the Ad4BP/SF-1 (Nr5a1) gene. In the present study, we examined the reporter gene expression in females and found that the reporter gene is turned on in postnatal ovaries. A comparison of the expressions of the EGFP and marker genes revealed that EGFP is expressed in not all but rather a proportion of steroidogenic theca and in interstitial gland cells in the ovary. This finding was further supported by experiments using BAC transgenic mice in which reporter gene expression recapitulated endogenous Ad4BP/SF-1 gene expression. In conclusion, our observations from this study strongly suggest that ovarian theca and interstitial gland cells in mice consist of at least two cell types..
21. Atsushi Yokoyama, Katsuhide Igarashi, Tetsuya Sato, Kiyoshi Takagi, Maky Otsuka, Yurina Shishido, Takashi Baba, Ryo Ito, Jun Kanno, Yasuyuki Ohkawa, Ken-ichirou Morohashi, Akira Sugawara, Identification of Myelin Transcription Factor 1 (MyT1) as a Subunit of the Neural Cell Type-specific Lysine-specific Demethylase 1 (LSD1) Complex, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M114.566448, 289, 26, 18152-18162, 2014.06, Regulation of spatiotemporal gene expression in higher eukaryotic cells is critical for the precise and orderly development of undifferentiated progenitors into committed cell types of the adult. It is well known that dynamic epigenomic regulation (including chromatin remodeling and histone modifications by transcriptional coregulator complexes) is involved in transcriptional regulation. Precisely how these coregulator complexes exert their cell type and developing stage-specific activity is largely unknown. In this study we aimed to isolate the histone demethylase lysine-specific demethylase 1 (LSD1) complex from neural cells by biochemical purification. In so doing, we identified myelin transcription factor 1 (MyT1) as a novel LSD1 complex component. MyT1 is a neural cell-specific zinc finger factor, and it forms a stable multiprotein complex with LSD1 through direct interaction. Target gene analysis using microarray and ChIP assays revealed that the Pten gene was directly regulated by the LSD1-MyT1 complex. Knockdown of either LSD1 or MyT1 derepressed the expression of endogenous target genes and inhibited cell proliferation of a neuroblastoma cell line, Neuro2a. We propose that formation of tissue-specific combinations of coregulator complexes is a critical mechanism for tissue-specific transcriptional regulation..
22. Takashi Baba, Hiroyuki Otake, Tetsuya Sato, Kanako Miyabayashi, Yurina Shishido, Chia-Yih Wang, Yuichi Shima, Hiroshi Kimura, Mikako Yagi, Yasuhiro Ishihara, Shinjiro Hino, Hidesato Ogawa, Mitsuyoshi Nakao, Takeshi Yamazaki, Dongchon Kang, Yasuyuki Ohkawa, Mikita Suyama, Bon-Chu Chung, Ken-Ichirou Morohashi, Glycolytic genes are targets of the nuclear receptor Ad4BP/SF-1, Nature Communications, 10.1038/ncomms4634, 5, 3634-3634, 2014.04, Genetic deficiencies in transcription factors can lead to the loss of certain types of cells and tissue. The steroidogenic tissue-specific nuclear receptor Ad4BP/SF-1 (NR5A1) is one such gene, because mice in which this gene is disrupted fail to develop the adrenal gland and gonads. However, the specific role of Ad4BP/SF-1 in these biological events remains unclear. Here we use chromatin immunoprecipitation sequencing to show that nearly all genes in the glycolytic pathway are regulated by Ad4BP/SF-1. Suppression of Ad4BP/SF-1 by small interfering RNA reduces production of the energy carriers ATP and nicotinamide adenine dinucleotide phosphate, as well as lowers expression of genes involved in glucose metabolism. Together, these observations may explain tissue dysgenesis as a result of Ad4BP/SF-1 gene disruption in vivo. Considering the function of estrogen-related receptor a, the present study raises the possibility that certain types of nuclear receptors regulate sets of genes involved in metabolic pathways to generate energy carriers..
23. Ken Ichirou Morohashi, Takashi Baba, Regulation of metabolism by orphan receptor regulone, Seikagaku, 86, 6, 719-725, 2014.01.
24. Kanako Miyabayashi, Yuko Katoh-Fukui, Hidesato Ogawa, Takashi Baba, Yuichi Shima, Noriyuki Sugiyama, Kunio Kitamura, Ken-ichirou Morohashi, Aristaless Related Homeobox Gene, Arx, Is Implicated in Mouse Fetal Leydig Cell Differentiation Possibly through Expressing in the Progenitor Cells, PLOS ONE, 10.1371/journal.pone.0068050, 8, 6, e68050, 2013.06, Development of the testis begins with the expression of the SRY gene in pre-Sertoli cells. Soon after, testis cords containing Sertoli and germ cells are formed and fetal Leydig cells subsequently develop in the interstitial space. Studies using knockout mice have indicated that multiple genes encoding growth factors and transcription factors are implicated in fetal Leydig cell differentiation. Previously, we demonstrated that the Arx gene is implicated in this process. However, how ARX regulates Leydig cell differentiation remained unknown. In this study, we examined Arx KO testes and revealed that fetal Leydig cell numbers largely decrease throughout the fetal life. Since our study shows that fetal Leydig cells rarely proliferate, this decrease in the KO testes is thought to be due to defects of fetal Leydig progenitor cells. In sexually indifferent fetal gonads of wild type, ARX was expressed in the coelomic epithelial cells and cells underneath the epithelium as well as cells at the gonad-mesonephros border, both of which have been described to contain progenitors of fetal Leydig cells. After testis differentiation, ARX was expressed in a large population of the interstitial cells but not in fetal Leydig cells, raising the possibility that ARX-positive cells contain fetal Leydig progenitor cells. When examining marker gene expression, we observed cells as if they were differentiating into fetal Leydig cells from the progenitor cells. Based on these results, we propose that ARX acts as a positive factor for differentiation of fetal Leydig cells through functioning at the progenitor stage..
25. Yuichi Shima, Kanako Miyabayashi, Shogo Haraguchi, Tatsuhiko Arakawa, Hiroyuki Otake, Takashi Baba, Sawako Matsuzaki, Yurina Shishido, Haruhiko Akiyama, Taro Tachibana, Kazuyoshi Tsutsui, Ken-ichirou Morohashi, Contribution of Leydig and Sertoli Cells to Testosterone Production in Mouse Fetal Testes, MOLECULAR ENDOCRINOLOGY, 10.1210/me.2012-1256, 27, 1, 63-73, 2013.01, Testosterone is a final product of androgenic hormone biosynthesis, and Leydig cells are known to be the primary source of androgens. In the mammalian testis, two distinct populations of Leydig cells, the fetal and the adult Leydig cells, develop sequentially, and these two cell types differ both morphologically and functionally. It is well known that the adult Leydig cells maintain male reproductive function by producing testosterone. However, it has been controversial whether fetal Leydig cells can produce testosterone, and the synthetic pathway of testosterone in the fetal testis is not fully understood. In the present study, we generated transgenic mice in which enhanced green fluorescence protein was expressed under the control of a fetal Leydig cell-specific enhancer of the Ad4BP/SF-1 (Nr5a1) gene. The transgene construct was prepared by mutating the LIM homeodomain transcription factor (LHX9)-binding sequence in the promoter, which abolished promoter activity in the undifferentiated testicular cells. These transgenic mice were used to collect highly pure fetal Leydig cells. Gene expression and steroidogenic enzyme activities in the fetal Leydig cells as well as in the fetal Sertoli cells and adult Leydig cells were analyzed. Our results revealed that the fetal Leydig cells synthesize only androstenedione because they lack expression of Hsd17b3, and fetal Sertoli cells convert androstenedione to testosterone, whereas adult Leydig cells synthesize testosterone by themselves. The current study demonstrated that both Leydig and Sertoli cells are required for testosterone synthesis in the mouse fetal testis. (Molecular Endocrinology 27: 63-73, 2013).
26. Ken-Ichirou Morohashi, Takashi Baba, M. Tanaka, Steroid hormones and the development of reproductive organs., Sexual Development, 10.1159/000342272, 7, 1-3, 61-79, 2012.12, It has been more than 150 years since the physiological function of androgen was reported for the first time in fowl. This finding has served as a basis for many studies focusing on steroid hormones from various aspects. These studies have significantly enhanced our knowledge about the structures of steroid hormones, their synthetic pathways, enzymes involved in the synthetic pathways, steroid hormone-specific receptors, actions of steroid hormones through receptor binding, and the differentiation of steroidogenic cells. However, there are still many attractive and important issues in these areas, some of which are currently being addressed. In this review, we trace the history and findings of the previous studies on steroid hormones, summarize our present understanding in this area, and discuss issues that remain to be elucidated..
27. Yuichi Shima, Kanako Miyabayashi, Takashi Baba, Hiroyuki Otake, Yukako Katsura, Sanae Oka, Mohamad Zubair, Ken-inchirou Morohashi, Identification of an Enhancer in the Ad4BP/SF-1 Gene Specific for Fetal Leydig Cells (vol 153, pg 417, 2012), ENDOCRINOLOGY, 10.1210/en.2012-1948, 153, 11, 5686-5686, 2012.11.
28. Yuko Katoh-Fukui, Kanako Miyabayashi, Tomoko Komatsu, Akiko Owaki, Takashi Baba, Yuichi Shima, Tomohide Kidokoro, Yoshiakira Kanai, Andreas Schedl, Dagmar Wilhelm, Peter Koopman, Yasushi Okuno, Ken-ichirou Morohashi, Cbx2, a Polycomb Group Gene, Is Required for Sry Gene Expression in Mice, ENDOCRINOLOGY, 10.1210/en.2011-1055, 153, 2, 913-924, 2012.02, Mice lacking the function of the polycomb group protein CBX2(chromobox homolog 2; also known as M33) show defects in gonadal, adrenal, and splenic development. In particular, XY knockout (KO) mice develop ovaries but not testes, and the gonads are hypoplastic in both sexes. However, how CBX2 regulates development of these tissues remains largely unknown. In the present study, we used microarray, RT-PCR, and immunohistochemical analyses to show that the expression of Sry, Sox9, Lhx9, Ad4BP/SF-1, Dax-1, Gata4, Arx, and Dmrt1, genes encoding transcription factors essential for gonadal development, is affected in Cbx2 KO gonads. Male-to-female sex reversal in Cbx2 KO mice was rescued by crossing them with transgenic mice displaying forced expression of Sry or Sox9. However, testes remained hypoplastic in these mice, indicating that the size and the sex of the gonad are determined by different sets of genes. Our study implicates Cbx2 in testis differentiation through regulating Sry gene expression. (Endocrinology 153: 913-924, 2012).
29. Keiko Nohara, Takashi Baba, Hikari Murai, Yayoi Kobayashi, Takehiro Suzuki, Yukiyo Tateishi, Michiyo Matsumoto, Noriko Nishimura, Tomoharu Sano, Global DNA methylation in the mouse liver is affected by methyl deficiency and arsenic in a sex-dependent manner, ARCHIVES OF TOXICOLOGY, 10.1007/s00204-010-0611-z, 85, 6, 653-661, 2011.06, Arsenic, a carcinogen, is assumed to induce global DNA hypomethylation by consuming the universal methyl donor S-adenosylmethionine (SAM) in the body. A previous study reported that a methyl-deficient diet (MDD) with arsenic intake greatly reduced global DNA methylation (the content of 5-methylcytosine) in the liver of male C57BL/6 mice. In the present study, we investigated the DNA methylation level, SAM content, and expression of DNA methyltransferases (DNMTs) in the liver of male and female C57BL/6 mice fed a methyl-sufficient diet (MSD), an MDD, or an MDD + arsenic. The DNA methylation level was accurately determined by measuring the content of genomic 5-methyldeoxycytidine (5medC) by high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) using stable-isotope-labeled 5medC and deoxycytidine (dC) as internal standards. The results of this study revealed that while the MDD and arsenic tended to reduce the genomic 5meC content in the male mice livers, the MDD + arsenic significantly increased the 5meC content in the female mice livers. Another unexpected finding was the small differences in 5meC content among the groups. The MDD and MDD + arsenic suppressed DNMT1 expression only in the male mice livers. In contrast, SAM content was reduced by the MDD and MDD + arsenic only in the livers of female mice, showing that the changes in 5meC content were not attributable to SAM content. The sex-dependent changes in 5meC content induced by methyl deficiency and arsenic may be involved in differences in male and female susceptibility to diseases via epigenetic modification of physiological functions..
30. Keiko Nohara, Takashi Baba, Hikari Murai, Yayoi Kobayashi, Takehiko Suzuki, Yukiyo Tateishi, Michiyo Matsumoto, Noriko Nishimura, and Tomoharu Sano, Genomic DNA methylation in the mouse liver is affected by methyl deficiency and arsenic in a sex- and a dietary-fat-content-dependent manner, Arch. Toxicol., 85, 653-661, 2011.01.
31. Masatomo Kusaka, Yuko Katoh-Fukui, Hidesato Ogawa, Kanako Miyabayashi, Takashi Baba, Yuichi Shima, Noriyuki Sugiyama, Yukihiko Sugimoto, Yasushi Okuno, Ryuji Kodama, Akiko Iizuka-Kogo, Takao Senda, Toshikuni Sasaoka, Kunio Kitamura, Shinichi Aizawa, Ken-ichirou Morohashi, Abnormal Epithelial Cell Polarity and Ectopic Epidermal Growth Factor Receptor (EGFR) Expression Induced in Emx2 KO Embryonic Gonads, ENDOCRINOLOGY, 10.1210/en.2010-0915, 151, 12, 5893-5904, 2010.12, The gonadal primordium first emerges as a thickening of the embryonic coelomic epithelium, which has been thought to migrate mediodorsally to form the primitive gonad. However, the early gonadal development remains poorly understood. Mice lacking the paired-like homeobox gene Emx2 display gonadal dysgenesis. Interestingly, the knockout (KO) embryonic gonads develop an unusual surface accompanied by aberrant tight junction assembly. Morphological and in vitro cell fate mapping studies showed an apparent decrease in the number of the gonadal epithelial cells migrated to mesenchymal compartment in the KO, suggesting that polarized cell division and subsequent cell migration are affected. Microarray analyses of the epithelial cells revealed significant up-regulation of Egfr in the KO, indicating that Emx2 suppresses Egfr gene expression. This genetic correlation between the two genes was reproduced with cultured M15 cells derived from mesonephric epithelial cells. Epidermal growth factor receptor signaling was recently shown to regulate tight junction assembly through sarcoma viral oncogene homolog tyrosine phosphorylation. We show through Emx2 KO analyses that sarcoma viral oncogene homolog tyrosine phosphorylation, epidermal growth factor receptor tyrosine phosphorylation, and Egfr expression are up-regulated in the embryonic gonad. Our results strongly suggest that Emx2 is required for regulation of tight junction assembly and allowing migration of the gonadal epithelia to the mesenchyme, which are possibly mediated by suppression of Egfr expression. (Endocrinology 151: 5893-5904, 2010).
32. Ken-ichirou Morohashi, Yuichi Shima, Kanako Miyabayashi, Hiroyuki Otake, Takashi Baba, Mohamad Zubair, Implication of Ad4BP/SF-1 into adrenal development, ENDOCRINE JOURNAL, 57, S309-S309, 2010.03.
33. Yuko Sato, Takashi Baba, Mohamad Zubair, Kanako Miyabayashi, Yoshiro Toyama, Mamiko Maekawa, Akiko Owaki, Hirofumi Mizusaki, Tatsuya Sawamura, Kiyotaka Toshimori, Ken-Ichirou Morohashi, Yuko Kato-Fukui, Importance of forkhead transcription factor Fkhl18 for development of testicular vasculature, MOLECULAR REPRODUCTION AND DEVELOPMENT, 10.1002/mrd.20888, 75, 9, 1361-1371, 2008.09, Forkhead transcription factors are characterized by a winged helix DNA binding domain, and the members of this family are classified into 20 subclasses by phylogenetic analyses. Fkhl18 is structurally unique, and is classified into FoxS subfamily. We found Fkhl18 expression in periendothelial cells of the developing mouse fetal testis. In an attempt to clarify its function, we generated mice with Fkhl18 gene disruption. Although KO mice developed normally and were fertile in both sexes, we frequently noticed unusual blood accumulation in the fetal testis. Electron microscopic analysis demonstrated frequent gaps, measuring 100-400 nm, in endothelial cells of blood vessels. These gaps probably represented ectopic apoptosis of testicular periendothelial cells, identified by caspase-3 expression, in KO fetuses. No apoptosis of endothelial cells was noted. Fkhl18 suppressed the transcriptional activity of FoxO3a and FoxO4. Considering that Fas ligand gene expression is activated by Foxs, the elevated activity of Foxs in the absence of Fkhl18 probably explains the marked apoptosis of periendothelial cells in Fkhl18 KO mice..
34. Maki Fukami, Yuka Wada, Michiyo Okada, Fumiko Kato, Noriyuki Katsumata, Takashi Baba, Ken-ichirou Morohashi, Jocelyn Laporte, Motoo Kitagawa, Tsutomu Ogata, Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M703289200, 283, 9, 5525-5532, 2008.02, Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wildtype CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure..
35. Baba, T.; Shima, Y.; Owaki, A.; Mimura, J.; Oshima, M.; Fujii-Kuriyama, Y.; Morohashi, K. -i., Disruption of aryl hydrocarbon receptor (AhR) induces regression of the seminal vesicle in aged male mice, Sex. Dev., 10.1159/000117714, 2, 1, 1-11, 2008.01.
36. Hiromi Kurokawa, Daisuke Saito, Shuhei Nakamura, Yuko Katoh-Fukui, Kohei Ohta, Takashi Baba, Ken-ichiro Morohashi, Minoru Tanaka, Germ cells are essential for sexual dimorphism in the medaka gonad, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.0609932104, 104, 43, 16958-16963, 2007.10, To further elucidate the roles of germ cells in the sex differentiation of gonads, we have used the medaka, a teleost fish, to generate mutants that lack germ cells from the onset of gonadogenesis by the morpholino-mediated knockdown of cxcr4. The resulting germ-cell-deficient medaka show female-to-male sex reversal of their secondary sex characteristics, accompanied by increased levels of androgen and reduced levels of estrogen. A failure to maintain granulosa cells or estrogen-producing cells also occurs at early stages of sex differentiation in the cxcr4 morphants, before the initiation of gonadal morphogenesis. In contrast, androgen-producing cells are unaffected in germ-cell-deficient medaka of either sex. In addition, a single tube-like gonad that expresses male-specific genes is formed in these mutants irrespective of the genetic sex. Significantly, each of these mutant phenotypes occurs in a somatic cell-autonomous manner, suggesting that gonadal somatic cells are predisposed toward male development in the absence of germ cells. This highlights the importance of germ cells in the sexual dimorphism of the gonads..
37. Yoshiaki Fujii-Kuriyama, Junsei Mimura, Takashi Baba, Ken-ichirou Morohashi, Fumiaki Ohtake, Shigeaki Kato, Yasuhito Kobayashi, Kaname Kawajiri, PHYSIOLOGICAL AND TOXICOLOGICAL FUNCTIONS OF ARYLHYDROCARBON (DIOXIN) RECEPTOR IN MICE, DRUG METABOLISM REVIEWS, 39, 23-24, 2007.01.
38. Yoshiaki Fujii-Kuriyama, Takashi Baba, Junsei Mimura, Masayuki Yamamoto, Ken-ichiro Morohashi, Physiological roles of AhR in murine reproductive and immunological processes, TOXICOLOGY LETTERS, 10.1016/j.toxlet.2006.06.068, 164, S31-S31, 2006.09.
39. Takashi Baba, Junsei Mimura, Naohito Nakamura, Nobuhiro Harada, Masayuki Yamamoto, Ken-Ichirou Morohashi, Yoshiaki Fujii-Kuriyama, Intrinsic function of the aryl hydrocarbon (dioxin) receptor as a key factor in female reproduction., Molecular and cellular biology, 25, 22, 10040-51, 2005.11, Dioxins exert a variety of adverse effects on organisms, including teratogenesis, immunosuppression, tumor promotion, and estrogenic action. Studies using aryl hydrocarbon receptor (AhR)-deficient mice suggest that the majority of these toxic effects are mediated by the AhR. In spite of the adverse effects mediated by this receptor, the AhR gene is conserved among a number of animal species, ranging from invertebrates to vertebrates. This high degree of conservation strongly suggests that AhR possesses an important physiologic function, and a critical function is also supported by the reduced fertility observed with AhR-null female mice. We demonstrate that AhR plays a crucial role in female reproduction by regulating the expression of ovarian P450 aromatase (Cyp19), a key enzyme in estrogen synthesis. As revealed by in vitro reporter gene assay and in vivo chromatin immunoprecipitation assay, AhR cooperates with an orphan nuclear receptor, Ad4BP/SF-1, to activate Cyp19 gene transcription in ovarian granulosa cells. Administration to female mice of an AhR ligand, DMBA (9,10-dimethyl-1,2-benzanthracene), induced ovarian Cyp19 gene expression, irrespective of the intrinsic phase of the estrus cycle. In addition to elucidating a physiological function for AhR, our studies also suggest a possible mechanism for the toxic effects of exogenous AhR ligands as endocrine disruptors..
40. Yoshiaki Fujii-Kuriyama, Takashi Baba, Junsei Mimura, Masayuki Yamamoto, Ken-ichirou Morohashi, Role of AhR in reproduction of female mice and expression of biological effects of endocrine disruptors, Proceedings of Annual Meeting of the Physiological Society of Japan Proceedings of Annual Meeting of the Physiological Society of Japan, S40-S40, 2004.11, Endocrine disruptors are usually considered to express their biologically adverse effects through taking over the nuclear receptors which have their cognate ligands, resulting in untimely activation or suppression of their transcriptional activities. 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD), one of most toxic manmade chemicals, binds arylhydrocarbon receptor (AhR or dioxin receptor) with an extremely high affinity and expresses pleiotropic biological activities..
41. Yoshiaki Fujii-Kuriyama, Junsei Mimura, Takashi Baba, Keiko Numayama-Tsuruta, Kazuhiro Sogawa, Action mechanism of dioxin receptor, Japan Society for Bioscience Biotechnology and Agrochemistry, 10.1271/nogeikagaku1924.76.1073, 76, 11, 1073-1075, 2002.11.
42. Yoshiaki Fujii-Kuriyama, Junsei Mimura, Takashi Baba, Keiko Numayama-Tsuruta, Kazuhiro Sogawa, Action mechanism of dioxin receptor, Nippon Nogeikagaku Kaishi, 10.1271/nogeikagaku1924.76.1073, 76, 11, 1073-1075, 2002.01.
43. T Baba, J Mimura, K Gradin, A Kuroiwa, T Watanabe, Y Matsuda, J Inazawa, K Sogawa, Y Fujii-Kuriyama, Structure and expression of the Ah receptor repressor gene., The Journal of biological chemistry, 276, 35, 33101-10, 2001.08, The aryl hydrocarbon receptor (AhR) repressor (AhRR) gene has been isolated and characterized from a mouse genomic library. The gene is distributed as 11 exons in a total length of about 60 kilobase pairs. Fluorescence in situ hybridization analysis has shown that the AhRR gene is located at mouse chromosome 13C2, at rat chromosome 1p11.2, and at human chromosome 5p15.3. The AhRR gene has a TATA-less promoter and several transcription start sites. In addition, putative regulatory DNA sequences such as xenobiotic responsive element (XRE), GC box, and NF-kappaB-binding sites have been identified in the 5'-upstream region of the AhRR gene. Transient transfection analyses of HeLa cells with reporter genes that contain deletions and point mutations in the AhRR promoter revealed that all three XREs mediated the inducible expression of the AhRR gene by 3-methylcholanthrene treatment, and furthermore, GC box sequences were indispensable for a high level of inducible expression and for constitutive expression. Moreover, by using gel mobility shift assays we were able to show that the AhR/Arnt heterodimer binds to the XREs with very low affinity, which is due to three varied nucleotides outside the XRE core sequence. We have also shown that Sp1 and Sp3 can bind to the GC boxes. Finally, both transient transfection analysis and gel mobility shift assay revealed that the AhRR gene is up-regulated by a p65/p50 heterodimer that binds to the NF-kappaB site when the cells has been exposed to 12-O-tetradecanoylphorbol-13-acetate, and this inducible expression was further enhanced by cotreatment of 12-O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene..