Shinya Kawabe

Androgen/androgen receptor pathway regulates expression of the genes for cyclooxygenase-2 and amphiregulin in periovulatory granulosa cells☆

It is well known that the androgen/androgen receptor (AR) pathway is involved in both male and female fertility in mammals. AR knockout female mice are reported to exhibit various abnormalities in follicle development, and a subfertile phenotype. In exogenous gonadotropin-induced superovulation, serum androgen levels were robustly elevated in female mice at the periovulatory stage after human chorionic gonadotropin (hCG) treatment. At this stage, ovarian AR proteins were strongly expressed in cumulus cells. Because these results suggested that the androgen/AR pathway is involved in ovulation, we investigated the expression of ovulation-related genes in the mouse ovary treated with the nonaromatizable androgen, 5α-dihydrotestosterone (DHT). DHT treatment induced the expression of the genes for cyclooxyganase-2 (Cox-2 or prostaglandin endoperoxidase synthase 2) and the epidermal growth factor-like factor, amphiregulin (Areg), in the ovary, whereas their hCG-induced expression was suppressed by the AR antagonist flutamide. These genes were also induced by DHT in AR-expressing primary granulosa and granulosa tumor-derived cells. Reporter assays, electrophoretic shift mobility assays and chromatin immunoprecipitation assays demonstrated that androgen response sequence(s) existing upstream of each gene were responsible for androgen responsiveness and were occupied by the AR in periovulatory granulosa cells. Our results suggest that the androgen/AR pathway is involved in the ovulatory process via expression of the Cox-2 and Areg genes in periovulatory granulosa cells.

Differentiation of mesenchymal stem cells and embryonic stem cells into steroidogenic cells using steroidogenic factor-1 and liver receptor homolog-1.

Previously, we have demonstrated that mesenchymal stem cells could be differentiated into steroidogenic cells through steroidogenic factor-1 and 8bromo-cAMP treatment. Use of liver receptor homolog-1, another of the nuclear receptor 5A family nuclear receptors, with 8bromo-cAMP also resulted in the differentiation of human mesenchymal stem cells into steroid hormone-producing cells. The same approaches could not be applied to other undifferentiated cells such as embryonic stem cells or embryonal carcinoma cells, because the over-expression of the nuclear receptor 5A family is cytotoxic to these cells. We established embryonic stem cells carrying tetracycline-regulated steroidogenic factor-1 gene at the ROSA26 locus. The embryonic stem cells were first differentiated into a mesenchymal cell lineage by culturing on collagen IV-coated dishes and treating with pulse exposures of retinoic acid before expression of steroidogenic factor-1. Although the untreated embryonic stem cells could not be converted into steroidogenic cells by expression of steroidogenic factor-1 in the absence of leukemia inhibitory factor due to inability of the cells to survive, the differentiated cells could be successfully converted into steroidogenic cells when expression of steroidogenic factor-1 was induced. They exhibited characteristics of adrenocortical-like cells and produced a large amount of corticosterone. These results indicated that pluripotent stem cells could be differentiated into steroidogenic cells by the nuclear receptor 5A family of protein via the mesenchymal cell lineage. This approach may provide a source of cells for future gene therapy for diseases caused by steroidogenesis deficiencies.

Stem cell differentiation into steroidogenic cell lineages by NR5A family.

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 (LRH-1), 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.

cDNA cloning and expression of grp94 in the Pacific oyster Crassostrea gigas

The 94-kDa glucose-regulated protein (GRP94) is an endoplasmic reticulum (ER) chaperone. We cloned the first mollusk grp94 from a cDNA library of the Pacific oyster Crassostrea gigas. Analysis of C. gigas grp94 (cggrp94) clone containing 3212 bp DNA revealed that the cDNA contains a 2391 bp open reading frame that encodes a 797 amino acid protein of 91.6 kDa. The deduced amino acid sequence of cgGRP94 is 67%, 68%, and 67% homologous to the GRP94 of Homo sapiens, GP96 of Strongylocentrotus purpuratus, and GP96 of Xenopus laevis, respectively. CgGRP94 contains an N-terminal 22 amino acid sequence, which is characteristic of a signal sequence. It also contains a HATPase_c domain. In addition, it contains the KDEL (-Lys-Asp-Glu-Leu) peptide motif at the C-terminus, which suggests that cgGRP94 localizes in the ER. Northern blot analysis showed that cggrp94 mRNA is expressed at high levels in the gill which cggrp94 mRNA is induced during air exposure condition. Expression patterns of cggrp94 mRNA differed between gill and mantle, and cggrp94 mRNA was induced at high temperature during air exposure condition. These indicate that cggrp94 mRNA is induced by hypoxia and heat shock stress, and there are different strategies for air exposure condition between gill and mantle.

Novel isoforms of heat shock transcription factor 1 are induced by hypoxia in the Pacific oyster Crassostrea gigas

The Pacific oyster Crassostrea gigas inhabits the intertidal zone and shows tolerance to various stress conditions such as hypoxia and heat shock. However, little is known about the cellular mechanism of responses to these stresses. Heat shock transcription factor 1 (HSF1) regulates the transcription of several genes, including heat shock proteins (HSPs). In this study, we cloned HSF1 from the oyster and investigated its response to air-exposure. The cDNA of oyster Hsf1 contains 2,931 bp, of which 1,389 bp encode a protein of 463 amino acid residues. Moreover, we found that the oyster has seven novel alternatively spliced isoforms, Hsf1b-h, consisting of insertion A with 48 bp, insertion B with 42 bp and/or insertion C with 42 bp. We determined the sequences of oyster genomic DNA containing Hsf1 insertions A, B and C. The results indicated that eight isoforms of Hsf1 are generated from a single Hsf1 gene by alternative splicing without frameshifting. Real-time PCR analysis showed that Hsf1a is expressed constitutively, and the expression of Hsf1b-h and Hsp70 mRNA is induced by air exposure and/or hypoxia. In addition, we found that 11 putative hypoxia response elements, which are hypoxia-inducible factor 1 (HIF-1) binding sequences, are located in the Hsf1 promoter region. These data suggest that the oyster has an HIF-HSF pathway in which HSPs are induced in an HIF-dependent manner, and that it also has a novel mechanism of alternative splicing of Hsf1 in response to hypoxia.

Nuclear Receptor 5A (NR5A) Family Regulates 5-Aminolevulinic Acid Synthase 1 (ALAS1) Gene Expression in Steroidogenic Cells

5-Aminolevulinic acid synthase 1 (ALAS1) is a rate-limiting enzyme for heme biosynthesis in mammals. Heme is essential for the catalytic activities of P450 enzymes including steroid metabolic enzymes. Nuclear receptor 5A (NR5A) family proteins, steroidogenic factor-1 (SF-1), and liver receptor homolog-1 (LRH-1) play pivotal roles in regulation of steroidogenic enzymes. Recently, we showed that expression of SF-1/LRH-1 induces differentiation of mesenchymal stem cells into steroidogenic cells. In this study, genome-wide analysis revealed that ALAS1 was a novel SF-1-target gene in differentiated mesenchymal stem cells. Chromatin immunoprecipitation and reporter assays revealed that SF-1/LRH-1 up-regulated ALAS1 gene transcription in steroidogenic cells via binding to a 3.5-kb upstream region of ALAS1. The ALAS1 gene was up-regulated by overexpression of SF-1/LRH-1 in steroidogenic cells and down-regulated by knockdown of SF-1 in these cells. Peroxisome proliferator-activated receptor-γ coactivator-1α, a coactivator of nuclear receptors, also strongly coactivated expression of NR5A-target genes. Reporter analysis revealed that peroxisome proliferator-activated receptor-γ coactivator-1α strongly augmented ALAS1 gene transcription caused by SF-1 binding to the 3.5-kb upstream region. Finally knockdown of ALAS1 resulted in reduced progesterone production by steroidogenic cells. These results indicate that ALAS1 is a novel NR5A-target gene and participates in steroid hormone production.

Transcriptional regulation of human ferredoxin 1 in ovarian granulosa cells.

Ferredoxin 1 (FDX1; adrenodoxin) is an iron-sulfur protein that is involved in various metabolic processes, including steroid hormone synthesis in mammalian tissues. We investigated the transcriptional regulation of FDX1 in ovarian granulosa cells. Previously, we reported that the NR5A family, including steroidogenic factor-1 (SF-1) and liver receptor homolog-1 could induce differentiation of human mesenchymal stem cells (hMSCs) into steroidogenic cells. A ChIP assay showed that SF-1 could bind to the FDX1 promoter in differentiated hMSCs. Luciferase reporter assays showed that transcription of FDX1 was synergistically activated by the NR5A family and 8Br-cAMP treatment through two SF-1 binding sites and a CRE-like sequence in a human ovarian granulosa cell line, KGN. Knockdown of FDX1 attenuated progesterone production in KGN cells. These results indicate transcription of FDX1 is regulated by the NR5A family and cAMP signaling, and participates in steroid hormone production in ovarian granulosa cells.

Molecular cloning of calnexin and calreticulin in the Pacific oyster Crassostrea gigas and its expression in response to air exposure.

Calnexin (CNX) and calreticulin (CRT) are endoplasmic reticulum (ER) chaperones. CNX is a type I transmembrane protein and CRT is a soluble CNX homologue. In the ER, CNX and CRT are important for Ca(2+) homeostasis and protein maturation. Here, we describe the full-length cDNA of the first mollusk CNX (cgCNX) and a second mollusk CRT (cgCRT) from the oyster Crassostrea gigas. CgCNX, containing 3255bp, was composed of a 1764bp open reading frame (ORF) that encodes a 588-amino acid protein. CgCRT, containing 1727bp, was composed of a 1242bp ORF that encodes a 414-amino acid protein. CgCNX and cgCRT contains an N-terminal 21- and 16-amino acid sequence, respectively, which is characteristic of a signal sequence. At the C-terminus, cgCRT also contains the KDEL (-Lys-Asp-Glu-Leu) peptide motif suggesting that cgCRT localizes in the ER. Northern blot analysis showed that both cgCNX and cgCRT mRNAs are induced by air exposure. The expression patterns of cgCNX mRNA differed from those of cgCRT during air exposure. This suggests that these two molecular chaperones have different roles in the response to air exposure.

Transcriptional regulation of genes related to progesterone production [Review]

Steroid hormones are synthesized from cholesterol in various tissues, mainly in the adrenal glands and gonads. Because these lipid-soluble steroid hormones immediately diffuse through the cells in which they are produced, their secretion directly reflects the activity of the genes related to their production. Progesterone is important not only for luteinization and maintenance of pregnancy, but also as a substrate for most other steroids. Steroidogenic acute regulatory protein (STAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4) isomerase (3β-HSD) are well-known proteins essential for progesterone production. In addition to them, glutathione S-transferase A1-1 and A3-3 are shown to exert Δ(5)-Δ(4) isomerization activity to produce progesterone in a cooperative fashion with 3β-HSD. 5-Aminolevulinic acid synthase 1, ferredoxin 1, and ferredoxin reductase also play a role in steroidogenesis as accessory factors. Members of the nuclear receptor 5A (NR5A) family (steroidogenic factor 1 and liver receptor homolog 1) play a crucial role in the transcriptional regulation of these genes. The NR5A family activates these genes by binding to NR5A responsive elements present within their promoter regions, as well as to the elements far from their promoters. In addition, various NR5A-interacting proteins including peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear receptor subfamily 0, group B, member 1 (DAX-1), and CCAAT/enhancer-binding proteins (C/EBP) are involved in the transcription of NR5A target genes and regulate the transcription either positively or negatively under both basal and tropic hormone-stimulated conditions. In this review, we describe the transcriptional regulation of genes related to progesterone production.

Establishment and characterization of a novel orthotopic mouse model for human uterine sarcoma with different metastatic potentials

Uterine sarcomas are rare and aggressive gynecologic tumors with a poor prognosis because of recurrence and metastasis. However, the mechanisms of uterine sarcoma metastasis are largely unknown. To investigate this mechanism, we developed a novel uterine sarcoma tissue-derived orthotopic and metastatic model in KSN nude mice using a green fluorescent protein stably expressed uterine sarcoma cell line, MES-SA. Histological analysis showed that all orthotopic primary tumors were undifferentiated sarcoma. Primary tumors were characterized by high (18)F-fluorodeoxyglucose uptake with a positive correlation to the number of pulmonary metastases. In addition, we generated uterine sarcoma cell sublines with high or low metastatic potentials by serial in vivo selection. Microarray analysis between orthotopic tumors with high and low metastatic potentials revealed differential expression of genes related to cell proliferation and migration (TNNT1, COL1A2, and ZIC1). Our model would be useful to compensate for the limited clinical cases of uterine sarcoma and to investigate the molecular mechanisms of metastatic uterine sarcoma.