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M Kobayashi and R Horiuchi

ABSTRACT

We have elucidated the action of tri-iodothyronine (T3) on the synthesis and secretion of seven major plasma proteins in a human hepatoblastoma cell line, Hep G2, and established an in vitro experimental model of human liver cells for the study of the mechanism of the action of thyroid hormone.

Hep G2 cells cultured in serum-free medium were treated with various concentrations of T3. During the first 24 h of T3 treatment, accumulation of α-fetoprotein in the medium was decreased in a dose-dependent manner (10−11-10−8 m), and the inhibitory effect was enhanced during the second 24 h of T3 treatment. On the other hand, α1-antitrypsin accumulation in the medium during the second 24 h of hormone treatment was decreased by T3 (10−9–10−8 m), although no change in accumulation was observed during the first 24 h of T3 treatment.

The newly synthesized [35S]Met-labelled α1-acid glycoprotein was increased by T3 and reached 3·4-fold within 37 h of 10−8 m T3 treatment. The stimulatory effect increased time-dependently (4·6fold after 61 h). In contrast, the synthesis of α-fetoprotein was reduced to half of that of the control after T3 treatment for 37 h.

Although the content of newly synthesized [35S]α1-antitrypsin was not affected by 10−8 m T3 treatment during 3 days of hormone treatment, the accumulation of α1-antitrypsin in the medium decreased to 87%; in contrast, total cellular newly synthesized α1-antitrypsin increased to 105–130% of that of the control. From these results, it is suggested that α1-antitrypsin secretion might be suppressed by T3 treatment. However, T3 did not affect the accumulation of albumin, transferrin, fibronectin and α2-macroglobulin in the medium throughout the experiments.

It was shown that T3 has diverse control mechanisms on the synthesis and secretion of plasma proteins in Hep G2 cells: stimulation (α1-acid glycoprotein) or inhibition (α-fetoprotein) of plasma protein synthesis, and inhibition of protein secretion (α1-antitrypsin).

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M. Suzuki, S. Hyodo, M. Kobayashi, K. Aida and A. Urano

ABSTRACT

Gonadotrophin-releasing hormone (GnRH) is considered to have an important role in the control of reproduction in salmonid fish, although we do not have any direct evidence. To clarify this problem by molecular techniques, we first determined the nucleotide sequence of the mRNA encoding the precursor of salmon-type GnRH (sGnRH) from the masu salmon, Oncorhynchus masou.

The masu salmon sGnRH precursor was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP) which was connected to sGnRH by a Gly-Lys-Arg sequence. The amino acid sequence of sGnRH and Gly-Lys-Arg were highly conserved when compared with the corresponding regions of African cichlid sGnRH and mammalian GnRH precursors. However, the GAP region was markedly divergent, with a 66% amino acid similarity to African cichlid GAP and an 8·3–15% similarity to mammalian GAPs. Northern blot analysis indicated the presence of a single mRNA species of about 600 bases in the olfactory bulb and telencephalon and in the diencephalon. The signal was more intense in the former regions.

An in-situ hybridization study further revealed that sGnRH neurones were distributed in the olfactory nerve, the ventral part of the olfactory bulb, the ventral part of the telencephalon, the lateral preoptic area and the preoptic nucleus. The sGnRH neurones were thus longitudinally scattered between the olfactory nerve and the lateral preoptic area in the rostroventral part of brain. The intensity of the hybridization signals and the size of hybridization-positive somata were much greater in the olfactory nerve and the rostral olfactory bulb than in the other regions. Preoptic sGnRH neurones were scarcely detected in immature masu salmon, whereas they were more frequently observed in maturing animals. It is possible that the olfactory and the preoptic sGnRH neurones have different physiological roles in salmonid fish.

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M Ashihara, M Suzuki, K Kubokawa, Y Yoshiura, M Kobayashi, A Urano and K Aida

ABSTRACT

Salmon gonadotropin-releasing hormone (sGnRH) is considered to have an important role in the control of reproduction in salmonid fish. As a basis for understanding the physiological functioning of sGnRH at the molecular level, we characterized the nucleotide sequences of two types of cDNAs encoding the precursors of sGnRH in sockeye salmon (ss), Oncorhynchus nerka, by a cloning strategy based on reverse transcription-PCR. The two types of cDNAs are referred to as ss-pro-sGnRH-I and -II, and consisted of 435 and 481 bases respectively. Both precursors are predicted to contain a signal peptide, the hormone and a GnRH-associated peptide that is attached to the hormone via a Gly-Lys-Arg sequence. The presence of two types of mRNAs hybridizing with either cDNA was confirmed by Northern blot analysis of brain RNA from sockeye salmon, masu salmon, O. masou, and rainbow trout, O. mykiss. The ss-pro-sGnRH-I cDNA had 97·2% and 82·8% overall identity with sGnRH cDNA from masu salmon and putative sGnRH cDNA deduced from the gene of the Atlantic salmon, Salmo salar respectively, whereas the ss-pro-sGnRH-II cDNA had 80·0% and 91·2% overall identity with the former and the latter respectively. The nucleotide sequences of ss-sGnRH-I and -II cDNAs showed less similarity (79·3%). These results indicated that each salmonid species possesses two differing sGnRH genes. The results of Southern blot analysis using genomic DNA extracted from individuals support this evidence in sockeye salmon, masu salmon and rainbow trout.

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H Watanabe, E Takahashi, M Kobayashi, M Goto, A Krust, P Chambon and T Iguchi

Recent studies have revealed that hundreds of genes in the uterus are activated by estrogen. Their expression profiles differ over time and doses and it is not clear whether all these genes are directly regulated by estrogen via the estrogen receptor. To select the genes that may be regulated by estrogen, we treated mice with several doses of estrogen and searched for those genes whose dose–response expression pattern mirrored the uterine growth pattern. Among those genes, we found that the dose-dependent expression of the adrenomedullin (ADM) gene correlated well with the uterotrophic effect of estrogen. ADM expression is induced early after estrogen administration and is restricted to the endometrial stroma. The spatiotemporal gene expression pattern of ADM was similar to that of receptor-modifying protein 3 (RAMP3). RAMP3 is known to modify calcitonin gene-related receptor (CRLR) so that it can then serve as an ADM receptor. Chromatin immunoprecipitation assays indicated that the estrogen receptor binds directly to the ADM promoter region and RAMP3 intron after estrogen administration. It was also shown that neither the ADM nor RAMP3 gene could be activated in estrogen receptor-α-null mouse. Although uterine ADM expression has been reported to occur in the myometrium, our observations indicate that estrogen-induced ADM is also expressed in the uterine stroma and that such variable, spatiotemporally regulated ADM expression contributes to a wider range of biological effects than previously expected.

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H Watanabe, A Suzuki, M Kobayashi, E Takahashi, M Itamoto, DB Lubahn, H Handa and T Iguchi

In order to understand early events caused by estrogen in vivo, temporal uterine gene expression profiles at early stages were examined using DNA microarray analysis. Ovariectomized mice were exposed to 17beta-estradiol and the temporal mRNA expression changes of ten thousand various genes were analyzed. Clustering analysis revealed that there are at least two phases of gene activation during the period up to six hours. One involved immediate-early genes, which included certain transcription factors and growth factors as well as oncogenes. The other involved early-late genes, which included genes related to RNA and protein synthesis. In clusters of down-regulated genes, transcription factors, proteases, apoptosis and cell cycle genes were found. These hormone-inducible genes were not induced in estrogen receptor (ER) alpha knockout mice. Although expression of ERbeta is known in the uterus, these findings indicate the importance of ERalpha in the changes in gene expression in the uterus.

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S Miyagawa, A Suzuki, Y Katsu, M Kobayashi, M Goto, H Handa, H Watanabe and T Iguchi

Developmental exposure to a synthetic estrogen, diethylstilbestrol (DES), induces carcinogenesis in human and laboratory animals. In mice, neonatal DES treatment induces persistent proliferation and keratinization of the vaginal epithelium, even in the absence of the ovaries, resulting in cancerous lesions later in life. To understand the mechanisms underlying this persistent cell proliferation and differentiation, we characterized the gene expression patterns in the neonatally DES-exposed mouse vagina using DNA microarray and real-time quantitative RT-PCR. We found that genes related to cellular signaling, which are candidates for mediating the persistent proliferation and differentiation, were altered, and genes related to the immune system were decreased in the neonatally DES-exposed mouse vagina. We also noted high expression of interleukin-1 (IL-1)-related genes accompanied by phosphorylation of JNK1. In addition, expression IGF-I and its binding proteins was modulated and led to phosphorylation of IGF-I receptor and Akt, which is one of the downstream factors of IGF-I signaling. This led us to characterize the expression as well as the phosphorylation status of IL-1 and IGF-I signaling pathway components which may activate the phosphorylation cascade in the vagina of mice exposed neonatally to DES. These findings give insight into persistent activation in the vagina of mice exposed neonatally to DES.

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H Watanabe, A Suzuki, M Kobayashi, DB Lubahn, H Handa and T Iguchi

Administration of physiological and non-physiological estrogens during pregnancy or after birth is known to have adverse effects on the development of the reproductive tract and other organs. Although it is believed that both estrogens have similar effects on gene expression, this view has not been tested systematically. To compare the effects of physiological (estradiol; E2) and non-physiological (diethylstilbestrol; DES) estrogens, we used DNA microarray analysis to examine the uterine gene expression patterns induced by the two estrogens. Although E2 and DES induced many genes to respond in the same way, different groups of genes showed varying levels of maximal activities to each estrogen, resulting in different dose-response patterns. Thus, each estrogen has a distinct effect on uterine gene expression. The genes were classified into clusters according to their dose-responses to the two estrogens. Of the eight clusters, only two correlated well with the uterotropic effect of different doses of E2. One of these clusters contained genes that were upregulated by E2, which included genes encoding several stress proteins and transcription factors. The other cluster contained genes that were downregulated by E2, including genes related to metabolism, transcription and detoxification processes. The expression of these genes in estrogen receptor-deficient mice was not affected by E2 treatment, indicating that these genes are affected by the E2-bound estrogen receptor. Thus, of the many genes that are affected by estrogen, it was suggested that only a small number are directly involved in the uterotropic effects of estrogen treatment.

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K. Ichikawa, K. Hashizume, Y. Nishii, T. Takeda, M. Kobayashi, S. Suzuki and T. Yamada

ABSTRACT

Human thyroid hormone receptor (c-erb A protein) produced by Escherichia coli expression vector plasmid was purified sequentially using polyethylenimine precipitation of DNA, hydroxylapatite column chromatography, ammonium sulphate precipitation, Sephacryl S-300 gel filtration and mono Q-Sepharose column chromatography. These column procedures resulted in 41.3-fold purification of 3,5,3′-tri-iodo-l-thyronine (T3) binding activity over the initial E. coli extract. Purified protein as well as crude preparation showed high-affinity binding to T3. The c-erb A protein enriched by column purification was further purified by electroelution after electrophoresis. Rabbit antibody against the c-erb A protein was prepared and used for the Western blotting analysis. The antibody recognized c-erb A protein but not the bacterial proteins in crude E. coli extract. When partially purified rat hepatic nuclear thyroid hormone receptor was analysed, a 56kDa receptor was specifically recognized by the antibody.

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X T Chang, T Kobayashi, H Kajiura, M Nakamura and Y Nagahama

ABSTRACT

A cDNA clone encoding the complete tilapia (a teleost fish, Oreochromis niloticus) cytochrome P450 aromatase (P450arom) was isolated from an ovarian follicle cDNA library. The deduced amino acid sequence (522 amino acid residues) had 72·2% and 59·5% homology with rainbow trout and catfish P450arom respectively, and about 50% homology with mammalian and avian P450arom. Expression of this cDNA in COS-7 cells produced a protein that converted exogenous testosterone to estrogens. Northern blots using a tilapia P450arom cDNA fragment and Western blots using an antiserum against a tilapia P450arom polypeptide fragment revealed a single P450arom mRNA (2·6 kb) and a single protein (59 kDa) in tilapia ovarian tissue respectively. These analyses also revealed that the levels of both P450arom mRNA and protein were low in early vitellogenic follicles, increased in midvitellogenic follicles, and declined to non-detectable levels in post-vitellogenic follicles. Changes in the ability of follicles to convert exogenous testosterone to estrogens (aromatase activity) were similar to those of P450arom mRNA and protein. These observations indicated that the capacity of tilapia ovarian follicles to synthesize estradiol-17β is closely related to the contents of P450arom mRNA and protein within them.

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M Tanaka, M Suzuki, T Kawana, M Segawa, M Yoshikawa, M Mori, M Kobayashi, N Nakai and T R Saito

In addition to the known four alternative first exons E11, E12, E13 and E14 of the rat prolactin receptor (PRL-R) gene, a novel first exon, E15, was identified by cDNA cloning of the 5′-end region of PRL-R mRNA in the rat liver. Genomic fragments containing E15 and its 5′- or 3′-flanking regions were also cloned from rat kidney genomic DNA. A sequence search for E15 revealed that E15 is located 49 kb upstream of exon 2 of the PRL-R gene in rat chromosome 2q16. RT-PCR analysis revealed that E15 was preferentially expressed in the liver, brain and kidney. Expression profiles of E12-, E13- and E15-PRL-R mRNAs in the liver of male and female rats at 5 days of age and those at 8 weeks of age were examined by RT-PCR. The levels of E12-PRL-R mRNA in the female rat increased remarkably in rats at 8 weeks of age compared with those at 5 days of age, and the levels of E15-PRL-R mRNA in the male rat decreased markedly at 8 weeks of age compared with those at 5 days of age. In the female rat, the levels of E12-PRL-R mRNA at 8 weeks of age decreased with ovariectomy performed at 4 weeks of age and recovered with the administration of β-oestradiol. On the contrary, the levels of E15-PRL-R mRNA increased with ovariectomy and decreased with the oestrogen treatment. In the male rat liver, the levels of E12-PRL-R mRNA at 8 weeks of age increased strikingly with castration performed at 4 weeks of age and became undetectable with the administration of testosterone. The levels of E15-PRL-R mRNA increased slightly with castration and were restored by testosterone treatment. Removal of gonadal tissues and sex steroid hormone treatment had no effect on the expression levels of E13-PRL-R mRNA in both female and male rat livers. These results indicated that the expression of the PRL-R gene in the liver is regulated by the differential effects of sex steroid hormones on the transcription of the multiple first exons including the novel one.