We have previously reported that the epidermal growth factor (EGF) family growth factor, epiregulin, is expressed in rat ovarian granulosa cells by induction with pregnant mare serum gonadotropin (PMSG). In this study, we report that amphiregulin, another member of the EGF family, was also induced in the rat ovary by gonadotropin treatment. Northern blot analysis revealed that PMSG treatment induced the expression of both epiregulin and amphiregulin mRNA after 24 h, but the expression then decreased 48 h after treatment. Further treatment with human chorionic gonadotropin (hCG) rapidly induced the expression of both epiregulin and amphiregulin genes and maximal levels were reached 4 h after hCG treatment. A marginal increase in amphiregulin mRNA levels was also observed 6 h after PMSG treatment. In situ hybridization revealed that epiregulin and amphiregulin mRNAs were localized in the granulosa cells of large antral follicles. These spatio-temporal expression patterns were similar to those of cyclo-oxygenase-2 (COX-2) and progesterone receptor (PR). In adult cycling rats, epiregulin and amphiregulin were strongly induced at 1800 and 2000 h on proestrus coinciding with the preovulatory LH surge. An in situ hybridization study also showed that epiregulin and amphiregulin mRNAs were detectable in the granulosa cells of preovulatory ovarian follicles at 2000 h on proestrus, where transcripts of COX-2 and PR were co-localized with those of epiregulin and amphiregulin. These observations suggested that the EGF family members, epiregulin and amphiregulin, may play a role in the ovulatory process of cycling rats as well as in the induction of ovulation in immature rats.
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T Sekiguchi, T Mizutani, K Yamada, T Kajitani, T Yazawa, M Yoshino, and K Miyamoto
T Monden, M Yamada, S Konaka, T Satoh, H Ezawa, T Iwasaki, and M Mori
ABSTRACT
To gain insight into the mechanism underlying the epidermal growth factor (EGF)-induced changes in responsiveness to TRH and in the numbers of TRH receptors (TRH-Rs) in the pituitary, we investigated the transcriptional regulation by EGF of the TRH-R gene in GH4C1 cells. Northern blot analyses and binding studies revealed that EGF reduced both TRH binding and TRH-R mRNA levels in a dose- and time-dependent manner, while no significant changes were observed in β-actin mRNA levels. Addition of actinomycin D caused an acute increase in the basal TRH-R mRNA level, and the rate of decrease of the TRH-R mRNA was identical in control and EGF-treated groups, suggesting that the stability of the TRH-R mRNA was not significantly affected in EGF-treated cells. Incubation with cycloheximide also induced an increase in the basal TRH-R mRNA level and completely reversed the EGF-induced reduction of TRH-R mRNA levels. Furthermore, a nuclear run-on assay demonstrated that the rate of transcription of the TRH-R gene was significantly inhibited in cells treated with EGF. We conclude that (1) EGF decreases the expression of the TRH-R mRNA largely by reducing its rate of transcription, and this action requires the synthesis of new proteins, and (2) inhibitors of protein and RNA synthesis cause a significant increase in the basal TRH-R mRNA level, suggesting that there may be a short-lived protein suppressing the TRH-R mRNA level in the pituitary.
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.
N. Takasu, I. Komiya, Y. Nagasawa, T. Asawa, Y. Shimizu, and T. Yamada
ABSTRACT
The role of calcium in cytoplasmic pH (pHi) changes was studied using 2′,7′-bis(2-carboxyethyl)-5-(and 6-)carboxyfluorescein, an internalized fluorescent pH indicator, in cultured porcine thyroid cells. The Ca2+ ionophores A23187 and ionomycin stimulated thyroid cell alkalinization. An increase in cytoplasmic free calcium resulted in activation of Na+/H+ exchange which alkalinized the cells.
H Wang, Y Horikawa, L Jin, T Narita, S Yamada, N Shihara, K Tatemoto, M Muramatsu, T Mune, and J Takeda
To clarify tissue-specificity of pancreatic β cells, comparison of mRNA expression in various conditions of the tissue of multiple organisms is important. Although the developed methodologies for mRNA monitoring such as microarray, rely on the growth of dbEST (database of expressed sequence tag), a large number of unknown genes in the genome, especially in the rat, have not been shown to be expressed. In this study, we have established the first database of ESTs from rat pancreatic islet and RINm5F cells. Two cDNA libraries were constructed using mRNAs from rat pancreatic islet and RINm5F cells to cover a wider spectrum of expressed genes. Over 40 000 clones were randomly selected from the two libraries and partially sequenced. The sequences obtained were subjected to BLAST database analyses. This large-scale sequencing generated 40 710 3′-ESTs. Clustering analysis and homology search of nucleotide and peptide databases using both 3′- and 5′-ESTs revealed 10 406 non-redundant transcripts representing 4078 known genes or homologs and 6328 unknown genes. To confirm actual expression, the unknown sequences were further subjected to dbEST search, resulting in the identification of 5432 significant matches to those from other sources. Interestingly, of the remaining sequences showing no match, 779 were found to be encoded by exon–intron organization in the corresponding genomic sequences, suggesting that these are newly found as actually expressed in this study. Since many genes are up- or down-regulated in differing conditions, applications of the expression profile should facilitate identification of the genes involved in cell-specific functions in normal and disease states.
