| Co-Investigator(Kenkyū-buntansha) |
MATSUMOTO Keishi Osaka Univ., Dept. of Patholagy. Professor, 医学部, 教授 (70028299)
KOUHARA Haruhiko Osaka Univ., The Third Dept. of Int. Med., 医学部・附属病院, 医員
KASAYAMA Soji Osaka Univ., The Third Dept. of Int. Med., 医学部・附属病院, 医員
KOGA Masafumi Osaka Univ., The Third Dept. of Int. Med., Assistant Prof., 医学部, 助手 (00186652)
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| Research Abstract |
The cultured cell lines derived from mouse mammary carcinoma and mouse Leyding cell tumor have found to contain various nuclear receptors belonging to steroid/thyroid/vitamin receptor superfamily. The cells, termed as SC-3, derived from mouse mammary carcinoma were growth-stimulated by both glucocorticoid and androgen. In addition, these hormones were also able to induce an identical secretory protein with a molecular weight of 24K as well as a fibroblast growth factor-like polypeptide. These results strongly suggest that both glucocorticoid and androgen receptor can activate an identical gene (s). Very interestingly, androgen-induced enhancement was partially inhibited by simultaneous stimulation with glucocorticoid. in addition, the unphysiological high concentrations of glucocorticoid was required to elicit the responses. These findings lead us to speculate that glucocorticoid in SC-3 cells has abnormal characters. To address these aspects more directly, MMTV-CAT reporter plasmid wa
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s transfected into SC-3 cells, and subsequently stimulated with various concentrations of glucocorticoid alone or in combination with androgen. To induce CAT activity, high concentrations (10^<-7>-10^<-6>M) of dexamethasone were again needed. However, co-transfection of glucocorticoid receptor expression vector resulted in a marked CAT induction at a physiological concentration (10^<-8>M) of dexamethasone. Testosterone stimulation also induced CAT activities. This androgeninduced C -10^<-6>M) of dexamethasone were again needed. However, co-transfection of glucocorticoid receptor expression vector resulted in a marked CAT induction at a physiological concentration (10^<-8>M) of dexamethasone. Testosterone stimulation also induced CAT activities. This androgeninducedAT activity was almost completely inhibited by a physiological concentration (10^<-8>M) of dexamethasone. Thus, glucocor-ticoid receptor in SC-3 cells is a suppressor at a physiological concentration of dexamethasone in terms of gene expression. Polymerase chain reaction (PCR) method revealed tthat glucocorticoid receptor in SC-3 cells contain a point mutation in C domain from valine to glycine. We conclude that this point mutation provides this glucocorticoid receptor with unique functions.
The cells, termed as B-1 F, derived from mouse Leydig cell tumor were growth-stimulated by estrogen. B-1 F cells were found to contain a unique estrogen receptor which is tightly associated with nuclei as a unliganded but transformed (5S state) form. When ERE_2-tk-CAT reporter plasmid was transfected into B-1 F cells, a physiological concentration of estradiol was required to induce maximum CAT activity. Interestingly, stimulation of transfected B-1 F cells with 10^<-7>M retinoic acid also resulted in CAT induction. B-1 F cells contained retinoic acid receptor-alpha. This retinoic acid-dependent CAT induction was blocked by antiestrogen, suggesting that retinoic acid-induced activation of ERE_2 is mediated through estrogen-receptor. Actually, retionic acid can stimulate the growth of B-1 F cells. To clarify this signal transmission mechanism, the molecular structure of both retionic acid and estrogen receptor was analyzed by PCR method. Any mutation of retionoic acid receptor could not be detected, but estrogen receptor was found to contain a point mutation in D domain from glutamine to lysine. This mutation created a nuclear transfer signal similar to that identified in SV-40 large T antigen. Therefore, we would conclude that this mutation of estrogen receptor cause its unique molecular structure and its interaction with retinoic acid receptor. Less
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