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Elucidation of novel mechanism of thyroid hormone action

Elucidation of novel mechanism of thyroid hormone action
阐明甲状腺激素作用的新机制
批准号:
09671044
负责人:
MURATA Yoshiharu
金额:
$2.3万
依托单位:
依托单位国家:
日本
项目类别:
Grant-in-Aid for Scientific Research (C)
财政年份:
1997
资助国家:
日本
项目状态:
已结题
起止时间:
1997 至 1998

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中文摘要
翻译
ZAKI-4是我们最近在人类皮肤成纤维细胞中发现的甲状腺激素反应基因。添加甲状腺激素的活性形式3,3 ',5-三碘甲状腺原氨酸(T_3)可增强皮肤成纤维细胞中ZAKII-4基因的转录。然而,转录激活完全阻断预处理放线菌酮,蛋白质合成的翻译抑制剂。结果表明,T_3激活ZAKI-4基因的转录需要从头蛋白质合成。T_3可能诱导了某些因子增强ZAKI-4基因的转录。T_3是通过与核受体结合,调节靶基因的表达而发挥作用的,因此,T_3诱导因子对ZAKI-4基因转录的促进作用可能为甲状腺激素的作用机制提供新的线索。因此,我们设计了本研究项目,以阐明T_ 关于我们 通过使用ZAKI-4基因的5 ′-侧翼区的ZAKI-4基因的3依赖性转录激活。我们通过引物延伸法鉴定了转录起始位点。利用PromoterFinderTM技术,我们在ZAKI-4的5'侧翼区克隆了一个约2.8kb的DNA片段,命名为alphaE 2 -5。我们构建了一个嵌合荧光素酶报告基因与alphaE 2 -5,并将此构建体转染到ZAKI-4表达的人胶质瘤细胞系,U-251-MG。与alphaE 2 -5,荧光素酶活性增加5倍,观察到,alphaE 2 -5含有一个区域,介导的ZAKI-4的启动子活性。我们还对启动子区进行了精确的分析,发现-28 ~-4区域介导了启动子的活性以及T_3依赖的转录调控。通过使用U-251 MG的核提取物的电泳迁移率变动分析(EMSA),我们证实了提取物含有与TFZ-1结合的蛋白质。以TFZ-1为探针,用Southwestern印迹法进一步分析了该蛋白,结果表明该蛋白的表观分子量为45 kDa,加入10 μ M T_3后表达量增加<-7>。这些结果表明TMZ 45蛋白是T_3介导ZAKI-4转录调控的因子。目前,我们正在尝试用酵母单杂交系统克隆TMZ 45的cDNA。少
英文摘要
ZAKI-4 is a thyroid hormone-responsive gene that we have recently identified in human skin fibroblasts. Addition of 3, 3', 5-triiodothyronine (T_3), an active form of thyroid hormone, enhances the transcription of the ZAKII-4 gene in skin fibroblasts. However, transcriptional activation was completely blocked by the pretreatment with cycloheximide, a translation inhibitor of protein synthesis. The result indicates that de novo protein synthesis is required to activate the transcription of the ZAKI-4 gene by T_3. It appears that T_3 induces some factor(s) which enhances ZAKI-4 gene transcription. Since it has generally been accepted that T_3 exerts its effect through binding to its nuclear receptor and regulating expression of target genes, the identification of the T_3 induced factor(s) that enhance the transcription of the ZAKI-4 gene could uncover a novel mechanism of thyroid hormone action. Therefore, we designed the current research project in order to elucidate the mechanism of T_ … More 3 dependent transcriptional activation of ZAKI-4 gene by using a 5'-flanking region of the ZAKI-4 gene. We identified the transcription start site by the primer extension method. Using PromoterFinderTM, we cloned a DNA fragment (about 2.8 kb, designated as alphaE2-5) in the 5' flanking region of ZAKI-4. We constructed a chimeric luciferase reporter gene with alphaE2-5 and transfected this construct to a ZAKI-4 expressing human glioma cell line, U-251-MG.With alphaE2-5, a 5-fold increase in luciferase activity was observed, indicating that alphaE2-5 contains a region which mediates the promoter activity of ZAKI-4. We have also precisely analyzed the promoter region and found that a region from -28 to -4 mediated the promoter activity as well as the T_3 dependent transcriptional regulation. By electrophoretic mobility shift assay (EMSA) using the nuclear extract from U-251MG, we confirmed that the extract contained a protein which binds to TFZ-1. We further analyzed the protein by South Western blotting with TFZ-1 as a probe and demonstrated that the apparent molecular mass of the protein is 45 kDa and expression increased by the addition of 10^<-7>M T_3. These results suggest that the protein designated as TMZ45 is the factor which mediates transcriptional regulation of ZAKI-4 by T_3. Currently we are trying to clone the cDNA for TMZ45 using the yeast one hybrid system. Less
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S.Yamaguchi, Y.Murata, T.Nagaya, Y.Hayashi, S.Ohmori, Y.Nimura, H.Seo: "Glucocorticoids increase retinoid-X receptor alpha (RXRalpha) expression and enhance thyroid hormone action in primary cultured rata hepatocytes." J.Mol.Endocrinol. 22. 81-91 (1999)
S.Yamaguchi、Y.Murata、T.Nagaya、Y.Hayashi、S.Ohmori、Y.Nimura、H.Seo:“糖皮质激素可增加原代培养的大鼠肝细胞中视黄醇 X 受体 α (RXRα) 的表达并增强甲状腺激素的作用
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T.NAGAYA: "Intracellular proteolytic cleavage of 9-cis-retinoic acid receptor α by cathepsin L-type protease is a potential mechanism for modulating thyroid hormone action" The Journal of Biological Chemistry. 273・50. 33166-33173 (1998)
T.NAGAYA:“组织蛋白酶 L 型蛋白酶对 9-顺式视黄酸受体 α 的细胞内蛋白水解是调节甲状腺激素作用的潜在机制”《生物化学杂志》273·50(1998)。
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A.Siddiq, T.Miyazaki, Y.Kanou, Y.Takagishi, M.Inouye, Y.Murata: "Tissue distribution of novel thyroid hormone-responsive gene, ZAKI-4, in the rat and its ontogenic expression in the brain." Environ.Med.42(1). 54-56 (1998)
A.Siddiq、T.Miyazaki、Y.Kanou、Y.Takagishi、M.Inouye、Y.Murata:“新型甲状腺激素反应基因 ZAKI-4 在大鼠中的组织分布及其在大脑中的个体发育表达。
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S.YAMAGUCHI: "Glucocorticoids increase retinoid-X receptor alpha(RXRα) expression and enhance thyroid hormone action in primary cultured rat hepatocytes." Journal of Molecular Endocrinology. 22. 81-91 (1999)
S.YAMAGUCHI:“糖皮质激素可增加原代培养大鼠肝细胞中视黄醇 X 受体 α (RXRα) 的表达并增强甲状腺激素的作用。”《分子内分泌学杂志》22. 81-91 (1999)。
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