Transcription factors involved in the gene transcription of calcium-regulating hormone

参与钙调节激素基因转录的转录因子

• 批准号: 18570058
• 负责人: SUZUKI Masakazu
• 金额: $ 2.63万
• 依托单位: Shizuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570058/
• 关键词: Rainbow trout; Calcitonin; Ultimobranchial gland; Thyroidal C cells; Transcription factors

Calcitonin (CT) inhibits bone resorption in mammals, and is used as a therapeutic medicine for the treatment of bone disorders. In mammals, this hormone is secreted by the C-cells scattered in the thyroid, but as for the other vertebrates, CT-producing cells form the ultimobranchial gland. However, little is known about the molecular mechanisms intensifying CT gene expression in these cells.In the first experiment, I have characterized mRNAs expressed in the ultimobranchial gland of rainbow trout (Oncorhynchus mykiss) by suppression-subtractive hybridization. Sequence analysis of 110 cDNA fragments indicated the existence of three types of keratins, Is (88), He (E3), and IIs (S2), and ictacalcin, a calcium-binding protein reported to be expressed in the epidermis in mammals, suggesting that the CT cell has characteristics of the epithelial cell. Cytoskeletal protein 4.1G was also identified. Furthermore, I found a secretory glycoprotein, clusterin, which is implicated in various physio … More logical processes in other animals. RT-PCR analysis showed that clusterin gene is expressed in all the tissues examined in rainbow trout, with the highest level in the ultimobranchial gland. Immunofluorescene staining with an anti-clusterin antibody revealed positive labels over the ultimobranchial gland. Intense immunoreativity was observed along the apical membrane of CT cells facing the follicular lumen, suggesting that clusterin accumulates in the follicles of ultimobranchial gland. Positive labels were also detected along the apical membrane of the follicular epithelial cells of the thyroid, at goblet cells in the intestine, and at mucous cells in the gills.As for transcription factors expressed in the ultimobranchial gland, Nkx2.1d, Paxl, and FoxF1 had been identified by cDNA cloning. It was recently reported that FoxA2 (HNF-3beta) is expressed in mammalian CT-producing cell lines, CA77, and increases transcriptional activity of rat CT/CT gene-related peptide (CGRP) gene. However, that effect of FoxA2 does not seem to be strong enough. In the second experiment, therefore, I tried cloning the cDNAs for winged helix/forkhead transcription factor Foxa (HNF-3) or similar factors from the ultimobranchial gland of rainbow trout. The cDNA encoding FoxA2 was isolated, and RT-PCR analysis showed the expression of FoxA2 mRNA in the UB, liver, and intestine. FoxA2 is considered to exist in the parenchymal cells of UB.To investigate whether FoxA2 activate stranscription from the CT promoter, I further cloned a DNA fragment encompassing the 2 kbp upstream region and 5'-noncoding region of the exon I of salmon-type CT-Ib gene from the rainbow trout, using PCR. In addition, I cloned a 3.8 kbp DNA fragment ranging from the 3'-noncoding region of exon IV to downstream region of CT/CGRP gene from medaka, using PCR. I inserted these DNA fragments into a luciferase reporter vector, and performed luciferase assay. However, transcriptional activity was not increased by co-transfection of FoxA1 and/or FoxA2 with the luciferase vector. Less

降钙素（CT）抑制哺乳动物的骨吸收，并用作治疗骨病的治疗药物。在哺乳动物中，这种激素由分散在甲状腺中的C细胞分泌，但对于其他脊椎动物，CT产生细胞形成后鳃腺。然而，很少有人知道的分子机制，加强CT基因在这些cells.In第一个实验中，我的特点是在后鳃腺虹鳟鱼（虹鳟鱼mykiss）通过抑制消减杂交的mRNA表达。110个cDNA片段的序列分析表明，存在三种类型的角蛋白，Is（88），He（E3），和IIs（S2），和ictacalcin，钙结合蛋白，报道在哺乳动物表皮中表达，这表明CT细胞具有上皮细胞的特征。还鉴定了细胞骨架蛋白4.1G。此外，我还发现了一种分泌性糖蛋白，丛生蛋白，它与各种生理学过程有关。 ...更多信息 其他动物的逻辑过程。RT-PCR分析表明，clusterin基因在虹鳟鱼的所有组织中均有表达，后鳃腺中表达量最高。免疫荧光染色与抗clusterin抗体显示阳性标签后鳃腺。沿着CT细胞顶膜朝向滤泡腔方向呈强免疫反应性，提示丛生素在后鳃腺滤泡内聚集。在后鳃腺中表达的转录因子中，Nkx2.1d、Pax 1和FoxF 1均为阳性。最近报道，FoxA 2（HNF-3 β）在哺乳动物CT产生细胞系CA 77中表达，并增加大鼠CT/CT基因相关肽（CGRP）基因的转录活性。然而，FoxA 2的效果似乎不够强。因此，在第二个实验中，我尝试从虹鳟鱼的后鳃腺中克隆翅螺旋/叉头转录因子Foxa（HNF-3）或类似因子的cDNA。RT-PCR分析显示FoxA 2 mRNA在UB、肝和肠中均有表达。为了研究FoxA 2是否能激活CT启动子的转录，我进一步用PCR方法克隆了虹鳟鱼CT-Ib基因外显子I上游2kbp和5 ′端非编码区的DNA片段。另外，本研究还利用PCR技术从青鳉鱼中克隆了一个3.8kbp的DNA片段，该片段位于青鳉鱼CT/CGRP基因外显子IV的3 '端非编码区至下游区域之间。我将这些DNA片段插入到荧光素酶报告载体中，并进行荧光素酶测定。然而，转录活性没有增加的FoxA 1和/或FoxA 2与荧光素酶载体的共转染。少

项目成果

Functional analysis of Nkx2.1 and Pax9 for calcitonin gene transcription.

Nkx2.1 和 Pax9 降钙素基因转录的功能分析。

• 发表时间: 2007
• 期刊: Gen Comp Endocrinol (In press)
• 作者: Suzuki M;Katagiri N;Ueda M;Tanaka S
• 通讯作者: Tanaka S

ニジマス鯉後腺で発現しているmRNAの解析

虹鳟鱼鲤腺mRNA表达分析

• 发表时间: 2006
• 作者: 土岐晋吾;上田誠;田中滋康;鈴木雅一
• 通讯作者: 鈴木雅一

甲状腺の分化に関連する遺伝子Nkx2.1の分子進化

甲状腺分化相关基因Nkx2.1的分子进化

• 发表时间: 2006
• 期刊: 生体の科学 57
• 作者: 鈴木雅一;田中滋康
• 通讯作者: 田中滋康

ニジマスにおけるクラスタリンの分布に関する分子組織学的解析

虹鳟鱼凝聚素分布的分子组织学分析

• 发表时间: 2007
• 作者: 福井亮司;中倉敬;田中滋康;鈴木雅一
• 通讯作者: 鈴木雅一

Transcription factors expressed in calcitonin-producing cells and their functions

降钙素产生细胞中表达的转录因子及其功能

• 发表时间: 2006
• 作者: Suzuki;M.;Katagiri;N.;Toki;S.;Ueda;M.;Tanaka;S
• 通讯作者: S