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

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。 关于我们 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。此外，我还利用PCR技术克隆了青鳉CT/CGRP基因3 '非编码区至下游区域的3.8 kbpDNA片段。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

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