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，被用作治疗骨疾病的治疗药物。在哺乳动物中，这种激素是由分散在甲状腺中的c细胞分泌的，但在其他脊椎动物中，产生ct的细胞是由鳃末梢腺分泌的。然而，对于增强这些细胞中CT基因表达的分子机制知之甚少。在第一个实验中，我通过抑制-减法杂交表征了虹鳟鱼（Oncorhynchus mykiss）鳃末腺中表达的mrna。对110个cDNA片段的序列分析表明，CT细胞存在Is（88）、He （E3）、IIs （S2）三种角蛋白，以及据报道在哺乳动物表皮中表达的钙结合蛋白ictacalcin，提示CT细胞具有上皮细胞的特征。细胞骨架蛋白4.1G也被鉴定出来。此外，我还发现了一种分泌糖蛋白，聚簇蛋白，它与其他动物的各种生理过程有关。RT-PCR分析显示，clusterin基因在虹鳟鱼的所有组织中均有表达，在鳃末梢腺中表达量最高。抗聚簇素抗体免疫荧光染色显示鳃末腺阳性标记。面向滤泡腔的CT细胞顶膜上可见强烈的免疫反应，提示簇蛋白在鳃末腺的滤泡内积聚。沿甲状腺滤泡上皮细胞的顶膜、肠的杯状细胞和鳃的粘液细胞也检测到阳性标记。在鳃末腺中表达的转录因子，通过cDNA克隆鉴定到Nkx2.1d、Paxl和FoxF1。最近有报道称FoxA2 （hnf -3 β）在哺乳动物CT产生细胞系CA77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，FoxA2的作用似乎还不够强。因此，在第二个实验中，我尝试从虹鳟鱼的鳃末梢腺中克隆翼螺旋/叉头转录因子Foxa （HNF-3）或类似因子的cdna。分离编码FoxA2的cDNA， RT-PCR分析显示FoxA2 mRNA在UB、肝脏和肠道中均有表达。FoxA2被认为存在于UB实质细胞中。为了研究FoxA2是否激活了CT启动子的转录，我进一步利用PCR技术从虹鳟中克隆了包含鲑鱼型CT- ib基因外显子I的2 kbp上游区域和5'-非编码区域的DNA片段。另外，我利用PCR技术从medaka中克隆了一个3.8 kbp的DNA片段，从IV外显子的3'-非编码区到CT/CGRP基因的下游区域。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，FoxA1和/或FoxA2与荧光素酶载体共转染并没有增加转录活性。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