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，被用作治疗骨病的药物。在哺乳动物中，这种激素是由散布在甲状腺中的C细胞分泌的，但对于其他脊椎动物来说，产生CT的细胞形成了多颧腺。然而，对于CT基因在这些细胞中表达增强的分子机制，我们知之甚少。在第一个实验中，我用抑制-消减杂交的方法鉴定了虹鱼(Oncorhynchus MykISS)多鳃腺中表达的mRNAs。对110个片段的序列分析表明，存在3种角蛋白，即IS(88)、He(E3)和IIs(S2)，以及据报道在哺乳动物表皮中表达的钙结合蛋白ICTCALIN，表明CT细胞具有上皮细胞的特征。细胞骨架蛋白4.1g也得到鉴定。此外，我发现了一种分泌型糖蛋白--聚集素，它与各种Physio…有关。在其他动物身上有更多的逻辑过程。RT-PCR分析表明，聚集素基因在虹鳟鱼的所有组织中都有表达，其中在多鳃腺中的表达水平最高。抗丛集素抗体的免疫荧光染色显示多鳃腺体上有阳性标记。CT细胞面对滤泡腔的顶膜上有较强的免疫反应，提示簇状蛋白在多鳃泡腺的滤泡中积聚。在甲状腺滤泡上皮细胞的顶膜、肠道的杯状细胞和鳃的粘液细胞上也检测到阳性标记。至于在上鳃腺中表达的转录因子，通过cDNA克隆鉴定了Nkx2.1d、Pax1和FoxF1。最近有报道FOXA2(HNF-3β)在哺乳动物CT产生细胞系CA77中表达，并能增强大鼠CT/CT基因相关肽(CGRP)基因的转录活性。然而，FOXA2的效果似乎还不够强。因此，在第二个实验中，我尝试从虹鱼的多鳃腺中克隆有翼螺旋/叉头转录因子FOXA(HNF-3)或类似因子的cDNA。分离出FOXA2基因的编码区，RT-PCR分析显示FOXA2基因在脐带、肝脏和肠道中均有表达。为了研究FOXA2是否激活了CT启动子的转录，我们进一步用聚合酶链式反应的方法，从虹鲑鱼中克隆了一个包含鲑鱼型CT-Ib基因外显子I的上游区和5‘端非编码区的DNA片段。此外，我还利用聚合酶链式反应的方法，从青竹的CT/CGRP基因下游克隆了一段3.8kbP的DNA片段，该片段位于外显子IV的3‘非编码区。我将这些DNA片段插入到荧光素酶报告载体中，并进行了荧光素酶检测。然而，FoxA1和/或FOXA2与荧光素酶载体共转染并不能提高转录活性。较少

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