|Budget Amount *help
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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
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