Ca2+-dependent regulation of calcitonin gene expression by the transcriptional repressor DREAM.
Calcitonin (CT), whose secretion from thyroid glands is regulated by increases in the concentration of extracellular Ca(2+), is a well-known hormone that regulates calcium homeostasis. However, the molecular mechanisms underlying the gene expression dependent on Ca(2+) have not been clarified. The downstream regulatory element (DRE) antagonist modulator (DREAM) was recently identified as a Ca(2+)-dependent transcriptional repressor. In the present study, we investigated the possible involvement of DREAM in the regulation of CT gene expression and secretion. A luciferase assay using TT cells, a thyroid carcinoma cell line, showed that a particular region in the CT gene promoter repressed the promoter activity under basal conditions but induced the activity when the Ca(2+) concentration was increased. We found two DRE sequences in a region located upstream from the transcription start site. Gel retardation assay confirmed that DREAM bound to the CT-DRE and also indicated that DREAM bound to the DRE in a Ca(2+)-dependent manner. We generated stable transfectants of TT cells with wild-type or mutant DREAM, which lacked the responsiveness to Ca(2+) changes. In contrast to the wild type, overexpression of the mutant DREAM inhibited the increase in CT secretion induced by a calcium ionophore. The addition of forskolin to increase cAMP activated the CT promoter, probably by the interaction of DREAM with cAMP-responsive element binding proteins, independent on the activation by Ca(2+). Together, these results suggest that DREAM plays an important role in human CT gene expression in a Ca(2+)- and cAMP-dependent manner.
Matsuda
Miho
M
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan.
Yamamoto
Tada-Aki
TA
Hirata
Masato
M
D015153:Blotting, Western; D002116:Calcitonin / Q000096:biosynthesis* / Q000235:genetics*; D002118:Calcium / Q000502:physiology*; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation; D007476:Ionophores / Q000494:pharmacology; D051605:Kv Channel-Interacting Proteins / Q000235:genetics / Q000502:physiology*; D008156:Luciferases / Q000378:metabolism; D009687:Nuclear Proteins / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013964:Thyroid Neoplasms / Q000378:metabolism; D014162:Transfection
D007476:Ionophores; C496749:KCNIP3 protein, human; D051605:Kv Channel-Interacting Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D002116:Calcitonin; D000242:Cyclic AMP; D008156:Luciferases; D005982:Glutathione Transferase; D002118:Calcium