| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Through the specific binding of a negative calcium responsive element (nCaRE) to its binding protein (nCaREB) in response to extracellular Ca (Ca^2+_e), nCaRE-bearing genes, such as the human parathyroid hormone (PTH) gene, are negatively regulated by Ca^2+_e. The Ku antigen mediated negative gene regulation by Ca^2+_e by interacting with a redox factor protein, refl. Though sequence-nonspecific DNA binding activity of the ku antigen has been well characterized, the mechanism of its sequence-specific DNA binding remained obscure, Here, we report that the specific binding of the Ku antigen to another protein, refl, leads to DNA-protein complex formation with a novel sequence-specificity and thereby regulates gene expression. We next examined whether the recently-identified membrane calcium-sensing receptor was involved in this type of gene regulation. Though cultured cells transfected with the calcium-sensing receptor expression vector potentiated the binding between nCaRE and nCaREB as exhibited repressed nCaRE-bearing reporter activity, these findings were observed irrespective of the extracellular calcium concentrations.
On the other hand, we have found that one of the nCaREs, oligo B,is very well conserved among such vasoactive genes as the vasopressin and atrial natriuretic polypeptide genes. Further, the oligo B in the former gene is conserved throughout evolution. We demonstrate that the binding between oligo B and its binding nuclear proteins including a redox factor 1 (refl) was reduced by hyperosmolarity generated by sodium chloride (NaCl) but not by urea. Such attenuated binding was reversed by dephosphorylating some of the nuclear proteins by a potato acid phosphatase, suggesting that NaCl treatment elicited phosphorylation of these nuclear proteins to weaken their binding activity to oligo B.Furthermore, these nuclear events let to hyperosmolarity-mediated transcriptional stimulation of the genes bearing this DNA element in the cultured cells.
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