A REDOX FACTOR PROTEIN, REF1, IS INVOLVED IN NEGATIVE GENE-REGULATIONBY EXTRACELLULAR CALCIUM

A rise in extracellular calcium (Ca-e(2+)) suppresses not only secreti on of parathyroid hormone (PTH) but also expression of the PTH gene to ensure constant plasma Ca2+ level. A nuclear protein(s) in a wide var iety of cells bound to the specific DNA elements (negative Ca2+ respon sive elements, nCaREs) in the human PTH gene in sequence-specific and Ca(2+)e concentration-dependent manners. Our Southwestern cloning reve aled that a redox factor protein (ref1), which was known to activate s everal transcription factors via alterations of their redox state, bel onged to an nCaRE binding protein. The level of ref1 mRNA as well as o f its protein was elevated by an increase in Ca-e(2+) concentration. I n gel shift assay, anti-ref1 antibody eliminated formation of the nCaR E-protein complex. We also found that there was another protein(s) int eracting with nCaREs and ref1. Further, experiments with an antisense- ref cDNA expression vector introduced into cultured cells suggested th at DNA (nCaRE)-ref1 interaction led to Ca-e(2+)-mediated transcription al suppression. Thus, it is concluded that ref1 possesses transcriptio n repressor activity in addition to its function as a transcriptional auxiliary protein.