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A redox factor protein, ref1, is involved in negative gene regulation by extracellular calcium
T Okazaki1, U Chung, T Nishishita
1Fourth Department of Internal Medicine, University of Tokyo School of Medicine, Japan.
Abstract:
A rise in extracellular calcium (Ca2+e) suppresses not only secretion of parathyroid hormone (PTH) but also expression of the PTH gene to ensure constant plasma Ca2+ level. A nuclear protein(s) in a wide variety of cells bound to the specific DNA elements (negative Ca2+ responsive elements, nCaREs) in the human PTH gene in sequence-specific and Ca2+e concentration-dependent manners. Our Southwestern cloning revealed that a redox factor protein (ref1), which was known to activate several transcription factors via alterations of their redox state, belonged to an nCaRE binding protein. The level of ref1 mRNA as well as of its protein was elevated by an increase in Ca2+e concentration. In gel shift assay, anti-ref1 antibody eliminated formation of the nCaRE-protein complex. We also found that there was another protein(s) interacting with nCaREs and ref1. Further, experiments with an antisense-ref cDNA expression vector introduced into cultured cells suggested that DNA (nCaRE)-ref1 interaction led to Ca2+e-mediated transcriptional suppression. Thus, it is concluded that ref1 possesses transcription repressor activity in addition to its function as a transcriptional auxiliary protein.
Insights
High extracellular calcium (Ca2+e) suppresses parathyroid hormone (PTH) gene expression. A nuclear protein, redox factor 1 (ref1), binds to specific DNA elements (nCaREs) and acts as a transcription repressor, maintaining calcium homeostasis.
Area of Science:
- Molecular Biology
- Endocrinology
- Gene Regulation
Background:
- Extracellular calcium (Ca2+e) levels regulate parathyroid hormone (PTH) secretion and gene expression to maintain calcium homeostasis.
- Specific DNA sequences, termed negative calcium-responsive elements (nCaREs), in the human PTH gene are involved in this regulation.
Purpose of the Study:
- To identify nuclear proteins that bind to nCaREs and elucidate their role in Ca2+e-mediated PTH gene suppression.
- To investigate the function of redox factor 1 (ref1) in the context of PTH gene regulation.
Main Methods:
- Southwestern cloning to identify nCaRE-binding proteins.
- Gel shift assays using anti-ref1 antibodies to confirm protein-DNA interactions.
- Experiments with antisense-ref cDNA expression vectors in cultured cells to assess ref1's transcriptional activity.
Main Results:
- Redox factor 1 (ref1) was identified as a nuclear protein that binds to nCaREs in a sequence-specific and Ca2+e concentration-dependent manner.
- Increased Ca2+e levels led to elevated ref1 mRNA and protein levels.
- Anti-ref1 antibodies disrupted the nCaRE-protein complex, and antisense experiments indicated that ref1 binding to nCaREs mediates Ca2+e-induced transcriptional suppression of the PTH gene.
Conclusions:
- Ref1 acts as a transcription repressor for the human PTH gene, in addition to its known role as a transcriptional auxiliary protein.
- The interaction between ref1 and nCaREs is a key mechanism for Ca2+e-mediated suppression of PTH gene expression, contributing to calcium homeostasis.