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26 REGULATION OF FGF23 BY 1,25-DIHYDROXYVITAMIN D3: A SECONDARY MECHANISM INVOLVING HISTONE H4 ACETYLATION.
  1. H. A. Hopper IV1,
  2. G. K. Whitfield1,
  3. M. R. Haussler1
  1. 1Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ.

Abstract

FGF23 is a newly recognized regulator of serum phosphate, acting reciprocally to 1,25-dihydroxyvitamin D3 (1,25D), the hormonally active form of vitamin D. The hormones also act to regulate each other, with FGF23 down-regulating the production of 1,25D, and 1,25D acting via its nuclear receptor to up-regulate FGF23 production.

Purpose To elucidate the mechanism whereby FGF23 gene transcription is up-regulated by the vitamin D receptor bound to 1,25D.

Methods An initial approach to address this question was to construct a luciferase reporter construct containing the FGF23 proximal promoter. A second approach was to search mammalian genomic sequences for conserved vitamin D response elements (VDREs) and also conserved binding sites for other transcription factors in the vicinity of the FGF23 gene. Finally, chromatin immunoprecipitation (ChIP) experiments were performed using antibodies directed against modified histones to investigate the role of chromatin remodeling in 1,25D-stimulated FGF23 expression. A collaborator used real-time PCR (rt-PCR) to test whether 1,25D regulates two transcription factors for which conserved binding sites were found.

Results The FGF23 reporter construct yielded a strong level of expression but very weak up-regulation (1.5X) by 1,25D. These data led us to believe that either our promoter segment did not contain the relevant control elements or that in vivo regulatory events (eg, chromatin remodeling) could not be replicated in our plasmid reporter construct. Other factors led us to conclude that 1,25D regulation of FGF23 might be a secondary effect: (a) genomic searches failed to yield conserved VDREs and (b) cyclohexamide experiments implied that protein synthesis was required for the 1,25D effect. We identified conserved binding sites for c-Ets-1 and GATA-3 in the FGF23 proximal promoter; however, we were unable to show 1,25D regulation by either factor using real-time PCR. Finally, ChIP experiments using various antisera to modified histones showed that histone H4 acetylation is increased in the FGF23 promoter in response to 1,25D.

Conclusions Our results, so far, are consistent with FGF23 up-regulation occurring via a secondary mechanism. Unfortunately, a transcription factor that fits the criteria of a primary 1,25D target has not yet been identified. We have nevertheless been able to demonstrate that histone modification is part of the mechanism for FGF23 activation and are hopeful that further ChIP and bioinformatics approaches will allow us to focus on a specific region of the FGF23 gene where key regulatory elements may reside.

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