Complex Transcriptional Regulation of the Fibroblast Growth Factor‐23 Gene by 1,25‐Dihydroxyvitamin D3: Evidence for Primary and Secondary Mechanisms

Abstract

Fibroblast growth factor 23 (FGF23) is a circulating phosphaturic hormone synthesized in osteocytes and osteoblasts that corrects hyperphosphatemic states by inhibiting renal phosphate reabsorption via the fibroblast growth factor receptor‐1 and its klotho coreceptor. In a closed endocrine loop, 1,25‐dihydroxyvitamin D3 (1,25D) induces FGF23 expression in bone, with the phosphaturic peptide in turn acting at the kidney to feedback repress CYP27B1 to suppress production of 1,25D. FGF23‐null mice cannot correct the severe hyperphosphatemia and ectopic calcification that results from the 1,25D‐stimulated calcium and phosphate intestinal absorption and renal reabsorption. Furthermore, abnormal concentrations of FGF23 have been linked to several potentially fatal pathologies, including chronic kidney disease and cardiovascular events. FGF23 synthesis is induced by the 1,25D‐bound vitamin D receptor (VDR), resulting in FGF23‐signaled suppression of the phosphate transporters NaPi‐IIa and NaPi‐ IIc, decreasing renal phosphate reabsorption and resulting in urinary phosphate excretion. Previous reports document upregulation of FGF23 mRNA by 1,25D; however the exact molecular mechanism(s) for this phenomenon remain unknown. In preliminary research, we identified five functional vitamin D responsive elements (VDREs) in the human FGF23 gene region remote to the promoter that may directly control FGF23 gene transcription. However, these VDREs displayed only modest induction activity, implying that a secondary mechanism for transactivation by 1,25D must exist to explain the observed high magnitude induction of FGF23 mRNA and protein by vitamin D, in vivo, and in cultured osteoblasts. In the present study, we disprove our hypothesis that membrane and extracellular matrix proteins, namely klotho and/or osteopontin, induced by 1,25D‐liganded VDR, secondarily generate a signaling cascade to increase FGF23 production. Instead, by truncation and point mutagenesis, we characterized a novel cis‐regulatory module in the proximal promoter of mouse FGF23, located between ‐400 and ‐200 bp from the transcriptional start site. A ‐0.6 kb construct of the murine FGF23 promoter demonstrates a 4‐fold induction in response to 1,25D when transfected into human leukemia (K562) cells. Mutation of one of the ETS1 sites at ‐346 bp within this construct, or an adjacent VDRE/Nurr1 site, reduces the transcriptional response to 1,25D to that of a minimal ‐0.06 kb promoter construct. Thus, the composite ETS‐VDRE/Nurr1 cis‐element identified herein may function in 1,25D‐elicited induction of FGF23 in a primary mechanistic manner via a novel VDRE located at ‐334 bp in the murine FGF23 promoter, and/or in a secondary fashion through 1,25D‐primary transcriptional activation of ETS1 and Nurr1. Also, we demonstrate a significant 1.7‐ to 2.0‐fold increase in transcription of a ‐1.0 kb murine FGF23 promoter construct in response to high calcium exposure of MC3T3 mouse osteoblastic cells. Thus, the FGF23 proximal promoter harbors cis‐elements that drive responsiveness to 1,25D and calcium, agents that induce FGF23 to curtail the pathologic consequences of their excess. Our results contribute to the understanding of the complex transcriptional regulation of FGF23 by providing evidence for cis‐regulatory elements within the FGF23 proximal promoter that are responsive to primary and secondary actions of 1,25D and calcium. Such findings may influence future therapeutic modalities in the treatment of FGF23‐related pathologies.