ABSTRACT: The active form of vitamin D, 1,25(OH)2 vitamin D3 (‘1,25D’), is required for maintaining normal immune responses, cancer resistance, vascular function, and musculoskeletal structure and function. The balanced synthesis of 1,25D from liver-produced 25(OH)D3 is exquisitely directed in final metabolizing steps solely in the kidney proximal tubule (PT) by the catabolic enzyme vitamin D 24-hydroxylase (CYP24A1) and anabolic vitamin D 1α-hydroxylase (CYP27B1). FGF23 and 1,25D itself, synergistically lower 1,25D by increasing CYP24A1 and suppressing CYP27B1, in a directly converse manner to the actions of PTH. We previously found that the necessary balance of maintaining vitamin D metabolism relies more on catabolic Cyp24a1 expression as the critical factor controlling circulating 1,25D. Using scRNAseq after temporal FGF23 injections into normal mice, we found that the E twenty-six (ETS) family member ETV1 was stimulated specifically in PT S1-S2 cells in parallel with increased Cyp24a1, and a 10-fold induction of ETS occupancy across the genome. In vivo, FGF23 induced localization of ETV1 to megalin+ PT nuclei. Importantly, ETV1 was rapidly recruited to genomic enhancers within the Cyp24a1 promoter and a kidney-specific distal enhancer (DS1) responsible for both FGF23 and PTH activities that overlapped with VDR binding. In a converse manner, PTH suppressed nuclear ETV1 protein in vivo, and opposed FGF23 increases by completely removing ETV1 from the Cyp24a1 genomic enhancers (xx sites down to xx sites). In vitro, a human ETV1 cDNA drove CYP24A1 mRNA in HEK-mKL cells, and ETV1 protein was upregulated by FGF23, but not by 1,25D. However 1,25D strongly enhanced co-precipitated ETV1-VDR complexes. Conditional targeting of kidney epithelial Etv1 in mice resulted in ‘FGF23 resistance’ with elevated FGF23 and 1,25D, and almost complete blockade of Cyp24a1 mRNA responses, which altered 1,25D metabolism as determined by MS. FGF23 was shown to inhibit an ancient system of E-3 Ubiquitin ligase Rfwd2 (COP1) nuclear localization, enhancing ETV1 protein stability. Further, a ‘PTH mimetic’ SIK inhibitor (SIKi) abolished ETV1 production by enhancing COP1-mediated degradation, whereas COP1 shRNA rescued SIKi-mediated ETV1 suppression. In sum, our findings demonstrate that ETV1 acts as a novel ‘unifying’ TF positively regulating FGF23/KL bioactivity with VDR on Cyp24a1 and opposing PTH actions, thus opening novel pathways critically needed to address severe diseases involving FGF23 and vitamin D.
