SAT-793 - ETV1 Unifies FGF23 and PTH Endocrine Regulation of Calcitriol

Calcium and phosphate concentrations are controlled by levels of active vitamin D3 (1,25(OH)2D3, 1,25D) which is produced in the proximal tubule of the kidney by the CYP27B1 enzyme and is degraded by the CYP24A1 enzyme. These enzymes are regulated through endocrine signaling. Parathyroid hormone (PTH) activates Cyp27b1 and suppresses Cyp24a1 while fibroblast growth factor 23 (FGF23) suppresses Cyp27b1 and activates Cyp24a1. 1,25D also exerts endocrine control over itself by suppressing Cyp27b1 and activating Cyp24a1. In vivo, homeostatic levels of the vitamin D hormone are determined by the balance of PTH and FGF23. Genomic actions of PTH and 1,25D are known to act through CREB and VDR respectively, however; the mechanism of PTH suppression of Cyp24a1 and genomic control by FGF23 have remained a critical knowledge gap. To identify this factor(s), we examined kidney expression data to determine candidate transcription factors (TFs), and performed ChIP-seq in vivo on the candidate TFs. In vitro, we tested their gene expression and activation by FGF23, 1,25D, and PTH signaling in the human embryonic kidney cells overexpressing the membrane Klotho receptor (HEK-mKL). We found that the E twenty-six (ETS) family member ETV1 binds to the genome at PT cell-specific genomic enhancers for Cyp24a1 and increased gene (8-fold) and protein expression after FGF23 treatment in the mouse kidney. Genome-wide, >80% of FGF23-stimulated ETV1 genomic binding overlapped with 1,25D-induced VDR binding. In vivo, FGF23+1,25D cotreatment synergistically upregulated Cyp24a1, and VDR and ETV1 were found to physically interact as co-IP increased their association. In vitro, Cyp24a1 gene expression revealed an mRNA increase from 4- to 50-fold with FGF23 and 1,25D individually to >400-fold in combination in the HEK-mKL cells. ETV1 bound to the Etv1 gene promoter as well, driving its own production. Mutation of an ETS binding site (EBS) prevented the activation of Cyp24a1 promoter in vitro by FGF23 and opposed the synergistic increase (1,25D+FGF23). With this mutation, activation of Cyp24a1 was inhibited regardless of ETV1 overexpression. Surprisingly, when mice were treated with PTH, ETV1 was completely removed from Cyp24a1 enhancers even with FGF23 pre-treatment. In fact, ETV1 genomic binding was reduced from 4,790 sites to just 22 sites, a remarkable depletion. These activities were corroborated in vitro as reporters revealed that inducing the PTH pathway similarly inhibits Cyp24a1 activation. Taken together, our data indicate that ETV1 binds to specific EBS to control Cyp24a1 in response to FGF23 in partnership with 1,25D-induced VDR. Strikingly, ETV1 was also involved in PTH-mediated suppression of Cyp24a1. These studies help answer a long-standing mechanistic question of FGF23 genomic actions in kidney and point to ETV1 as being at the nexus of PTH and FGF23 signaling that controls vitamin D endocrinology.

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