Volume 86, Issue 6, Pages 1106-1115 (December 2014) Parathyroid hormone activates the orphan nuclear receptor Nurr1 to induce FGF23 transcription  Tomer Meir, Karina Durlacher, Zheng Pan, Gail Amir, William G. Richards, Justin Silver, Tally Naveh-Many  Kidney International  Volume 86, Issue 6, Pages 1106-1115 (December 2014) DOI: 10.1038/ki.2014.215 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 FGF23 mRNA levels are increased by PTH, Nurr1, and PKA activation. (a, b) PTH increases Nurr1 expression before FGF23. UMR106 cells were incubated with PTH (1–34) (solid line) or vehicle (dashed line), and mRNA levels for (a) FGF23 and (b) Nurr1 were measured by qRT-PCR. (c) Activation of PKA increases FGF23 expression. FGF23 and Nurr1 mRNA levels after exposure of UMR106 cells to PTH (1–34) (black) or the PKA activator forskolin (gray) for 24h compared with vehicle (white). (d) Nurr1 overexpression increases FGF23 mRNA. UMR106 cells were transfected with Nurr1 expression plasmid and FGF23 mRNA levels were measured at 30h. mRNA levels are presented as mean±s.e., n=4, *P<0.05. Similar results were found in three repeat experiments for (a–c). PKA, protein kinase A; PTH, parathyroid hormone; qRT-PCR, quantitative reverse transcriptase-PCR. Kidney International 2014 86, 1106-1115DOI: (10.1038/ki.2014.215) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 PTH increases Nurr1 and FGF23 gene transcription. (a, b) Actinomycin D prevents the increase in FGF23 and Nurr1 mRNA levels by PTH. UMR106 cells were incubated with PTH (1–34) or vehicle supplemented with actinomycin D. mRNA levels for (a) FGF23 and (b) Nurr1 were measured by qRT-PCR. PTH (solid line), vehicle (dashed line), and PTH and vehicle together with actinomycin D (dotted and dash-dotted lines). (c) PTH enhances FGF23 promoter activity. UMR106 cells were transfected with a reporter plasmid containing the firefly luciferase gene driven by the mouse FGF23 8-kb promoter sequence (schematically presented below) and a renilla luciferase expression plasmid used as a control for transfection efficiency. Luciferase expression indicating FGF23 promoter activity 6h after the addition of PTH (black), vehicle (white), or the biological inactive PTH peptide (3–34) (diagonal lines). (d) Nurr1 overexpression enhances FGF23 promoter activity. UMR106 cells were transfected with the FGF23 promoter-firefly luciferase plasmid and a renilla luciferase expression plasmid as in 2C, together with a Nurr1 overexpression or a control plasmid, PTH, or vehicle added and FGF23 promoter activity measured. Vehicle (white), PTH (1–34) (black), Nurr1 overexpression (dotted bar), and Nurr1 overexpression with added PTH (gray bar). Results are presented as mean±s.e., n=4, *P<0.05. Similar results were found in three repeat experiments. PTH, parathyroid hormone; qRT-PCR, quantitative reverse transcriptase-PCR. Kidney International 2014 86, 1106-1115DOI: (10.1038/ki.2014.215) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Nurr1 knockdown prevents the effect of PTH to induce FGF23 expression. (a, b) UMR106 cells were infected with lentiviral particles containing Nurr1 shRNA or scrambled shRNA as control. The cells were transfected with the FGF23 8-kb promoter-firefly luciferase plasmid, incubated with PTH for 5h, and assayed for (a) endogenous FGF23 mRNA expression and (b) luciferase promoter activity . Results are presented as mean±s.e., n=3–4, *P<0.05 PTH vs. vehicle. PTH, parathyroid hormone. Kidney International 2014 86, 1106-1115DOI: (10.1038/ki.2014.215) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Nurr1 increases FGF23 promoter activity by binding to response elements in the proximal region of the FGF23 promoter. (a) Identification of functional NBREs in the FGF23 promoter. UMR106 cells were transfected with plasmids containing firefly luciferase driven by the FGF23 8-kb promoter sequence or partially deleted promoter sequences (schematically presented beside the graphs). PTH (1–34) (black bar), vehicle (white), or the biological inactive PTH (3–34) (diagonal lines) were added for 6h, and the FGF23 promoter activity was measured. Firefly luciferase activity was corrected to renilla luciferase as a transfection efficiency control. (b) UMR106 cells transfected with an FGF23 proximal promoter 1275-bp element fused to a firefly luciferase sequence were incubated with vehicle (white), PTH (black), or co-transfected with Nurr1 overexpression plasmid with vehicle (dotted) or PTH (gray). Results are presented as mean±s.e., n=4–5, *P<0.05. (c) Promoter analysis and preservation among species. An alignment of human, rat, and mouse genomic DNA containing 650bp of FGF23 promoter and 100bp of 5′-untranslated region with the human transcriptional start site indicated by an arrow. Complete conservation between the species is indicated by dark shading, and light shading indicates conservation between two of the three species. Nurr1-binding elements are highlighted in black. NBRE, NGFI-B response element; PTH, parathyroid hormone. Kidney International 2014 86, 1106-1115DOI: (10.1038/ki.2014.215) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Nurr1 binds the FGF23 promoter. Chromatin immunoprecipitation followed by qRT-PCR using primers directed to sequences in the FGF23 promoter or the transcribed region (intron 2-exon 3) as negative control. There was binding to the NBRE-containing proximal promoter regions but not to the control region. The results represent four independent experiments combined, and are presented as IP/input ratio, mean±s.e., *P<0.05. NBRE, NGFI-B response element. Kidney International 2014 86, 1106-1115DOI: (10.1038/ki.2014.215) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Nurr1 and FGF23 are increased in experimental uremia, and this is prevented by a calcimimetic. (a) Nurr1 and FGF23 mRNA levels in calvaria from control and adenine high-phosphorus diet rats given daily injections of the calcimimetic R568 or vehicle. Calvaria mRNA levels for FGF23 (left) and Nurr1 (right) were measured by qRT-PCR. mRNA levels are shown for control rats (white), uremic rats (black), and uremic rats given R568 (gray). (b, c) Calvaria Nurr1 protein levels are decreased in uremic rats after R568. (b) Western blot analysis and (c) quantification of Nurr1 protein levels in calvaria of uremic rats with and without R568 administration. Results are presented as mean±s.e., n=6, *P<0.05. (d) Representative immunohistochemistry staining for Nurr1 in femurs from control, adenine-fed uremic rats, and adenine-fed uremic rats given R568. The arrows mark some of the positively stained osteocytes (magnification × 400). qRT-PCR, quantitative reverse transcriptase-PCR. Kidney International 2014 86, 1106-1115DOI: (10.1038/ki.2014.215) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Model for the PTH-FGF23 feedback loop. PTH binds to the PTH receptor (PTH1R) on osteocytes and osteoblasts to activate PKA, which increases Nurr1 expression. Nurr1 induces FGF23 transcription by binding to defined NBREs in the FGF23 promoter. FGF23 in turn inhibits PTH production but not in CKD. In CKD, the hyperparathyroidism increases Nurr1 and then FGF23 expression. Calcimimetics decrease PTH, Nurr1, and FGF23. PTH and FGF23 are both phosphaturic. cAMP, cyclic adenosine monophosphate; CKD, chronic kidney disease; NBRE, NGFI-B response element; PKA, protein kinase A; PTH, parathyroid hormone. Kidney International 2014 86, 1106-1115DOI: (10.1038/ki.2014.215) Copyright © 2014 International Society of Nephrology Terms and Conditions