Rapid genomic changes by mineralotropic hormones and kinase SIK inhibition drive coordinated renal Cyp27b1 and Cyp24a1 expression via CREB modules

Abstract

Vitamin D metabolism centers on kidney regulation of Cyp27b1 by mineralotropic hormones, including induction by parathyroid hormone (PTH), suppression by fibroblast growth factor 23 (FGF23) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), and reciprocal regulations for Cyp24a1. This coordinated genomic regulation results in production of endocrine 1,25(OH)₂D₃, which, together with PTH and FGF23, controls mineral homeostasis. However, how these events are coordinated is unclear. Here, using in vivo chromatin immunoprecipitation sequencing in mouse kidney, we demonstrate that PTH activation rapidly induces increased recruitment of phosphorylated (p-133) CREB (pCREB) and its coactivators, CBP (CREB-binding protein) and CRTC2 (CREB-regulated transcription coactivator 2), to previously defined kidney-specific M1 and M21 enhancers near the Cyp27b1 gene. At distal enhancers of the Cyp24a1 gene, PTH suppression dismisses CBP with only minor changes in pCREB and CRTC2 occupancy, all of which correlate with decreased genomic activity and reduced transcripts. Treatment of mice with salt-inducible kinase inhibitors (YKL-05-099 and SK-124) yields rapid genomic recruitment of CRTC2 to Cyp27b1, limited interaction of CBP, and a transcriptional response for both Cyp27b1 and Cyp24a1 that mirrors the actions of PTH. Surprisingly, we find that 1,25(OH)₂D₃ suppression increases the occupancy of CRTC2 in the M1 enhancer, a novel observation for CRTC2 and 1,25(OH)₂D₃ action. Suppressive actions of 1,25(OH)₂D₃ and FGF23 at the Cyp27b1 gene are associated with reduced CBP recruitment at these CREB-module enhancers that disrupts full PTH induction. Our findings show that CRTC2 contributes to transcription of both Cyp27b1 and Cyp24a1, demonstrate salt-inducible kinase inhibition as a key modulator of vitamin D metabolism, and provide molecular insight into the coordinated mechanistic actions of PTH, FGF23, and 1,25(OH)₂D₃ in the kidney that regulate mineral homeostasis.

Keywords: 1,25(OH)(2)D(3); ChIP-Seq; Cyp24a1; Cyp27b1; FGF23; cytochrome P450; gene regulation; parathyroid hormone; salt-inducible kinases; vitamin D.

Figure 1

Pathways and enhancers that control the expression of Cyp27b1 and Cyp24a1 in the kidney.A, schematic diagram for the regulation of vitamin D metabolism and serum calcium and phosphate homeostasis in the kidney. Pathways either increase (+) or decrease (−) in response to changing calcium and phosphate levels. Our genetic models (black) and previously existing models (gray) are overlaid on or near the pathways they disrupt. Schematic representation of PTH, FGF23, and 1,25(OH)₂D₃ actions on Cyp27b1 (B) and Cyp24a1 (C) through tissue-specific enhancers M1 and M21, which control Cyp27b1 as well as DS1 (-21 kb to -32 kb) and DS2 (-35 kb to -37 kb) enhancers that control Cyp24a1. Figures adapted from recent studies (, 15). DS, DownStream region of Cyp24a1; FGF23, fibroblast growth factor 23; 1,25(OH)₂D_3, 1,25-dihydroxyvitamin D₃; PTH, parathyroid hormone.

Figure 2

Time course of PTH actions in mouse kidney near the Cyp27b1 gene.A, gene expression in the kidney for Cyp27b1 in 8- to 9-week-old WT C57BL/6 mice injected with 230 mg/kg bw PTH for 0, 1, 3, or 6 h. Data are displayed as relative quantitation (RQ, mean ± SEM) compared with Gapdh. n = 4 for each time point. One-way ANOVA with multiple comparison Tukey post test: ∗p < 0.05 time point versus 0 h. ChIP-Seq analysis near Cyp27b1 for (B) pCREB, (C) CBP, (D) CRTC2, (E) H3K27ac, or (F) H3K9ac from WT mice injected with 230 mg/kg bw PTH for 0 (Veh only), 15, 30, or 60 min (n = 3). Overlaid triplicate and averaged ChIP-Seq tracks where Veh (0 h) are shown in yellow, treatments shown in blue, and overlapping data appear as green. Regions of interest are highlighted in gray boxes (M1, CP [Cyp27b1 promoter], M21(a–c), tissue specific) or red (MP [Mettl1/Mettl21b promoters], nontissue specific). Genomic region displayed is chr10:126,469,260 to 126,490,800, and maximum height of tag sequence density for each data track indicated on the Y-axis (normalized to input and 10⁷ tags). Fold change table (right) was calculated for triplicate tag density in each peak region versus Veh. ∗p < 0.05 paired t test: treatment versus vehicle. nc, no change (<1.5-fold). bw, body weight; CBP, CREB-binding protein; ChIP-Seq, chromatin immunoprecipitation sequencing; CRTC, CREB-regulated transcription coactivator; H3K9ac, histone acetylation at histone H3 lysine 9; H3K27ac, histone acetylation at histone H3 lysine 27; pCREB, phosphorylated (p-133) CREB; PTH, parathyroid hormone.

Figure 3

Time course of PTH actions in mouse kidney near the Cyp24a1 gene.A, gene expression in the kidney for Cyp24a1 in 8− to 9–week-old WT C57BL/6 mice injected with 230 mg/kg bw PTH for 0, 1, 3, or 6 h. Data are displayed as relative quantitation (RQ, mean ± SEM) compared with Gapdh. n = 4 for each time point. One-way ANOVA with multiple comparison Tukey post-test: ∗p < 0.05 time point versus 0 h. ChIP-Seq analysis near Cyp24a1 for (B) pCREB, (C) CBP, (D) CRTC2, (E) H3K27ac, or (F) H3K9ac from WT mice injected with 230 mg/kg bw PTH for 0 (Veh only), 15, 30, or 60 min (n = 3). Additional details as for Figure 2. Genomic region displayed is chr2: 170,278,379 to 170,324,235. bw, body weight; CBP, CREB-binding protein; ChIP-Seq, chromatin immunoprecipitation sequencing; CRTC, CREB-regulated transcription coactivator; H3K9ac, histone acetylation at histone H3 lysine 9; H3K27ac, histone acetylation at histone H3 lysine 27; pCREB, phosphorylated (p-133) CREB; PTH, parathyroid hormone.

Figure 4

SIK inhibition increases Cyp27b1 and decreases Cyp24a1 expression.A, gene expression of Cyp27b1 (left) and Cyp24a1 (right) in the kidney and B, Fgf23 (left) and Tnfsf11 (right) in the L5 vertebrae from WT mice treated with vehicle (Veh, 3 h), 230 mg/kg PTH (1 h), 30 mg/kg YKL-05-099 (3 h), or 40 mg/kg SK-124 (3 h). Data are displayed as relative quantitation (RQ, mean ± SEM) compared with Gapdh. n = 6 for each treatment. One-way ANOVA with multiple comparison Tukey post-test: ∗p < 0.05 treatment versus Veh. #p < 0.05 treatment versus PTH. parathyroid hormone; SIK, salt-inducible kinase.

Figure 5

SIK inhibitors YKL-05-099 and SK-124 have PTH-like actions on the genome near Cyp27b1 and Cyp24a1.A, gene expression in the kidney for Cyp27b1 after WT mouse treatment with 30 mg/kg bw YKL-05-099 or 40 mg/kg bw SK-124 for 0, 0.5, 1, 3, 6, 12, 24 h (left) or 3 h YKL-05-099 at 7.5, 15, and 30 mg/kg bw and SK-124 at 40, 80, and 120 mg/kg bw (right). Data are displayed as relative quantitation (RQ, mean ± SEM) compared with Gapdh. n = 6 for time point. One-way ANOVA with multiple comparison Tukey post-test: ∗p < 0.05 time point or treatment versus 0 h. ChIP-Seq analysis near Cyp27b1 for (B) pCREB, (C) CBP, and (D) CRTC2 from WT mice injected with 230 mg/kg bw PTH for 30 min (PTH30), 30 mg/kg bw YKL-05-099 for 1 h, or 40 mg/kg bw SK-124. PTH30 data tracks are same as for Figure 2. Additional details as for Figure 2. Genomic region displayed is chr10: 126,469,260 to 126,490,800. E, Cyp24a1 expression in the mouse kidney after treatments described in A. ChIP-Seq analysis near Cyp24a1 for (F) pCREB, (G) CBP, and (H) CRTC2 as described above in B–D. PTH30 data tracks same as for Figure 3. Additional details as for Figure 2. Genomic region displayed is chr2: 170,278,379 to 170,324,235. bw, body weight; CBP, CREB-binding protein; ChIP-Seq, chromatin immunoprecipitation sequencing; CRTC, CREB-regulated transcription coactivator; pCREB, phosphorylated (p-133) CREB; PTH, parathyroid hormone; SIK, salt-inducible kinase.

Figure 6

SIK inhibitors elevate serum PTH and/or serum iFGF23.A, gene expression in the L5 vertebrae for Fgf23 and Tnfsf11 after WT mouse treatment with 30 mg/kg bw YKL-05-099 or 40 mg/kg bw SK-124 for 0, 0.5, 1, 3, 6, 12, and 24 h. Serum measurements for (B) iFGF23 (pg/ml) and (C) PTH (pg/ml) for same dose and time as indicated for Figure 5. n = 6 for time point. One-way ANOVA with multiple comparison Tukey post-test: ∗p < 0.05 time point or treatment versus 0 h. bw, body weight; iFGF23, intact fibroblast growth factor 23; PTH, parathyroid hormone; SIK, salt-inducible kinase.

Figure 7

Activities of 1,25(OH)₂D₃and FGF23 at Cyp27b1. Gene expression of Cyp27b1 after time course with (A) 10 mg/kg bw 1,25(OH)₂D₃ at 0, 1, 3, 6, 12, and 24 h (n = 4) and (B) 50 mg/kg bw FGF23 at 0, 3, 6, and 12 h (n = 3–4) from our previous publication (10). ChIP-Seq analysis near Cyp27b1 for (C) VDR and RXR (1,25(OH)₂D₃versus Veh only), (D) pCREB, (E) CBP, (F) CRTC2, (G) H3K27ac, and (H) H3K9ac from WT mice injected with 10 mg/kg bw 1,25(OH)₂D₃ or 50 mg/kg bw FGF23 (1 h). Additional details as for Figure 2. Genomic region displayed is chr10: 126,469,260 to 126,490,800. bw, body weight; CBP, CREB-binding protein; ChIP-Seq, chromatin immunoprecipitation sequencing; CRTC2, CREB-regulated transcription coactivator 2; FGF23, fibroblast growth factor 23; H3K9ac, histone acetylation at histone H3 lysine 9; H3K27ac, histone acetylation at histone H3 lysine 27; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; pCREB, phosphorylated (p-133) CREB; RXR, retinoid X receptor; VDR, vitamin D receptor.

Figure 8

Activities of 1,25(OH)₂D₃and FGF23 at Cyp24a1. Gene expression of Cyp24a1 after time course with (A) 10 mg/kg bw 1,25(OH)₂D₃ at 0, 1, 3, 6, 12, and 24 h (n = 4) and (B) 50 mg/kg bw FGF23 at 0, 3, 6, and 12 h (n = 3–4) from our previous publication (10). ChIP-Seq analysis near Cyp24a1 for (C) VDR and RXR (1,25(OH)₂D₃versus Veh only), (D) pCREB, (E) CBP, (F) CRTC2, (G) H3K27ac, and (H) H3K9ac from WT mice injected with 10 mg/kg bw 1,25(OH)₂D₃ or 50 mg/kg bw FGF23 (1 h). Additional details as for Figure 2. Genomic region displayed is chr2: 170,278,379 to 170,324,235. bw, body weight; CBP, CREB-binding protein; ChIP-Seq, chromatin immunoprecipitation sequencing; CRTC2, CREB-regulated transcription coactivator 2; FGF23, fibroblast growth factor 23; H3K9ac, histone acetylation at histone H3 lysine 9; H3K27ac, histone acetylation at histone H3 lysine 27; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; pCREB, phosphorylated (p-133) CREB; RXR, retinoid X receptor; VDR, vitamin D receptor.

Figure 9

SRC1 and SRC3 recruitment at Cyp27b1 and Cyp24a1. ChIP-Seq analysis near Cyp27b1 for (A) SRC1, (B) SRC3 and Cyp24a1, (C) SRC1, (D) SRC3 from WT mice injected with 230 mg/kg bw PTH (30 min, PTH30), 30 mg/kg bw YKL-05-099 (1 h), 40 mg/kg SK-124 (1 h), 10 mg/kg bw 1,25(OH)₂D₃ (1 h), or 50 mg/kg bw FGF23 (1 h). Additional details as for Figure 2. Genomic regions displayed are chr10: 126,469,260 to 126,490,800 and chr2: 170,278,379 to 170,324,235. bw, body weight; ChIP-Seq, chromatin immunoprecipitation sequencing; 1,25(OH)₂D_3, 1,25-dihydroxyvitamin D₃; PTH, parathyroid hormone.

Figure 10

Transcriptional implications of treatments at Cyp27b1. ChIP-Seq analysis near Cyp27b1 for (A) BRD4, (B) RNA polymerase II, and (C) H3K36me3 from WT mice injected with 230 mg/kg bw PTH (30 min, PTH30), 30 mg/kg bw YKL-05 to 099 (1 h), 40 mg/kg bw SK-124 (1 h), 10 mg/kg bw 1,25(OH)₂D₃ (1 h), or 50 mg/kg bw FGF23 (1 h). Additional details as for Figure 2. Genomic region displayed is chr10: 126,469,260 to 126,490,800. bw, body weight; ChIP-Seq, chromatin immunoprecipitation; FGF23, fibroblast growth factor 23; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; PTH, parathyroid hormone.

Figure 11

Transcriptional implications of treatments at Cyp24a1. ChIP-Seq analysis near Cyp24a1 for (A) BRD4, (B) RNA polymerase II (scaled to 70), (C) RNA polymerase II (scaled to 570), and (D) H3K36me3 from WT mice injected with 230 mg/kg bw PTH (30 min, PTH30), 30 mg/kg bw YKL-05-099 (1 h), 40 mg/kg bw SK-124 (1 h), 10 mg/kg bw 1,25(OH)₂D₃ (1 h), or 50 mg/kg bw FGF23 for 1 h. Additional details as for Figure 2. Genomic region displayed is chr2: 170,278,379 to 170,324,235. bw, body weight; ChIP-Seq, chromatin immunoprecipitation sequencing; PTH, parathyroid hormone.

Figure 12

Summary figure of Cyp27b1 and Cyp24a1 regulation in the kidney. Salt-inducible kinases (SIKs) sequester CRTC2 associated with 14-3-3 proteins in the cytoplasm through phosphorylation of CRTC2. PTH signaling cascade activates Cyp27b1 expression (A) in part by PKA inactivation of SIKs, which allows nuclear translocation of unphosphorylated CRTC2 to complex with CBP and phosphorylated CREB to induce Cyp27b1 expression (green up arrow) through the M1 and M21(a–c) enhancers. SIK inhibition by SK-124 allows translocation of CRTC2 without apparent activation of the PKA pathway or increases of pCREB and CBP to activate Cyp27b1. 1,25(OH)₂D₃ acts through the VDR and RXR binding at the M1 and M21(a–c) enhancers to suppress Cyp27b1 (red down arrow). SRC1 and SRC3 are involved in PTH/SIK activation, whereas SRC1 is involved in 1,25(OH)₂D₃ and FGF23 suppression of Cyp27b1. CRTC2 is also increased on the genome by 1,25(OH)₂D₃ through an unknown mechanism. FGF23 signaling through the FGFR/Klotho (Kl) membrane receptor leads to suppression of Cyp27b1 through an unknown transcription factor (??) or signaling cascade. For Cyp24a1 expression, 1,25(OH)₂D₃ and FGF23 signaling uses SRC1, SRC3, and CRTC2 signaling for activation. PTH/SIK signaling suppresses Cyp24a1 expression (B). Enhancers −21 kb to −32 kb, DS1. enhancers −35 kb to −37 kb, DS2. CBP, CREB-binding protein; CRTC2, CREB-regulated transcription coactivator; DS, DownStream region; 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; pCREB, phosphorylated (p-133) CREB; PTH, parathyroid hormone; RXR, retinoid X receptor; VDR, vitamin D receptor.

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