Characterization of FGF23-Dependent Egr-1 Cistrome in the Mouse Renal Proximal Tubule

<div><p>Fibroblast growth factor 23 (FGF23) is a potent regulator of phosphate (Pi) and vitamin D homeostasis. The transcription factor, early growth response 1 (egr-1), is a biomarker for FGF23-induced activation of the ERK1/2 signaling pathway. We have shown that ERK1/2 signaling blockade suppresses renal egr-1 gene expression and prevents FGF23-induced hypophosphatemia and 1,25-dihydroxyvitamin D (1,25(OH)₂D) suppression in mice. To test whether egr-1 itself mediates these renal actions of FGF23, we administered FGF23 to <i>egr-1</i>^<i>-/-</i> and wild-type (WT) mice. In WT mice, FGF23 induced hypophosphatemia and suppressed expression of the renal Na/Pi cotransporters, Npt2a and Npt2c. In FGF23-treated <i>egr-1</i>^<i>-/-</i> mice, hypophosphatemic response was greatly blunted and Na/Pi cotransporter expression was not suppressed. In contrast, FGF23 induced equivalent suppression of serum 1,25(OH)₂D concentrations by suppressing renal <i>cyp27b1</i> and stimulating c<i>yp24a1</i> mRNA expression in both groups of mice. Thus, downstream of receptor binding and ERK1/2 signaling, we can distinguish the effector pathway that mediates FGF23-dependent inhibition of Pi transport from the pathway that mediates inhibition of 1,25(OH)₂D synthesis in the kidney. Furthermore, we demonstrate that the hypophosphatemic effect of FGF23 is significantly blunted in <i>Hyp</i>/<i>egr-1</i>^<i>-/-</i> mice; specifically, serum Pi concentrations and renal Npt2a and Npt2c mRNA expression are significantly higher in <i>Hyp</i>/<i>egr-1</i>^<i>-/-</i> mice than in <i>Hyp</i> mice. We then characterized the egr-1 cistrome in the kidney using ChIP-sequencing and demonstrate recruitment of egr-1 to regulatory DNA elements in proximity to several genes involved in Pi transport. Thus, our data demonstrate that the effect of FGF23 on Pi homeostasis is mediated, at least in part, by activation of egr-1.</p></div

Ingenuity Pathway analysis (IPA)-Top regulatory networks representing genes involved in cellular assembly and organization.

<p><i>A</i>. Network of common genes found in CHIP-seq and microarray datasets. Ubiquitin C (UBC) shown as the central regulator. <i>B</i>. Network of common genes found in CHIP-seq, microarray and Col4a3^-/- mice datasets, <i>C</i>. Network of common genes found in CHIP-seq and FGF23^Tg datasets and, <i>D</i>. Network of common genes found in microarray and FGF23^Tg datasets. The networks are built according to the identified interconnected pathways involving the majority of genes displaying direct interactions. Genes represented in gray belong to the dataset. Genes represented in white are intermediary regulators that do not belong to the cluster.</p

Schematic representation of the active regions identified by ChIP-seq analysis in normal mice kidney after treatment with FGF23 or vehicle.

<p><i>A-C</i>. Venn and Euler diagrams showing overlap of egr-1 binding sites between different treatment groups. <i>D</i>. Motif discovery using MEME. Consensus motif for egr-1, as determined by querying the 1,200 most enriched ChIP-seq loci.</p

List of genes present in the CHIP-seq datasets and reported in the literature to be involved in renal Pi transport.

<p>*Fold change represents egr-1 binding events in FGF23 treated 1-hr sample when compared to vehicle-treated sample. AR-Active Region, TSS-Transcription start site</p><p>List of genes present in the CHIP-seq datasets and reported in the literature to be involved in renal Pi transport.</p

Renal DNase I gene expression.

<p>A. Egr-1 ChIP-seq analysis showed recruitment of egr-1 to DNase I. Peak signal intensity for egr-1 binding up-stream (-7700 bp) of DNase I gene in FGF23-treated mice. A low signal is noted in the vehicle-treated sample. <i>B</i>. Renal DNase I mRNA abundance in WT and <i>egr-1</i>^<i>-/-</i> mice treated with vehicle or FGF23. <i>C</i>. Renal DNase I mRNA abundance in normal mice fed a normal (NP), low (LP) or high (HP) Pi diet for 5 days. Bars depict mean ± SEM (n = 5 mice/group). <i>* P < 0</i>.<i>05</i> compared to control group.</p

Effects of egr-1 gene deletion on phosphate homeostasis.

<p><i>Egr-1</i>^<i>-/-</i> and wild-type (WT) mice were treated with vehicle or FGF23. <i>A</i>. Serum phosphorus concentration. Bars depict percent change when compared to vehicle treated group (n = 8–10 mice/group). <i>B</i>. Renal Npt2a and Npt2c mRNA abundance were quantitated by real-time PCR, normalized to that of gus mRNA, and expressed as a percent relative to vehicle-treated mice. Bars depict mean ± SEM (n = 8–10 mice/group) * <i>P<0</i>.<i>05</i>, compared to <i>egr-1</i>^<i>-/-</i> and WT mice treated with vehicle. <i>C</i> and <i>D</i>. Renal Npt2a and Npt2c protein abundance in renal brush border membrane vesicle preparations normalized to β-actin.</p

Effects of egr-1 gene deletion on renal 1,25(OH)₂D metabolism.

<p><i>Egr-1</i>^<i>-/-</i> and wild-type (WT) mice were treated with vehicle or FGF23. <i>A</i>. Serum 1,25(OH)₂D concentrations. <i>B</i>. Renal c<i>yp27b1</i> and, <i>C</i>. Renal <i>cyp24a1</i> mRNA abundance were quantitated by real-time PCR, normalized to that of gus mRNA, and expressed as a percent relative to vehicle-treated WT mice. Bars depict mean ± SEM (n = 8–10 mice/group) * <i>P<0</i>.<i>05</i>, compared to <i>egr-1</i>^<i>-/-</i> and WT mice treated with vehicle.</p

List of vesicle trafficking genes that are present in both CHIP-seq and microarray datasets.

<p>*Fold change represents change in gene expression in microarray datasets in FGF23 treated 1-hr sample when compared to vehicle-treated sample. Fold change in ChIP-seq datasets not shown.</p><p>List of vesicle trafficking genes that are present in both CHIP-seq and microarray datasets.</p

Analysis of the egr-1 cistrome.

<p>Genome-wide distribution of egr-1 binding sites in normal mice kidney after treatment with FGF23 or vehicle. <i>A</i>. Visualization of egr-1-binding sites across all mouse chromosomes. <i>B</i>. Spatial distribution of egr-1 binding sites at the genome level and, <i>C</i>. near individual genes. <i>D</i>. The ChIP-seq and input data files were analyzed by seqMINER, which analyzes the signal profiles across mouse promoters (TSS), and clusters the profiles according to binding patterns. The result shows that the 3 clusters with the strongest signals (vehicle, FGF23 -1hr and FGF23-2hr) contained 1328, 3288, and 4811 genes (total = 9427), respectively. Vehicle and FGF23-treated samples did not show a difference in signal profile but differed markedly in signal intensity.</p

<i>CYP27B1</i> expression in extra-renal tissues of <i>fgf-23^+/+/</i>1α-Luc<i>^+/−</i> (wt) and <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> (ko) mice.

<p>Mice were sacrificed at 4 weeks of age and the organs removed for determination of <i>CYP27B1</i> mRNA expression, quantitated by real-time PCR, normalized to that of gus mRNA, and expressed as a percent relative to <i>fgf-23^+/+/</i>1α-Luc<i>^+/−</i> mice. Bars depict mean±SEM (n = 5 mice/group), *<i>P<0.05</i>, compared to <i>fgf-23^+/+/</i>1α-Luc<i>^+/−</i> mice. Sm.Int- small intestine, Lg.Int-large intestine.</p