FGF-23 Regulates CYP27B1 Transcription in the Kidney and in Extra-Renal Tissues

The mitochondrial enzyme 25-hydroxyvitamin D 1α-hydroxylase, which is encoded by the CYP27B1 gene, converts 25OHD to the biological active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D). Renal 1α-hydroxylase activity is the principal determinant

Effect of FGF-23 on CYP27B1 promoter activity in HEK293 cells.

<p>HEK293 cells were transfected with CYP27B1 promoter constructs and treated with FGF-23 (1–200 ng/ml) or vehicle for 21 hours. <b>A.</b> 1576 bp full-length CYP27B1 promoter-driven luciferase activity normalized to renilla luciferase activity and expressed as fold change to vehicle-treated samples. <b>B.</b> CYP27B1 promoter activity of the deletion constructs (−1576 bp, −1170 bp, −926 bp, −789 bp, −409 bp, −200 bp), normalized to renilla luciferase activity. Bars depict fold change of luciferase activity relative to vehicle-treated samples, expressed as mean±SEM (n = 3 experiments, each performed in triplicate). *P<0.05 when compared to the vehicle group.</p

Effect of FGF-23 on <i>CYP27B1</i> mRNA expression in cultured mouse aortic vascular smooth muscle cells (VSMC).

<p><b>A.</b> Activation of ERK1/2 signaling pathway was demonstrated in VSMC treated with FGF-23 (100 ng/ml) for 5–60 min. Phosphorylated ERK1/2 protein expression was detected by western blot analysis. Total Erk2 protein expression was used as loading control. <b>B. </b><i>CYP27B1</i> mRNA expression in VSMC treated with FGF-23 (100 ng/ml) for 21 hrs. mRNA expression was quantitated by real-time PCR, normalized to that of gus mRNA, and expressed as a percent relative to vehicle-treated group. Bars depict mean±SEM (n = 3 separate experiments in triplicate) *<i>P<0.05</i>, compared to vehicle-treated group.</p

Effects of MEK/ERK1/2 signaling blockade on <i>CYP27B1</i> in the kidney.

<p><i>Fgf-23^+/+/</i>1α-Luc<i>^+/−</i> (wt) and <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> (ko) transgenic mice were treated with vehicle or PD0325901 and administered a single injection of FGF-23 as described in <i>Methods</i>. <b>A.</b> Renal <i>CYP27B1</i> activity, expressed as luciferase activity per mg of tissue. Graph depicts fold change with respect to luciferase activity in vehicle-treated <i>fgf-23^+/+/</i>1α-Luc mice. <b>B.</b> Renal mitochondrial 1α-hydroxylase protein abundance normalized to β-actin (for western blotting, n = 1 mouse/lane). Lane 1–2 (vehicle-treated <i>fgf-23^+/+/</i>1α-Luc<i>^+/−</i> mice), 3–4 (vehicle-treated <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> mice), 5–6 (FGF-23-treated <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> mice), 7–8 (FGF-23+PD0325901-treated <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> mice). Bars depict mean±SEM (n = 5 mice/group. *<i>P<0.05</i>, compared to vehicle-treated <i>fgf-23^+/+/</i>1α-Luc<i>^+/−</i> mice; ^#<i>P<0.05</i>, compared to vehicle-treated <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> mice.</p

Pulmonary expression of <i>CYP27B1.</i>

<p><i>Fgf-23^+/+/</i>1α-Luc<i>^+/−</i> (wt), <i>fgf-23^+/−/</i>1α-Luc<i>^+/−</i> (het) and <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> (ko) mice were bred as described in <i>Methods.</i> Mice were sacrificed at 4 weeks of age and the lung removed and divided into two parts for determination of <b>A.. </b><i>CYP27B1</i> promoter activity, expressed as luciferase activity per mg of tissue. Graph depicts fold change with respect to luciferase activity in <i>fgf-23^+/+/</i>1α-Luc<i>^+/−</i> mice. <b>B.. </b><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.</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

Role of MEK/ERK1/2 pathway in FGF-23-mediated signaling in HEK293 cells.

<p>HEK293 cells were transfected with Egr-1 promoter or −200 bp CYP27B1 promoter plasmid and treated with FGF-23 (100 ng/ml) or vehicle for 21 hours. For MEK inhibition, HEK-293 cells were pre-treated with CI-1040 (0–10 µM) 30 min prior to FGF-23 treatment. <b>A.</b> Egr-1 promoter-driven luciferase activity normalized to renilla luciferase activity and expressed as fold change to vehicle-treated samples. <b>B.</b> CYP27B1 promoter activity of the −200 bp deletion construct normalized to renilla luciferase activity and expressed as fold change to vehicle-treated samples. Bars depict mean±SEM (n = 3 experiments, each performed in triplicate). *P<0.05 when compared to the vehicle group.</p

Cardiac and aortic expression of <i>CYP27B1</i>.

<p><i>Fgf-23^+/+/</i>1α-Luc<i>^+/−</i> (wt), <i>fgf-23^+/−/</i>1α-Luc<i>^+/−</i> (het) and <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> (ko) mice were bred as described in <i>Methods.</i> Mice were sacrificed at 4 weeks of age, the heart and aorta removed and divided into two parts for determination of <i>CYP27B1</i> promoter activity in heart <b>(A),</b> and aorta <b>(B)</b>, expressed as luciferase activity per mg of tissue. Graph depicts fold change with respect to luciferase activity in <i>fgf-23^+/+/</i>1α-Luc<i>^+/−</i> mice. <i>CYP27B1</i> mRNA expression in heart <b>(C),</b> and aorta <b>(D)</b>, 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.</p

Serum biochemical values in <i>fgf-23^+/+/</i>1α-Luc<i>^+/−</i> (wt) and <i>fgf-23^−/−/</i>1α-Luc<i>^+/−</i> (ko) transgenic mice.

*<p><i>P<0.05 when compared to fgf-23^+/+/</i>1α-Luc<i>^+/−</i> (wt) mice. Values expressed as Mean±SEM, n = 7 mice per group.</p