Figure 1.
A. Basal activity of the human CYP27B1 promoter constructs (−1576 bp, −1170 bp, −926 bp, −789 bp, −409 bp, −200 bp) in HEK293 cells.
1α-hydroxylase promoter-driven firefly luciferase activity is normalized to renilla luciferase activity in each individual sample and expressed as relative luciferase activity units (RLU). *P<0.05 when compared to HEK293 cells transfected with empty vector. # P<0.05 when compared to HEK293 cells transfected with 1.6 kb CYP27B1 promoter construct. B. Effect of forskolin (10−5 M) on CYP27B1 promoter activity in HEK293 cells. Bars depict fold induction of relative luciferase activity expressed as mean±SEM. *P<0.05 when compared to HEK293 cells treated with vehicle for each promoter deletion construct. (n = 3 experiments, each performed in triplicate).
Figure 2.
Effect of FGF-23 on CYP27B1 promoter activity in HEK293 cells.
HEK293 cells were transfected with CYP27B1 promoter constructs and treated with FGF-23 (1–200 ng/ml) or vehicle for 21 hours. A. 1576 bp full-length CYP27B1 promoter-driven luciferase activity normalized to renilla luciferase activity and expressed as fold change to vehicle-treated samples. 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.
Figure 3.
Role of MEK/ERK1/2 pathway in FGF-23-mediated signaling in HEK293 cells.
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. A. Egr-1 promoter-driven luciferase activity normalized to renilla luciferase activity and expressed as fold change to vehicle-treated samples. 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.
Figure 4.
Fgf-23+/+/1α-Luc+/− (wt), fgf-23+/−/1α-Luc+/− (het) and fgf-23−/−/1α-Luc+/− (ko) mice were bred as described in Methods. Mice were sacrificed at 4 weeks of age and the kidneys removed and divided into three parts for determination of A. CYP27B1 promoter activity, expressed as luciferase activity per mg of tissue. Graph depicts fold change with respect to luciferase activity in fgf-23+/+/1α-Luc+/− mice. B. CYP27B1 mRNA expression, quantitated by real-time PCR, normalized to that of gus mRNA, and expressed as a percent relative to fgf-23+/+/1α-Luc+/− mice. C. Renal mitochondrial 1α-hydroxylase protein abundance normalized to β-actin (for western blotting, n = 1 mouse/lane). Lane 1–2 (fgf-23+/+/1α-Luc+/− mice), 3–4 (fgf-23+/−/1α-Luc+/− mice), 5–6 (fgf-23−/−/1α-Luc+/− mice). Bars depict mean±SEM (n = 5 mice/group), *P<0.05, compared to fgf-23+/+/1α-Luc+/− mice.
Figure 5.
Effects of MEK/ERK1/2 signaling blockade on CYP27B1 in the kidney.
Fgf-23+/+/1α-Luc+/− (wt) and fgf-23−/−/1α-Luc+/− (ko) transgenic mice were treated with vehicle or PD0325901 and administered a single injection of FGF-23 as described in Methods. A. Renal CYP27B1 activity, expressed as luciferase activity per mg of tissue. Graph depicts fold change with respect to luciferase activity in vehicle-treated fgf-23+/+/1α-Luc mice. B. Renal mitochondrial 1α-hydroxylase protein abundance normalized to β-actin (for western blotting, n = 1 mouse/lane). Lane 1–2 (vehicle-treated fgf-23+/+/1α-Luc+/− mice), 3–4 (vehicle-treated fgf-23−/−/1α-Luc+/− mice), 5–6 (FGF-23-treated fgf-23−/−/1α-Luc+/− mice), 7–8 (FGF-23+PD0325901-treated fgf-23−/−/1α-Luc+/− mice). Bars depict mean±SEM (n = 5 mice/group. *P<0.05, compared to vehicle-treated fgf-23+/+/1α-Luc+/− mice; #P<0.05, compared to vehicle-treated fgf-23−/−/1α-Luc+/− mice.
Figure 6.
Cardiac and aortic expression of CYP27B1.
Fgf-23+/+/1α-Luc+/− (wt), fgf-23+/−/1α-Luc+/− (het) and fgf-23−/−/1α-Luc+/− (ko) mice were bred as described in Methods. Mice were sacrificed at 4 weeks of age, the heart and aorta removed and divided into two parts for determination of CYP27B1 promoter activity in heart (A), and aorta (B), expressed as luciferase activity per mg of tissue. Graph depicts fold change with respect to luciferase activity in fgf-23+/+/1α-Luc+/− mice. CYP27B1 mRNA expression in heart (C), and aorta (D), quantitated by real-time PCR, normalized to that of gus mRNA, and expressed as a percent relative to fgf-23+/+/1α-Luc+/− mice. Bars depict mean±SEM (n = 5 mice/group) *P<0.05, compared to fgf-23+/+/1α-Luc+/− mice.
Figure 7.
Effect of FGF-23 on CYP27B1 mRNA expression in cultured mouse aortic vascular smooth muscle cells (VSMC).
A. 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. CYP27B1 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) *P<0.05, compared to vehicle-treated group.
Figure 8.
Pulmonary expression of CYP27B1.
Fgf-23+/+/1α-Luc+/− (wt), fgf-23+/−/1α-Luc+/− (het) and fgf-23−/−/1α-Luc+/− (ko) mice were bred as described in Methods. Mice were sacrificed at 4 weeks of age and the lung removed and divided into two parts for determination of A. CYP27B1 promoter activity, expressed as luciferase activity per mg of tissue. Graph depicts fold change with respect to luciferase activity in fgf-23+/+/1α-Luc+/− mice. B. CYP27B1 mRNA expression, quantitated by real-time PCR, normalized to that of gus mRNA, and expressed as a percent relative to fgf-23+/+/1α-Luc+/− mice. Bars depict mean±SEM (n = 5 mice/group), *P<0.05, compared to fgf-23+/+/1α-Luc+/− mice.
Figure 9.
CYP27B1 expression in extra-renal tissues of fgf-23+/+/1α-Luc+/− (wt) and fgf-23−/−/1α-Luc+/− (ko) mice.
Mice were sacrificed at 4 weeks of age and the organs removed for determination of CYP27B1 mRNA expression, quantitated by real-time PCR, normalized to that of gus mRNA, and expressed as a percent relative to fgf-23+/+/1α-Luc+/− mice. Bars depict mean±SEM (n = 5 mice/group), *P<0.05, compared to fgf-23+/+/1α-Luc+/− mice. Sm.Int- small intestine, Lg.Int-large intestine.
Table 1.
Serum biochemical values in fgf-23+/+/1α-Luc+/− (wt) and fgf-23−/−/1α-Luc+/− (ko) transgenic mice.