FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis - PubMed (original) (raw)

FGF-23-Klotho signaling stimulates proliferation and prevents vitamin D-induced apoptosis

Damian Medici et al. J Cell Biol. 2008.

Abstract

Fibroblast growth factor 23 (FGF-23) and Klotho are secretory proteins that regulate mineral-ion metabolism. Fgf-23(-/-) or Klotho(-/-) knockout mice exhibit several pathophysiological processes consistent with premature aging including severe atrophy of tissues. We show that the signal transduction pathways initiated by FGF-23-Klotho prevent tissue atrophy by stimulating proliferation and preventing apoptosis caused by excessive systemic vitamin D. Because serum levels of active vitamin D are greatly increased upon genetic ablation of Fgf-23 or Klotho, we find that these molecules have a dual role in suppression of apoptotic actions of vitamin D through both negative regulation of 1alpha-hydroxylase expression and phosphoinositide-3 kinase-dependent inhibition of caspase activity. These data provide new insights into the physiological roles of FGF-23 and Klotho.

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Figures

Figure 1.

Figure 1.

Signaling events induced by FGF-23–Klotho. Immunoblotting showing that FGF-23 or Klotho alone have no effect on kinase activity in PTEC or FHs74Int cells. Combined effects of FGF-23 and Klotho show increased phosphorylation of ERK1/2, p38, JNK, AKT, IκB, and GSK-3β. α-Tubulin was used as a loading control.

Figure 2.

Figure 2.

FGF-23–Klotho signaling promotes cell proliferation. (A and B) ELISA analysis showing increased expression of the cell cycle proteins Cyclin D1 (A) and c-myc (B) upon exposure of cells to FGF-23 and Klotho. FGF-23 or Klotho alone had little effect on expression of these proteins. Addition of a small molecule Ras inhibitor nearly abolished up-regulation of these proteins by FGF-23–Klotho, whereas PI3K inhibitor had minimal effect. Graphs represent mean ± SD (n = 3). *, P < 0.001. (C) Flow cytometry analysis for BrdU incorporation under these conditions showing that cell proliferation correlated with the observed expression patterns for Cyclin D1 and c-myc. (D and E) Ki67 immunostaining showing that expression was lower in Fgf-23−/− mice and higher in FGF-23 transgenic mice. Intestinal tissue showed intense nuclear Ki67 staining (E), whereas the kidney tissue only stained in the cytoplasm. Bar, 100 μm. Graphs represent mean ± SD (n = 3). *, P < 0.05.

Figure 3.

Figure 3.

FGF-23–Klotho prevents vitamin D–induced apoptosis. (A) ELISA analysis of 1α-hydroxylase expression showing no significant changes in PTEC cells exposed to FGF-23 or Klotho alone but greatly decreased levels when exposed to both FGF-23 and Klotho. Small molecule inhibitors against Ras and PI3K were sufficient to provide marginal rescue of this decrease in expression. No significant changes were found for treatment of FHs74Int cells. Graphs represent mean ± SD (n = 3). *, P < 0.05. (B) Flow cytometry analysis for TUNEL staining of cells exposed to exogenous vitamin D showing that it caused extremely high levels of apoptosis. Addition of FGF-23 and Klotho was sufficient to rescue most of the vitamin D–induced apoptosis, whereas FGF-23 or Klotho alone did not. PI3K inhibitor prevented this rescue, whereas Ras inhibitor had no effect. (C) ELISA for active caspase-3 levels, showing the same patterns as observed with the TUNEL analysis. Graphs represent mean ± SD (n = 3). *, P < 0.001.

Figure 4.

Figure 4.

Genetic ablation of 1α-hydroxylase prevents apoptosis and atrophy in tissues of Fgf-23−/− mice. (A) Real-time quantitative PCR analysis showing that gene expression levels of 1α-hydroxylase from kidney tissues of Fgf-23−/− mice is much higher than those of wild-type (control) mice. Graphs represent mean ± SD (kidney, n = 6; intestine, n = 5 ). *, P < 0.01. (B and C) TUNEL staining of tissue sections from kidney and intestine of Fgf-23−/− mice showing far higher levels of apoptosis than those of wild-type mice. Fgf-23−/−/1α-hydroxylase−/− double knockout mice show a complete rescue of the apoptosis seen in tissues of Fgf-23−/− mice. Bar, 100 μm. Graphs represent mean ± SD (n = 3). *, P < 0.001.

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