Regulation of fibroblast growth factor-23 signaling by klotho

Abstract

The aging suppressor gene Klotho encodes a single-pass transmembrane protein. Klotho-deficient mice exhibit a variety of aging-like phenotypes, many of which are similar to those observed in fibroblast growth factor-23 (FGF23)-deficient mice. To test the possibility that Klotho and FGF23 may function in a common signal transduction pathway(s), we investigated whether Klotho is involved in FGF signaling. Here we show that Klotho protein directly binds to multiple FGF receptors (FGFRs). The Klotho-FGFR complex binds to FGF23 with higher affinity than FGFR or Klotho alone. In addition, Klotho significantly enhanced the ability of FGF23 to induce phosphorylation of FGF receptor substrate and ERK in various types of cells. Thus, Klotho functions as a cofactor essential for activation of FGF signaling by FGF23.

Figure 1. Klotho binds to multiple FGF receptors

Lysates of 293KL cells transfected with expression vectors for different FGFR isoforms were immunoprecipitated with Klotho using anti-FLAG antibody and probed either with FGFRs using anti-V5 antibody or with Klotho using KM2119 (upper two panels). The lysates were also immunoprecipitated with FGFRs using anti-V5 antibody and probed either with Klotho using KM2119 or with FGFRs using anti-V5 antibody (lower two panels). Antibodies used for immunoprecipitation (i.p.) and immunoblotting (i.b.) were indicated. The possibility that Klotho could bind to FGFR2b(L) was not excluded because of its low expression. Schemes for FGFR isoforms used in this study are shown above the data. The difference between b and c isoforms in FGFR1-3 resides in the C-terminal half of the third immunoglobulin-like (Ig3) domain as indicated in the scheme by white and black boxes, respectively. The difference in genomic structure among FGFR isoforms was summarized in supplemental Fig. 1.

Figure 2. FGF23 preferentially binds to the Klotho-FGFR complex

Agarose beads bound to FGFR, Klotho, or the Klotho-FGFR complex were incubated with conditioned medium containing mouse FGF23(R179Q) and probed with FGF23, FGFR (anti-V5 anti-body), or Klotho by immunoblotting.

Figure 3. FGF23 requires Klotho to activate FGF signaling

A, 293 cells or Klotho-expessing 293 cells (293KL) were stimulated with various concentration of recombinant human FGF23 or basic FGF (100 ng/ml) for 15 min. Activation of FGF signaling was determined by immunoblot analysis using anti-phospho-FRS2a antibody (p-FRS2a), anti-phospho-ERK1/2 antibody (p-ERK1/2), or anti-ERK1/2 antibody (ERK1/2). B, 293KL cells or 293KLDTM cells were stimulated with the conditioned medium containing mouse FGF23 (R179Q) (~10 ng/ml) for 15 min. The same volume of conditionedmedium from mock-transfected 293 cells was used as a negative control (FGF23−). C, detection of endogenous FGFRs bound to Klotho in 293KL cells. Lysates of 293, 293KL, and 293KLDTM cells were immunoprecipitated with Klotho using anti-FLAG antibody and then immunoblotted with antibodies against FGFR1, FGFR2, FGFR3, and Klotho. Lysates of 293 cells transfected with expression vectors for mouse FGFR1c, FGFR2c, and FGFR3c were immunoprecipitated with anti-V5 antibody and used as positive controls for immunoblotting of FGFR1, FGFR2, and FGFR3, respectively. D, a model for interaction between Klotho, FGFR, FGF23, and FGF signaling.

Figure 4. Klotho functions as a regulator of FGF23 signaling in various types of cells

PC12 and HeLa cells were infected with adenovirous expressing the full-length Klotho or GFP as a negative control and then stimulated with mouse FGF23 (R179Q) for 15 min. CHO cells and Klotho-expressing CHO cells (CHOKL) were also stimulated in the same way. Cell lysates were immunoblotted with anti-Klotho antibody (KM2119) to confirm Klotho expression. Activity of FGF signaling was analyzed in the same way as Fig. 3.