Klotho is a substrate for alpha-, beta- and gamma-secretase

Abstract

Klotho is an anti-aging protein with different functions of the full-length membrane protein and the secreted hormone-like form. Using overexpression and knock-down approaches as well as embryonic fibroblasts of knock-out mice we present evidence that Klotho is shedded by the alpha-secretases ADAM10 and 17 as well as by the beta-secretase beta-APP cleaving enzyme 1. The remaining membrane-bound fragment is a substrate for regulated intramembrane proteolysis by gamma-secretase. Our data suggest that therapeutic approaches targeting these proteases should be carefully analyzed for potential side effects on Klotho-mediated physiological processes.

Fig. 1

Kl is shedded by ADAM10 and BACE1. A) 293Kl cells were transiently transfected with ADAM10 (A10) or empty vector (vehicle) or with siRNAs as indicated or treated with PMA or TAPI-1 for 16h and shedding of Kl was assayed after 4h. After Western blotting, lysates and media were probed with Kl antibodies and relevant lysates with ADAM10 antibodies. B) mRNA from cells transfected with transfection reagent only (ctrl) or control siRNAs or two different siRNAs against ADAM10 was isolated and qPCR using primers specific for ADAM10 was performed. mRNA level in ctrl cells was set to 100% and levels in siRNA treated cells related to that. Results from two independent experiments are shown. C) 293Kl cells were transiently transfeced with empty vector (vehicle) or with BACE1 cDNA or with BACE1 cDNA and in addition with siRNAs as indicated or treated with BACE inhibitor. Thereafter shedding of Kl was assayed as in A). Lysates in addition were probed with BACE1 antibodies.

Fig. 2

Kl is shedded by ADAM10, 17 and BACE1. A) MEFs cells derived from KO mice as indicated were transiently transfected with Kl-flag and shedding was assayed after 24h. After Western blotting, lysates and media were probed with Kl antibody. Representative gels are shown. B) Quantitation of shedding. The ratio of shedded Kl in the medium versus Kl in cell lysates was determined in all MEF lines. In ctrl MEF cells this ratio was set to 100% and the ratios from the other cell lines related to that. Error bars depict standard deviation from 4-7 independent experiments. ***, p<0.001; **, p<0.01.

Fig. 3

Kl is processed by γ-secretase. A) 293Kl cells were incubated with or without two different γ-secretase inhibitors for 24h as indicated, lysed and immunoprecipitated with flag-antibody, separated on 10-20% Tris-Tricine gels, blotted and probed with flag-antibody. B) Kl-flag cDNA was transiently transfected in HEK293 cells stably expressing PS1wt (wt) or dominant negative PS1(D385N). Lysates were immunoprecipitated and processed as in A). C) MEFs derived from wild type mice (wt) or PS1/2 double knock-out mice were transiently transfected with Kl-flag and processed as in A). D) To demonstrate specificity of the antibody lysates from HEK293Kl cells were subjected to immunoprecipitation with flag antibody (IP flag) and with beads only (IP ctrl) and processed as in A). In the input lane 1/25^th of the lysate used for IP was loaded.