Figure 6.

Shedding of Klotho from cultured cells through ADAM10 activation. (A) Expression of ADAM10 (hatched bars) or ADAM17 (open bars) mRNA after incubation of HEK-293 cells with indicated siRNA (siADAM10 or siADAM17) or scrambled control siRNA sequence (dotted line). siADAM10 reduced ADAM10 mRNA to 0.18±0.45 compared with control whereas ADAM17 mRNA was 1.15±0.14 compared with control. siADAM17 reduced ADAM17 mRNA to 0.37±0.0.09 compared with control, whereas ADAM10 mRNA was 0.97±0.0.08 compared with control. Values shown are mean±SEM, n=3 for all conditions. (B) Expression of ADAM10 or ADAM17 protein in HEK-293 cells transiently expressing siRNAs as above. siADAM10 reduced ADAM10 protein to 0.30±0.0.7 compared with control, whereas ADAM17 protein was 1.11±0.11 compared with control. siADAM17 reduced ADAM17 protein to 0.43±0.04 compared with control, whereas ADAM10 protein was 1.16±0.0.06 compared with control. Values shown are mean±SEM, n=7 for all conditions. (C) ADAM10 activity–dependent Klotho shedding from HEK-293 cells transiently transfected with full-length Klotho, CaSR, and ADAM10- or ADAM17-specific siRNAs. Amount of Klotho shed is quantified as the ratio of amount of Klotho released from negative control scrambled siRNA transfected cells, defined as 1.0 (dotted line), and ADAM10 siRNA (hatched bar) or ADAM17 siRNA (open bar). ADAM10 knockdown significantly reduced Klotho levels to 0.50±0.04, consistent with inhibition by TAPI-1 and GI254023X (Figure 2), whereas ADAM17 knockdown had no effect (1.00±0.14). Values are mean±SEM, n=8. Significance was determined by ANOVA with Dunnett multiple comparisons to control scrambled siRNA. ***P<0.001, ****P<0.0001. (D) Representative immunoblots of Klotho shed during 3 hours in serum-free medium, with Ponceau-stained carrier BSA protein shown as loading control (medium, above horizontal bar) and transfected Klotho, endogenous ADAM10, ADAM17, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in HEK-293 cell lysates (below bar, cell lysate).