ADAM10 Releases a Soluble Form of the GPNMB/Osteoactivin Extracellular Domain with Angiogenic Properties
Figure 5
ADAM10 induces shedding of the GPNMB/OA ectodomain.
(A) Immunoblot analysis of ADAM10, ADAM12 and ADAM17 expression in BT549 and MDA-MB-453 cells. Arrow indicates band corresponding to Adam17 and asterisk denotes a doublet of non-specific bands. (B) siRNA-mediated knockdown of ADAM10, but not ADAM17, reduced shedding of GPNMB/OA in BT549 cells. Upper panels, immunoblot analysis for GPNMB/OA in the CM harvested from BT549-GPNMB/OA cells treated with the indicated control and ADAM-specific siRNAs. Lower panels, immunoblot analysis was performed to determine the degree of ADAM10 and ADAM17 knockdown. Arrow indicates band corresponding to Adam17 and asterisk denotes a doublet of non-specific bands. (C) A role for ADAM10 in GPNMB/OA ectodomain shedding is confirmed in MDA-MB-468 human breast cancer cells that endogenously express GPNMB/OA. An immunoblot for GPNMB/OA was performed on CM harvested from MDA-MB-468 breast cancer cells treated with control or ADAM10-specific siRNAs. Immunoblot analysis with antibodies specific for ADAM10 was performed to confirm knockdown of ADAM10 expression. Immunoblots for α-Tubulin were performed to control for protein loading in whole cell lysates (A, B, and C). Immunoblots for AMF/GPI were performed to control for protein loading in the CM samples (B, C). CM refers to conditioned media, Lysate indicates whole cell lysates prepared from these cells.