Abstract
ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.
Highlights
Introduction of Point MutationsThe Tyr-861 of focal adhesion kinase (FAK) was mutated into Phe-861 using the QuikChange site-directed mutagenesis kit from Stratagene according to the manufacturer’s instructions.Preparation of Cell Lysates and Western Blotting—Cell lysates were prepared and immunoblotted as described previously [21]
The expression of ADAM15 in T/C28a4 transfected cells remained below the level detected in the majority of distinct primary chondrocyte populations derived from 15 OA patients
Cells expressing full-length ADAM15 displayed a significantly reduced caspase-3/7 activation in response to apoptosis induction by camptothecin compared with vector-transfected control chondrocytes (Fig. 1A) [21]
Summary
ADAM15 confers apoptosis resistance to chondrocytes upon exposure to genotoxic stress. That ADAM15 expression leads to a significantly increased cell viability and apoptosis resistance of primary human OA chondrocytes after inducing DNA damage by the topoisomerase inhibitor camptothecin [21] This anti-apoptotic effect of ADAM15 was accompanied by an up-regulation of the anti-apoptotic protein X-linked inhibitor of apoptosis with a concomitantly reduced expression of activated caspase-3 [21]. We demonstrate that FAK phosphorylation at Tyr-397, Tyr-576, and Tyr-861 induced by camptothecin is considerably enhanced in ADAM15 expressing T/C28a4 chondrocytic cells This ADAM15-dependent reinforcement of FAK phosphorylation in response to an apoptosis-inducing stimulus is critically dependent on the cytoplasmic domain of ADAM15. ADAM15 leads to an amplified FAKSrc complex activation in response to genotoxic stress, thereby reinforcing counter-regulatory survival pathways This ADAM15-dependent mechanism is likely not confined to chondrocytic cell survival and relevant to apoptosis resistance in neoplastic growth
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