ADAM15 is a transmembrane-anchored multidomain glycoprotein that belongs to the large family of the disintegrin metalloproteinases (for review, see ref. 1). ADAM15 has been implicated in several conditions involving inflammatory (2) and neoplastic extracellular matrix (ECM) remodeling (3, 4) and neovascularization (5), and its overexpression could be closely associated with the progression of aggressive forms of prostate and breast cancer (for review, see ref. 4). However, ADAM15 up-regulation is not confined to tumorigenesis, but is also detectable in non-neoplastic conditions involving tissue remodeling, such as degenerative joint disease (6–8). It is markedly up-regulated even at early stages of osteoarthritis (OA), whereas its expression remains below the threshold of detectability in healthy cartilage (6). Catalytic activities of ADAM15 have been demonstrated in conjunction with ectodomain shedding (9–11) and type IV collagen cleavage (12), thereby implying at least a theoretical possibility of its contribution to chondrocyte-mediated ECM breakdown in OA cartilage. However, there is currently no direct experimental evidence for such a role of ADAM15 in proteolytic cartilage remodeling in vivo. Moreover, our previous studies, which revealed accelerated development of OA lesions in ADAM15-deficient mice compared with wild-type mice (7), suggest a homeostatic rather than a destructive role of ADAM15 in cartilage remodeling.
More recent data further suggest that ADAM15 can exert additional functions that are independent of its potential proteolytic activity and highly relevant to the cell–matrix interactions and outside-in signaling in OA chondrocytes ( 8). Thus, the pro domain of ADAM15 confers binding to cartilage-specific types II and VI collagens (7, 8). Moreover, the cytoplasmic tail of ADAM15 has a modulatory effect on the phosphorylation of focal adhesion kinase (FAK) during chondrocyte–collagen interaction (8). Since FAK plays a central role in the integration of growth factor receptor and ECM signals by forming a signaling scaffold involved in growth, differentiation, and survival pathways (13, 14), its modulation by up-regulation of ADAM15 in OA chondrocytes is likely to affect further downstream cascades that control cell fate and thus the integrity of the entire cartilage. As the replenishment of ECM molecules is accomplished exclusively by chondrocytes, any significant impairment of their vitality would inevitably result in the loss of functional cartilage matrix. Accordingly, substantial DNA damage (15–18) and other alterations indicative of cellular degeneration are indeed detectable in OA chondrocytes and are likely involved in the activation of effector pathways of programmed cell death. Thus, apoptosis of chondrocytes is a feature of OA cartilage, although its quantitative contribution to matrix degeneration is still a matter of debate (for review, see ref. 19).
Irrespective of these uncertainties, there appears to be an obvious discrepancy between the extent of detectable severe cellular damage and the relatively small fraction of apoptotic cells (<1%) traceable at any time in OA cartilage ( 15). It seems that instead of dying, a considerable proportion of OA chondrocytes remain preserved in a pre- or para-apoptotic phenotype (20) with a discoordinated gene expression pattern, for reasons that are thus far unknown. In the present investigation we obtained clear experimental evidence of the capacity of ADAM15 to exert antiapoptotic effects in stressed human chondrocytes in vitro, thereby suggesting its contribution to the preservation of a preapoptotic chondrocytic phenotype in OA cartilage. Our results with an ADAM15-transfected chondrocyte line as well as with primary human OA chondrocytes revealed a striking role of ADAM15 as a potent suppressor of apoptosis upon induction of cell death by various stimuli. This action of ADAM15 is accomplished by up-regulation of X-linked inhibitor of apoptosis (XIAP).
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Results of the present study provide substantial evidence of a novel function of ADAM15 as a protein with antiapoptotic properties in OA cartilage. OA is a degenerative joint disease characterized by the progressive loss of intact cartilage matrix and impairment of the phenotypic stability and cellular integrity of chondrocytes, which, in combination, lead to failure to maintain tissue homeostasis (for review, see ref. 30). Thus, the chondrocytes in OA cartilage are exposed to enhanced stress, e.g., by mechanical forces or by cytokine exposure that is associated with increased production of reactive oxygen (31, 32) which, in turn, has been incriminated in accelerated aging processes due to the accumulation of oxidatively damaged molecules (33).
In this regard, genomic DNA is an extraordinarily delicate target of oxidative damage that can cause significant changes in the transcription pattern of genes required for normal chondrocyte function and viability. Many studies show that OA chondrocytes exhibit significant DNA damage ( 15–18), which should normally render these cells susceptible to programmed cell death. Accordingly, experimental evidence also clearly suggests that apoptosis occurs in OA cartilage at an increased frequency compared with that in normal cartilage (for review, see ref. 19). However, considering the extent of detectable DNA damage in OA chondrocytes, the occurrence of apoptosis rather appears to be a relatively rare event in OA cartilage, affecting <1% of the total cell population (15), whereas a considerable proportion of the chondrocytes seem to survive in a pre- or para-apoptotic phenotype for reasons that are thus far unknown (20). In this respect, the present results revealing previously unrecognized antiapoptotic properties of ADAM15 suggest a mechanism that could contribute to the rescue of chondrocytes from genotoxic-stress–induced apoptosis. Thus, we have shown that ADAM15-transfected chondrocytes exhibit significantly lower caspase 3/7 activity and subsequent PARP cleavage compared with vector-transfected cells, following induction of apoptosis by camptothecin exposure as a model of genotoxic stress or other cell death stimuli, such as growth factor deprivation or loss of matrix contact.
Caspase 3 is crucial in virtually every model of apoptosis ( 34) and belongs to a subfamily of effector caspases, activated far downstream of apoptosis initiation. Among the natural substrates of caspase 3 are many proteins involved in cell maintenance and/or repair (35), and its selective pharmacologic inhibition has been shown to inhibit chondrocyte apoptosis and to maintain cell functionality (36). It is therefore intriguing that our results suggest that the up-regulation of ADAM15 in OA chondrocytes (2) is capable of activating an antiapoptotic pathway that finally interferes at the level of effector caspases. Our study provides unequivocal evidence of the ADAM15-dependent suppression of DNA damage–induced caspase 3 activity by up-regulation of XIAP, which belongs to the family of inhibitor of apoptosis proteins (37).
The pathways underlying ADAM15-induced XIAP up-regulation upon genotoxic stress seem to be rather complex, involving transcriptional and posttranscriptional mechanisms. The increase of XIAP protein in ADAM15-transfected chondrocytes is not mirrored by an equivalent rise in mRNA levels, in contrast to findings in control chondrocytes, which exhibit a concomitant decrease of XIAP protein and mRNA levels under identical conditions. Accordingly, it appears that ADAM15-mediated XIAP up-regulation is at least partially due to stabilizing protein interactions and/or interference with XIAP mRNA decay, representing already described paradigmatic posttranscriptional control mechanisms of XIAP expression ( 28, 29). In this context, it is of note that silencing of XIAP in ADAM15-expressing OA chondrocytes did not affect ADAM15 expression (data not shown), indicating that a potential reciprocal regulatory connection between XIAP and ADAM15 is rather unlikely.
XIAP is probably the most potent member of this family in terms of its unique ability among the mammalian IAPs to inhibit activated caspases and thereby to suppress both the mitochondrial and the death receptor–mediated pathways of apoptosis ( 37). XIAP directly inhibits both initiator caspase 9 and effector caspases 3 and 7. Accordingly, our investigations using RNA interference technology show that the specific knockdown of XIAP protein expression in ADAM15-transfected T/C-28a4 chondrocytes as well as in primary OA chondrocytes leads to an increase in caspase 3 activity upon exposure to camptothecin as an apoptosis-inducing stimulus. Corresponding results have been obtained in studies aimed at the development of therapeutic strategies to sensitize neoplastic cells to apoptosis induction, which have shown that XIAP-specific RNA interference renders breast cancer and chondrosarcoma cells more susceptible to chemotherapeutic agents (38, 39) and enhances radiosensitivity of grade II chondrosarcoma (40).
In light of the pivotal role of XIAP in apoptosis regulation, the literature on its expression and cellular distribution in cartilage is surprisingly sparse. To our knowledge, the present immunohistochemical investigation is the first to demonstrate intense and widely distributed cellular XIAP staining in all layers of OA cartilage, in contrast to its moderate expression in chondrocytes of normal cartilage, with limited detectability only in the superficial zone and the uppermost parts of the middle zone. Not only is XIAP expression in OA cartilage markedly up-regulated and widespread, but it seems to be coexpressed with ADAM15 in OA. Accordingly, the ex vivo detectable up-regulation of XIAP in ADAM15-positive chondrocytes might at least partially reflect the newly elucidated functional role of ADAM15 in an in vitro model of genotoxic stress–induced caspase 3 activation. Clearly such an interpretation remains somewhat restricted by the inherent limited transferability of in vitro studies to in vivo conditions. However, since the up-regulation of ADAM15 is already detectable in early OA ( 2), this mechanism might contribute to the escape of the stressed OA chondrocytes from apoptosis and could at least partially explain why aging ADAM15-deficient mice exhibit accelerated OA development compared with wild-type controls (7).
The precise sequence of events responsible for the up-regulation of XIAP in the stressed chondrocytes remains to be elucidated in future studies. However, a few possibilities can be envisaged. It might be hypothesized that ADAM15-mediated shedding of growth factor receptor ligands plays a role, analogous to scenarios that have been proposed for its role in tumorigenesis; e.g., E-cadherin shedding with subsequent transactivation of the ErbB receptor by soluble cadherin ( 11). However, such a hypothetical mechanism is critically dependent on the assumption that ADAM15 is proteolytically active, and is rather unlikely to explain our results in chondrocytes. The protease domain of ADAM15 is kept inactive by the pro domain, which needs to be cleaved by furin-type proconvertases for activation, as has been demonstrated for the homologous ADAMs 10 and 17 (41, 42). In our experiments, however, no convertase activity became detectable, since transfection of the T/C-28a4 chondrocytes with full-length ADAM15 always produced the intact molecule, without the appearance of any fragment corresponding to a furin-cleaved ADAM15 in Western blot analysis.
Thus, we suggest another scenario as potentially relevant to the antiapoptotic properties of ADAM15, relating to its recently elucidated function as a proadhesive molecule. ADAM15 binds via its pro domain to cartilage-specific collagens, thereby leading to reinforcement of integrin-mediated cartilage–matrix adhesion ( 7, 8). Moreover, during chondrocyte–collagen interaction ADAM15 can modulate the phosphorylation of FAK (8), which is a central signaling scaffold that integrates growth factor receptor and ECM signals into various connected downstream cascades, including crucial survival pathways. It remains an intriguing possibility that ADAM15 might exert its protective role for the stressed chondrocyte in OA cartilage by tuning outside- in signals via the modulation of FAK, which is also part of the mechanotransduction pathway (43).
However, the protective effect provided by ADAM15 to stressed OA chondrocytes could be a double-edged sword, since the advantage of rescue from cell death is likely to be at least partially counterbalanced by the preservation of an altered chondrocytic phenotype resulting from accumulated genotoxic damage that would otherwise inevitably be censored by programmed cell death. Accordingly, the ADAM15-mediated chondroprotection could help to delay the time frame until chondrocytes fail to maintain homeostasis in OA cartilage. However, the capacity of this compensation mechanism to help the postmitotic chondrocytes fulfill their function in the replenishment of ECM molecules for periods of time as prolonged as decades is presumably limited, thereby explaining the low but detectable rate of chondrocyte apoptosis in OA cartilage despite ADAM15 up-regulation. Thus, the novel role of ADAM15 as a potent suppressor of apoptosis, accomplished by up-regulating XIAP in response to different death-inducing stimuli, might be of prime relevance to the understanding of mechanisms leading to the altered preapoptotic phenotype of chondrocytes in OA cartilage. This warrants further elucidation in future studies.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Burkhardt had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Böhm, Burkhardt.
Acquisition of data. Böhm, Hess, Krause, Schirner, Ewald, Aigner.
Analysis and interpretation of data. Böhm, Aigner, Burkhardt.