ADAM15 belongs to a family of adamalysin (ADAM) metalloproteinase disintegrins (MDCs) that are membrane-anchored glycoproteins containing modular metalloproteinase, disintegrin, cysteine-rich, and epidermal growth factor–like domains, followed in most cases by a transmembrane region and a cytoplasmic tail (1). ADAMs have been implicated in fertilization, myogenesis, neurogenesis, and protein ectodomain shedding (for recent reviews, see refs. 1 and2) and are also thought to play roles in cell–cell or cell–matrix adhesion through interactions with integrins (3, 4) or syndecans (5, 6).
For ADAM15, a variety of potential functions have been postulated. Thus, cell–cell interaction studies using its recombinant extracellular domains provide experimental evidence for a role in integrin ligation (αvβ3, α5β1, α9β1) (3,7,8). The localization to adherens junctions in endothelial cell cultures and an enhancement of cell–cell interactions in ADAM15-overexpressing fibroblasts (9) indicate a role in cell adhesion. In addition, ADAM15 contains a catalytic site consensus sequence for zinc-dependent metalloproteinases, and the purified recombinant protein is catalytically active (10). The cytoplasmic domain of ADAM15 harbors potential signaling motifs, such as src homology 3 (SH3) ligand domains, and has been shown to interact with Src kinase family members (11, 12).
More recently, the generation of lines of mice with a targeted disruption of ADAM15 allowed an evaluation of its role in mouse development and adult homeostasis (13). ADAM15 deficiency did not affect fertility and viability and was not associated with any evident pathologic phenotype. However, reduced responses to experimental hypoxia and tumor-induced angiogenesis in the knockout mice indicated a role of ADAM15 in pathologic neovascularization.
Another pathologic condition that has been associated with ADAM15 expression is degenerative joint disease (14, 15). The hypothesis of a role of this MDC in promoting pathologic extracellular matrix remodeling in the joints had been proposed on the basis of a strong up-regulation by chondrocytes in human osteoarthritic and neoplastic cartilage specimens (14). This hypothetical catabolic effect of ADAM15 on joint integrity was further investigated in the present comparative study of aging ADAM15-deficient (ADAM15−/−) and wild-type (WT) mice representing a spontaneous age-dependent murine model of progressive joint degeneration. The histopathologic scores used for quantification of degenerative joint disease were significantly increased (up to 3-fold) in the ADAM15−/− mice compared with the respective age- and sex-matched WT controls, although the severity of osteoarthritic lesions was more pronounced in male than in female ADAM15−/− mice.
These results render unlikely the originally proposed catabolic effect of ADAM15 on cartilage metabolism, and they instead suggest a long-term protective role of ADAM15 in maintaining joint integrity. Accordingly, overexpression of ADAM15 in the human chondrocytic cell line T/C28a4 (16) results in increased adhesion and cell survival on matrices of types II and VI collagen, which represent the major constituents of the perichondrocytic collagenous meshwork in cartilage. This newly unraveled potential of ADAM15 to enhance extracellular survival signals might be a relevant compensatory mechanism to counteract chondrocytic stress factors in joint degeneration.
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- MATERIALS AND METHODS
The purpose of the present study was to gain insight into the role of ADAM15 in the development of osteoarthritis by a histopathologic study of the age-dependent development of degenerative cartilage and bone lesions in ADAM15-deficient mice (13) compared with WT controls. While an analysis of ADAM15−/− mice did not uncover any evident defects during development or adult homeostasis, this study did not include a thorough analysis of potential defects in cartilage or bone in adult mice.
A potential role of ADAM15 as a factor that promotes tissue remodeling in degenerative joint disease, most likely associated with a proteolytic effect of its catalytic domain on extracellular matrix molecules, was originally suggested by its strong up-regulation in synovial and cartilage specimens derived from human osteoarthritic lesions (14, 15), although alternative interpretations could not be formally excluded. In this respect, the monitoring of age-dependent morphologic changes in ADAM15−/− mice offered a unique opportunity to evaluate potential relationships between the expression of ADAM15 and the development of osteoarthritic lesions. However, instead of the expected amelioration of joint pathology in ADAM15−/− mice, we observed more severe signs of cartilage and bone degradation than in the respective age- and sex-matched WT controls. Accordingly, the results in the knockout model suggest a protective role of ADAM15 in the maintenance of joint integrity.
Thus, the targeted gene disruption seems to weaken the compensatory mechanisms of articular cartilage that are required to preserve long-term structural integrity and to withstand life-long mechanical stress in the aging knockout mice. While these results shed new light on the role of ADAM15 in the metabolism of aging cartilage, they also raise new intriguing questions concerning the underlying mechanisms of action.
The multidomain structure of ADAM15 harbors several functional domains that could hypothetically be involved in mediating protective effects on cartilage. In an initial attempt to establish an in vitro system for the investigation of potential mechanisms contributing to protective effects, full-length cDNA of human ADAM15 was stably transfected into the human chondrocytic cell line T/C28a4 (16). To investigate whether cell–matrix interactions might be modulated by ADAM15 overexpression, we analyzed the binding properties of ADAM15- and vector-transfected T/C28a4 cells to different extracellular matrix molecules. Interestingly, we noted a specific enhancement of in vitro adhesion to types II and VI collagen as physiologic components of the pericellular collagen meshwork in normal cartilage (20), but we noted no enhancement of in vitro adhesion to fibronectin. The improved collagen adhesion of the ADAM15-overexpressing T/C28a4 cells compared with the vector-transfected controls was accompanied by a prolongation of viability under conditions of serum starvation in vitro, indicating the reinforcement of matrix contact–dependent survival signals by ADAM15.
Chondrocyte attachment to collagen is known to involve the integrin-mediated transduction of signals that are critical for cell survival (21, 22), and we could confirm reported data on the expression of the respective collagen-binding integrins α1β1 and α2β1 (19) in T/C28a4 cells. Accordingly, a modulating interaction with outside-in signaling of integrins had to be considered as a hypothesis to explain the effect of ADAM15 on the reinforcement of cell adhesion. Additional circumstantial evidence supporting this assumption is derived from the known capability of ADAM15 to function as an integrin ligand (αvβ3 and α9β1) (3, 7, 8). However, a direct interaction with collagen-binding integrins is rather unlikely, since it has been shown that the recombinant extracellular domains of ADAM15 do not bind to α1β1 and α2β1 integrins (7). Likewise, a direct cis-acting effect of ADAM15 on the extracellular domains of the fibronectin-binding integrins αvβ3 and α5β1 can also be excluded as an explanation for the modulated cell–matrix attachment. Although both integrins have been shown to interact with the recombinant disintegrin domain of human (but not mouse) ADAM15 (3, 7, 8), fibronectin binding of T/C28a4 cells remained unaffected by overexpression of the human ortholog.
Therefore, the molecular mechanism that is critical for the enhancement of collagen binding in ADAM15-overexpressing chondrocytes remains to be elucidated in future studies. Nevertheless, the present in vitro studies revealed new facets of ADAM15 action that could be relevant as compensatory mechanisms to sustain chondrocyte–matrix adhesion and cell survival in cartilage degeneration. This hypothesis about the homeostatic effects of ADAM15 is entirely consistent with the observed aggravation of cartilage degeneration in aging ADAM15−/− mice compared with WT controls.
Moreover, the consequences of ADAM15 gene disruption on cartilage–matrix interactions suggested by the results of the transfection experiments with the T/C28a4 cells in the present investigation are consistent with more recent findings on accelerated, aging-dependent osteoarthritis development in α1 integrin–deficient mice (23). This knockout model of degenerative joint disease shares a surprisingly high degree of phenotypic similarity with the ADAM15-deficient mice. Whereas both α1 integrin and ADAM15 are physiologically expressed in hypertrophic cartilage of the growth plate, their roles do not seem to be critical for normal skeletal development in the respective knockout mice. ADAM15 and α1 integrin are both up-regulated in remodeling cartilage (14, 23), and the targeted disruption of the respective genes in mice leads to accelerated development of osteoarthritic lesions. For α1 integrin, the phenotypic consequences of the gene knockout occur somewhat earlier (23) and have been associated with the disturbance of its function in promoting adhesion-dependent cell survival and progression through the G1 phase of the cell cycle in response to mitogenic growth factors (24).
Interestingly, the transfection experiments in the present study indicate a reinforcing role of ADAM15 on the same effector mechanism, namely, adhesion-dependent cell survival. Moreover, for the intracellular SH3 ligand homology domains of ADAM15, interactions with adaptor molecules and Src kinase family members have been described (11, 12) that are also essential in the α1 signaling cascades. In light of the data described here, it will now be interesting to determine whether ADAM15-mediated signal transduction might lead to a convergence with the α1 integrin pathway to provide reinforced survival signals for chondrocytes in the remodeling osteoarthritic cartilage.