It is now well accepted that TNFα mediates the joint inflammation evident in many forms of arthritis (1
). TNFα accomplishes this in part by inducing MMPs and by inhibiting the expression of cartilage-specific genes, such as α2(I) collagen and aggrecan (1
) (). Thus, to understand how arthritis develops, one must understand the mechanisms by which TNFα and related cytokines exert their effects on gene expression. Herein, we showed that TNFα has a unique function, in that it induces cathepsin B cleavage of SirT1, resulting in a stable but enzymatically inactive SirT1 fragment of 75 kd. Thus, the cleavage and inactivation of SirT1 parallels the down-regulation of cartilage-specific gene expression and the up-regulation of the expression of matrix-degrading enzymes (MMPs) that occur following TNFα treatment of human chondrocytes.
The findings of the present study suggest that mediators of inflammation, such as TNFα, work in opposition to SirT1. Inactivation of SirT1 by TNFα may therefore be necessary for optimal development of the inflammatory response. Importantly, many other proinflammatory cytokines participating in arthritis (i.e., IL-1β, IL-6, etc) have been reported to activate cathepsin B (27
). Consistent with these findings, in this study, we observed the 75-kd SirT1 fragment in extracts of human chondrocytes exposed to IL-1β, indicating that it may mediate cathepsin B activation, similar to TNFα.
That a mediator of inflammation plays a role in inhibiting SirT1 is noteworthy when the biologic function of this protein deacetylase is considered. SirT1 plays a role in enhancing organism longevity by inhibiting the development of age-related diseases (9
). It may be that part of the antiaging function of SirT1 resides in its ability to inhibit inflammation. For example, SirT1 inhibits the inflammation associated with chronic obstructive pulmonary disease (31
), colitis (33
), and hepatic steatosis (34
) and can provide a vasoprotective antiinflammatory effect on the vascular endothelium (35
) and in adipocytes (36
). These antiinflammatory functions of SirT1 may be partly due to its ability to block the mediator of inflammation NF-κB via deacetylation (17
). When examined in a cartilage context, these functions of SirT1 are consistent with its role in maintaining chondrocyte viability and phenotype as well as cartilage-specific gene expression (13
). SirT1 could therefore be an important player in reducing the severity of arthritis. Taken together, the data would also suggest that prolonged inflammation may work against any SirT1-mediated enhancement of longevity by blocking SirT1 function.
The mechanism by which TNFα inhibits SirT1 activity appears to rely on an interesting posttranslational modification. While modifications such as phosphorylation and sumoylation have been reported to alter SirT1 enzymatic activity (37
), in this study, we demonstrated that SirT1 was subject to site-specific cleavage by cathepsin B, resulting in impaired enzymatic activity. While the cleavage did not appear to occur within the active site of SirT1, enzymatic activity was nevertheless reduced, suggesting that sequences outside the active site are essential for optimal activity. Recent evidence indicates that protein SENP1 associates with the carboxy-terminus of SirT1 and thereby enhances its activity via desumoylation (38
). It is therefore possible that cleavage of the carboxy-terminus of SirT1 reduces the ability of factors such as SENP1 to bind and activate SirT1. Additionally, evidence in yeast indicates that SirT1 forms homotrimeric complexes via an interaction domain within the N-terminal part of the protein (39
). Since the 75-kd fragment retains the N-terminal domain, it is possible that it is able to form these trimeric complexes, yet these complexes may be enzymatically inactive.
The mechanism by which cathepsin B is able to cleave SirT1 is interesting because cathepsins are normally confined to the lysosome. However, under certain conditions, these enzymes are able to translocate to the cytoplasm and nucleus, retaining their activity (23
). Our unpublished observations indicate that TNFα induces expression of cathepsin B and its translocation from the lysosome to the cytoplasm and nucleus. It has recently been demonstrated, for example, that nuclear histone H3 can be cleaved by cathepsin L even at neutral pH, under conditions of embryonic stem cell differentiation (41
). In this study, we showed that TNFα stimulation had the same effect on SirT1, which is typically confined to the nuclear compartment. Baici et al found that chondrocytes derived from OA cartilage possessed elevated levels of active cathepsin B as compared to normal chondrocytes (42
), which is also consistent withourunpublishedobservations,suggestingthatcathepsin B may play a pathologic role OA development.
In conclusion, our data indicate that the inflammatory cytokine TNFα mediates a proteolytic cleavage of SirT1, producing a stable 75-kd fragment that is incapable of binding chromatin and chromatin-associated coactivators, such as PGC-1α and SOX9. As a result of this cleavage, SirT1 additionally loses a significant level of its enzymatic activity, which may directly deter the expression of cartilage-specific genes. Considering that TNFα alone does not induce apoptosis in chondrocytes, the data would suggest that this fragment of SirT1 serves an alternate biologic function, perhaps even to protect cells against death during the inflammatory response. Current efforts are underway to identify this function.