a) Background. ? The work in my lab has revolved around the study of chondrocyte gene expression, cell proliferation and viability with attention focused on the changes that take place in human cartilage that lead to the arthritic diseases. We have sought to identify and analyze the factors that affect these aspects of cartilage biology. We have explored the roles of cartilage oligomeric matrix protein (COMP) and histone deacetylases in regulating chondrocyte viability and gene expression. We have shown that these proteins affect chondrocytes in distinct ways. For example COMP enhances survival and proliferation by regulating expression of a unique set of anti-apoptotic proteins. We have also found that distinct histone deacetylase are potent regulators of cartilage specific gene expression. These results provide us with the background to our present studies, enabling us to fill the gaps in our knowledge about the role of critical extracellular and intracellular proteins in cartilage biology. ? b) Objectives of Present Studies. ? Currently our objectives are three-fold. The first objective is to explore the role of the COMP protein in chondrocyte survival. The second objective is to define the role of histone deacetylateses in chondrocyte gene epxression. The third objective is to identify the mechanism by which specific histone deacetylase enzymes regulate chondrocyte survival. ? c) Results During the Past Year. ? The results of the past year are grouped according to the Objectives outlined above. ? (1) Analysis of the structure and function of the COMP Gene. Cartilage oligomeric matrix protein (COMP) is a specific component of cartilage, tendon and ligament. Mutations within the COMP gene lead to skeletal malformations and early onset arthritis. While COMP appears to play a crucial role in cartilage biology its exact role is unknown. We have analyzed the role of COMP in regulating cell survival and gene expression. We have utilized human chondrocytes in these studies. We find that COMP protects these cells against death, either in the presence or absence of TNF alpha (Tumor Necrosis Factor alpha) and is able to block activation of caspase 3, a critical effector caspase. This effect appears to be mediated by the IAP (Inhibitor of Apoptosis Protein) family of anti-apoptotic proteins since the levels of XIAP, Survivin, cIAP1 and cIAP2 are significantly elevated in the COMP expressing cells and downregulation of Survivin and XIAP protein levels by siRNAs blocks the ability of COMP to enhance survival. The mRNAs for most of the IAP family members were not affected by COMP, indicating that a translational/post-translational mechanism was involved in their induction. However in both HeLa cells and chondrocytes, COMP induced the Survivin mRNA level by 5-fold. Thus Survivin is the first gene identified to be upregulated transcriptionally by COMP. The carboxy terminal half of the protein comprising the Type 3 repeats and the RDG sequence (CaCTD domain) was sufficient to promote survival and to elevate the levels of the IAPs. Further, an RGD peptide was able to block the prosurvival effect of COMP and the induction of XIAP and Survivin, indicating that survival is likely mediated through integrin signaling. These data point to a new role for COMP in protecting cells against death and should help to shed light on the contribution of this important ECM molecule to cartilage biology and osteoarthritis. ? (2) Analysis of the role of chromatin modifying enzymes in cartilage specific gene expression. We have been able to identify the most abundant histone deacetylase enzymes (HDACs) that are expressed in primary human chondrocytes. We find that these enzymes are potent regulators of chondrocyte specific gene expression such that when select HDACs are overexpressed in human chondrocytes they can act as potent enhancers or inhibitors of cartilage gene expression. For example, SirT1 is a class III histone deacetylase that can extend organism life span during caloric restriction. However, little is know of the role it plays in human chondrocyte biology. We find that the SirT1 plays a very positive role in the regulation of both cartilage expression and chondrocyte survival. Further, we find that SirT1 protein levels are downregulated in the chondrocytes derived from osteoarthritic patients, compared to normal donors. Thus SirT1 may be an important new marker for OA. In addition, we find that other HDACs, namely HDAC1 and HDAC2, act in an opposite way to SirT1 in that they are potent repressors of cartilage gene expression. Further we find that HDAC1 and HDAC2 are elevated in OA cartilage. Thus a preliminary conclusion from our studies is that a misregulation of HDAC enzymes may contribute to the onset of OA.? (3) Analysis of the role of Sirt1 in chondrocyte survival. We find that SirT1 is a potent regulator of chondrocyte survival. When SirT1 is overexpressed in human chondrocytes it leads to activation of the IGF/Akt pathway, leading to a block to apoptosis. SirT1 accomplishes this function in part by induction of IGFBP5. ? (d) Conclusions and Significance With Future Directions.? In the last two to three decades, much progress has been made in the discovery of the causes and treatments of the various forms of arthritis. However, the initiating events for these diseases are not well understood and many of the treatment regimens are suboptimal at best. Since arthritis is a very debilitating disease that affects a large proportion of the population, much effort should be made at uncovering the root cause of the disease and identifying new treatments. I believe we have made excellent progress in defining new areas of research in cartilage biology and potential treatments of arthritic diseases. We have explored the role of COMP, a prominent cartilage protein, in the role of cell death and survival and are attempting to determine its role in the long-term progression of arthritis. Further, we have explored the role of HDACs and their corresponding inhibitors in chondrocyte gene expression and as a potential treatment of OA. The role of HDACs in cartilage biology is a completely untouched area that holds much promise for a better understanding of the regulatory mechanisms underlying chondrocyte gene expression and in the treatment of cartilage diseases through the use of specific HDAC inhibitors.
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