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 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 antiapoptotic 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 define the role of cartilage oligomeric matrix protein (COMP) in chondrocyte survival. The second objective is to identify the mechanism by which specific histone deacetylase enzymes regulate chondrocyte specific gene expression. Our third objective is to use genetic screens to identify genes whose expression enhances 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 have ectopically expressed COMP (wild-type and mutants) via plasmid and retroviral expression vectors and find that COMP enhances chondrocyte survival in vitro in human chondrocytes. By expressing specific domains of COMP in these human chondrocytes we will be able to isolate and identify regions of COMP that specify its differentiation-enhancing functions. We have also assessed the role of COMP in the regulation of gene expression and have found that COMP is able to upregulate the IAP family of antiapoptotic proteins. Future analysis will be directed at identifying the cellular receptors for COMP and determining its mode of action in terms of cell survival and gene expression. In addition, we will generate COMP transgenic mice where wild-type and mutant COMP proteins will be targeted to cartilage. In this way we can determine the extent of any developmental defects in these mice due to an effect on cartilage. These approaches should help to shed light on the contribution of this important ECM molecule to cartilage biology and osteoarthritis.? (2) We have been able to identify the prominent histone deacetylase enzymes (HDAC) in primary human chondrocytes. We find that these enzymes are potent regulators of chondrocyte specific gene expression such that when select HDAC enzymes are expressed in human chondrocytes they cause the cells to undergo dedfferentiation. Further, when chondrocytes are treated with HDAC inhibitors we find that this treatment dramatically upregulates chondrocyte gene expression. These data indicate that certain HDACs are capable of controlling chondrogenesis and maintenance of the chondrocyte phenotype. Future analysis will be directed at identifying the cellular targets for these HDACs in terms of differentiation. In addition, we will generate HDAC transgenic mice where specific HDAC proteins will be targeted to cartilage. In this way we can determine the extent of any developmental defects in these mice due to an effect of HDACs on chondrogenesis.? (3) It is well recognized that there are multiple causes for the initiation of OA, some of which involve the effect of cytotoxic factors directly on cartilage. We have therefore initiated functional screens in an attempt to identify genes that play critical roles in chondrocyte survival. To perform such a screen we have used human cDNA libraries cloned into a retroviral expression vector, which were then used to infect chondrosarcoma cells. In our first screens we have begun the process of selection for specific characteristics, such as for survival of cells treated with agents known to be toxic to cartilage. For example, we have assayed for proliferation in media containing gadolinium (Gd) an inhibitor of the mechanotransduction receptor and a toxin for chondrocytes. Through this approach we have identified novel genes whose products aid in cell survival. The results of these initial screens are very promising and have helped us identify a number of genes that one would not normally expect to be part of this cell survival process. In the future we will use the information derived from these screens along with retroviral gene transfer approaches to express these potential chondro-protective genes in cartilage. We will then determine the outcome of this expression on the survivability of chondrocytes in vitro and cartilage in vivo.? 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. Finally, our use of a genetic screen to identify genes that aid in chondrocyte survival may lead to the discovery of new factors that are involved in many aspects of cartilage biology.
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