Rheumatoid arthritis (RA) is a chronic inflammatory disease that often produces severe destruction of articular cartilage and bone. Considerable evidence indicates that bone erosions in RA are produced by osteoclasts (OCs). An essential factor for the differentiation and activation of osteoclasts is receptor-activator of NF-KB ligand (RANKL), which mediates its effects via a specific cell surface receptor, receptor-activator of NF-kB (RANK). Recently, a novel secreted form of RANKL (secRANKL) has been identified which appears to be regulated by a unique promoter. The studies outlined in this proposal are designed to test the hypothesis that that there is enhanced local expression of RANKL at sites of bone erosion in RA and that regulation of RANKL expression in cells at sites of bone erosion is a critical determinant of focal bone loss.
Specific Aim 1 will test the hypothesis that RANKL production by cells derived from RA synovium is critical in the pathogenesis of osteoclastic bone erosion.
This Aim will address the following questions: What are the exact cellular sources of RANKL at sites of bone erosion in RA? Where is RANKL expressed in relation to OPG expression and to RANK positive OC precursors? Retrieved human tissues will be utilized initially to answer these questions. Cellular expression profiles will be confirmed and the temporal expression of these factors will be determined in two murine models of inflammatory arthritis: collagen-induced arthritis (CIA), and the KJBxN model. Dispersed cells from RA synovium will be used to determine the expression of RANKL isoforms in relevant cell types. Finally, the activity of the secRANKL isoform in osteoclastogenesis will be determined in an in vitro co-culture model of osteoclastogenesis.
Specific Aim 2 will test the hypothesis that T cell-derived RANKL is required for bone erosion in RA. Arthritis will be generated in mice in which T cells provide the only source of RANKL, and in genetically engineered mouse strains lacking T cells, in order to definitively determine the role of T cell-derived RANKL in bone erosion.
Specific Aim 3 will identify regulatory elements responsible for the constitutive and inducible expression of the membrane-bound RANKL isoform in cell types present in RA, and will test the hypothesis that the NFAT family of transcription factors is critical in the inducible regulation of the memRANKL gene. RT-PCR analysis of RANKL expressing cell-types present in focal RA bone erosions demonstrates differential constitutive and inducible expression of the two known RANKL isoforms. Preliminary data also demonstrate a potential role of NFATs in RANKL regulation in T cells.
This Aim will provide data on the regulation of RANKL gene expression in cell types present in bone erosion in RA and may lead to new therapeutic strategies for preventing bone destruction in this disease.
