taken from application): Loss of articular cartilage in rheumatoid arthritis (RA) is associated with the normal migration of synoviocytes and increased secretion of matrix metalloproteinases (MMPs). Growth, adherence, and migration, as well as signal transduction pathways known to induce transcription of MMPs, are mediated by the Rho-type GTPases. The Ras-related GTPases, RhoA, RacI, and Cdc42, interact with the actin cytoskeleton to alter morphology. They can be activated by growth factors (e.g., PDGF), cytokines (e.g., IL-lb and TNF-a and integrins. In addition, activation of GTPases leads to alteration of downstream signal transduction pathways that ultimately induce gene transcription. The general hypothesis of this proposal is that rheumatoid fibroblast-like synoviocytes (FLS) mediate cartilage destruction that is dependent on Rho-type GTPases. Specifically, we hypothesize that activation of the Rho-type GTPases affects the physical interaction between synoviocytes and articular cartilage. In addition, GTPase activation triggers a signal transduction cascade resulting in increased production of MMPs that contribute to loss of cartilage matrix in RA. In this exploratory/developmental proposal, we will use a retro viral vector system to over-express constitutively active and dominant negative forms of the Rho-type GTPases, RhoA, RacI, and Cdc42 with polycistronic expression of enhanced green fluorescent protein. We will standardize methods for producing articular cartilage discs, both vital and devitalized, and refine our methods to assay for loss of extracellular matrix in co-culture. We will develop the methods necessary to assess changes in morphology and migration of untreated, PDGF-stimulated, and IL-lB or TNF-alpha-stimulated FLS during co-culture with articular cartilage using conventional and video microscopy. We will determine the basal, PDGF-stimulated, and IL-lB or TNF-alpha- stimulated activity of the Rho-type GTPases in unmodified rheumatoid FLS. Ultimately, we will determine the effects of the Rho-type GTPases on synoviocyte adherence to and migration over articular cartilage, production of MMPs, and destruction of the cartilage matrix.
| Crofford, Leslie J; McDonagh, Kevin T; Guo, Sutang et al. (2005) Adenovirus binding to cultured synoviocytes triggers signaling through MAPK pathways and induces expression of cyclooxygenase-2. J Gene Med 7:288-96 |
