Effects of Matrix Metalloproteinase-13 on Extracellular Matrix Molecule Synthesis and Small GTPase Activation in Articular Chondrocytes
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The degerative joint disease, osteoarthritis (OA), is a leading cause of disability in both humans and animals. OA involves the degradation of articular cartilage, which functions to dissipate tensile forces and cushion the joint1. Cartilage degeneration in OA is the result of multiple events including enzymatic degradation of matrix components and malfunction of the intracellular signaling pathways in chondrocytes 2. Traditionally, the catabolic cytokine interlukin-1, also called catabolin, has been used to induce an OA phenotype for in vitro experiments3, 4. Recent studies, however, indicate that matrix metalloproteinase-13 (MMP-13), also termed collagenase-3, is chiefly responsible for cartilage deterioration2, 4, 5. Though MMP-13 has been shown to play an integral role in OA, most studies to date have focused on proteoglycan cleavage mechanisms and the characterization of MMP-13 cell-surface binding and internalization 2, 3, 6-9. Thus it is the broad objective of this Honors Research Project to determine the effects of equine MMP-13 on the matrix molecule expression of chondrocytes and on the activation states of the small G-proteins of the Rho family (Cdc42, Rac, RhoA), and compare the results to those achieved after treatment with IL-1. To accomplish these aims, equine recombinant MMP-13 (eqrMMP-13) was cloned, inserted into a protein expression vector, and expressed as the active enzyme. To evaluate the expression of extracellular matrix molecules, chondrocytes were plated in monolayer, treated with eqrMMP-13, and total RNA was extracted and evaluated using quantitative RT-PCR. Assessment of the effect of MMP-13 GTPase activation was accomplished by treating chondrocytes in monolayer, retaining active GTP-bound GTPases from cell lysates using a GST fusion protein containing the binding domain of a down-stream target, in pull-down assays, and analyzing the retained proteins by polyacrylamide gel electrophoresis (PAGE) and western analysis, in addition to confocal microscopy. This study showed that MMP-13 induced similar changes in matrix molecule expression as IL-1 and significantly upregulated gene expression of the catabolic factor MMP-3, while down regulating gene expression of the anabolic factor collagen II B (Col2A1). Preliminary data from the pull-down assays indicate that IL-1 and MMP-13 decrease the activation status of Rac, and increase the activation status of RhoA. Confocal microscopy images support these findings. Together the results of this study suggest utilizing MMP-13 to induce the OA phenotype is an acceptable model of cartilage degradation in vitro. This may better mimic the native articular environment, and lead to more clinically translatable results than studies using IL-1 chondrocyte cultures.
dissertation or thesis