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Osteoarthritic-like cartilage damage by fibronectin fragments occurs through elevated intracellular kinase activity as triggered by changes in distribution of receptor, actin, vinculin and c-src kinase.

Fibronectin (Fn) fragments (Fn-f), which are elevated in cartilage and synovial fluids in osteoarthritis, can greatly augment cartilage destruction through upregulation of catabolic cytokines and matrix metalloproteinases (MMPs). The precursor, native Fn, is inactive. How Fn-f cause changes in signal transduction that lead to enhanced chondrolytic activities is unknown. Since receptor-binding Fn peptides decrease focal contacts, disturb cytoskeletal elements such as actin and decrease Fn receptor aggregation in fibroblasts, we have proposed that Fn-f have similar effects in chondrocytes and that these changes in receptor and cytoskeletal elements are the cause of altered signal transduction that eventually leads to upregulation of cytokines and MMPs. Our first objective was to investigate whether Fn-f alter actin and focal contacts and disrupt the Fn receptor, [alpha]5[beta]1, in chondrocytes and to compare with native Fn. Our second objective was to test whether specific kinases, which are involved in integrin signaling, cytokine upregulation and MMPs expression, are upregulated by Fn-f. To investigate, fluorescent confocal microscopy was used to visualize changes caused by added Fn-for Fn. Bovine chondrocyte monolayer cultures were treated with unlabeled or with FITC-labeled Fn or Fn-fs for 4 hrs or with BSA as a negative control. Cells were then fixed by paraformaldehyde, permeabilized and probed with antibody against [alpha]5 integrin subunit to test for changes in Fn receptor or with rhodamine phalloidin to test for changes in actin or with antibody to vinculin to test for changes in focal contacts. Changes in intracellular kinase activity were examined by Western Blotting by use of antibodies specific to total and phosphorylated kinase forms. The targets included c-src which can activate focal adhesion kinase (FAK), PYK2, a soluble form of FAK, and the MAP kinases ERK 1/2, p38 and SAPK/JNK, which can upregulate cytokines that upregulate MMPs. The transcription factor, NF-kB, involved in cytokine upregulation was also investigated. The effects of Fn and Fn-fs on kinetics and effects as a function of concentration were examined. The effects of several kinase inhibitors on upregulation of MMPs were also tested. We found that Fn-f disrupted cortical actin, caused diffusion and internalization of the Fn receptor and appeared to alter distribution of the focal contact protein, vinculin. There are dramatic effects of increasing concentrations of Fn-f(0-200 nM) on actin and on c-src (p-src is phosphorylated form) kinase. The Fn-f enhanced diffusion of cortical actin and that this movement was associated with movement of p-src away from the plasma membrane. Images obtained using confocal microscopy emphasize this association. The Fn-f also activated c-src, PYK2, ERK1/2, p38 and SAPK/JNK and NF-kB while native Fn only enhanced ERK 1/2 and FAK. For c-src, Fn appeared to enhance phosphorylation at an inactivation site of c-src, while the 29-kDa Fn-f did not but had a weak effect on enhancing phosphorylation at the activation site. This is consistent with our tests of the c-src inhibitor, PP2, which blocked Fn-f mediated upregulation of MMP-13. Our data suggest that Fn-f disrupt Fn receptor and enhance diffusion of cortical actin, the latter of which is associated with movement of c-src away from the plasma membrane where it can be inactivated. Active c-src might then activate PYK2, which may lead to activation of the inflammatory kinases (p38 and SAPK/JNK) and transcription factor NF-eB, and eventually cause cartilage damage. In contrast, we propose that exogenous Fn inactivates c-src and thus, cannot activate p38 and SAPK/JNK. Since the Fn-f system is a model of osteoarthritis, further knowledge of the mechanism may be applicable to therapeutic intervention in catabolic processes. Future work will more precisely delineate the mechanism and test additional kinase inhibitors for their interventive potential. Supported by ND EPSCoR and National Science Foundation Grant (#01322899).

Ding L., Guo D.P., Singh B., and Homandberg G.A.

Department of Biochemistry and Molecular Biology, University of North Dakota, Grand Forks, ND
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Title Annotation:A. Rodger Denison Student Research Competition: COMMUNICATIONS: GRADUATE DIVISION
Author:Ding, L.; Guo, D.P.; Singh, B.; Homandberg, G.A.
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2004
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