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A model of osteoarthritic cartilage damage: action of Fibronectin Fragments.

Osteoarthritis (OA) is a degenerative joint disease in which the cartilage that covers the ends of bones in the joint deteriorates, causing pain and loss of movement as bone begins to rub against bone. Previous studies from our laboratory showed that Fibronectin Fragments (Fn-fs), the degradation products of a ubiquitous extracellular matrix protein-Fibronectin (Fn), greatly augment cartilage destruction through upregulation of catabolic cytokines and matrix metalloproteinases (MMPs). This leads to severe cartilage matrix damage as observed in OA. The precursor, native Fn, is inactive. How Fn-fs 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-fs have similar effects in chondrocytes and that these changes in receptor and cytoskeletal elements alter the location of upstream kinases near receptors which leads to altered signal transduction and emergence of inflammatory MAP kinase pathways. Our first objective was to compare Fn-fs to Fn in terms of activation of kinases that are involved in integrin signaling, catabolic cytokine upregulation and MMP expression. The second objective was to investigate if blocking of some of these kinases leads to blocking of MMP expression. The third objective was to investigate whether the most cartilage-damaging N-terminal 29-kDa Fn-f alters actin structure and disrupts the Fn receptor, alphasbetal, as has been observed upon addition of small tetrapeptides to fibroblasts. The fourth objective is to ultimately determine whether these changes in cytoskeletal structure might explain changes in kinase location, which in turn, turn on/off kinase activity.

Chondrocyte monolayer cultures were derived from the cartilage of bovine metacarpophalangeal joints. Changes in intracellular kinase activity were examined by Western Blotting by use of antibodies specific to total and phosphorylated kinase forms. The targets included focal adhesion kinase (FAK), Pyk2 (a soluble form of FAK), c-Src which can phosphorylate FAK, and the MAP kinases ERK1/2, p38 and JNK. The transcription factor, NF-kB, (involved in catabolic 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 downregulation of MMPs were also tested. 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 3% paraformaldehyde, permeabilized and probed with antibody against [alpha.sub.5] integrin subunit to test for changes in Fn receptor or with rhodamine phalloidin to test for changes in actin. FITC- or TRITC labeled phospho-specific antibody against Pyk2, FAK, c-Src or JNK were used to target the location of activated kinases.

Fibronectin fragments activated c-Src, Pyk2, ERK1/2, p38 and JNK and NF-kB while native Fn only enhanced activation of FAK and ERK 1/2. For c-src, Fn appeared to enhance phosphorylation at an inactivation site, while the 29-kDa Fn-f instead, weakly enhanced phosphorylation at an activation site. Inhibitors of PYK2, MEK, JNK and PKC-delta blocked Fn-f mediated upregulation of MMP-3 and MMP- 13. N-terminal 29-kDa Fn-f disrupted Fn receptor ([alpha.sub.5][beta.sub.1]) aggregation and actin structure, the latter of which was associated with movement of c-Src away from the plasma membrane. The movements of activated Pyk2, FAK or JNK are under investigation.

We propose a tentative model of cartilage damage by Fn-fs: after Fn-fs bind to receptor, receptor aggregation is disrupted and actin distribution is altered. This is associated with movement of c-Src or other factors in such a fashion that Pyk2 activity is enhanced (Movement of other upstream kinases or factors is under investigation). This leads to activation of MAP kinases (p38, JNK and ERK1/2) and transcription factor NF--B. This would lead to NF--B translocation into the nucleus, upregulation of catabolic cytokines and MMPs and consequent matrix proteoglycan (PG) degradation. In contrast, binding of native Fn does not alter receptor or actin, does not alter c-Src distribution and Pyk2 is not activated. Thus, the inflammatory MAP kinases-p38 and JNK are not activated and cartilage damage does not occur. Since the Fn-f system is a model of OA and represents a unique dysregulation of integrin signaling that may occur in other pathologies as well, further knowledge of the mechanism may be applicable to therapeutic intervention. Future work will more precisely delineate the mechanism and test additional kinase inhibitors for their interventive potential.

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

Department of Biochemistry and Molecular Biology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, USA.
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Title Annotation:Collegiate Communications--Graduate
Author:Ding, Lei; Guo, D.P.; Singh, B.; Homandberg, G.A.
Publication:Proceedings of the North Dakota Academy of Science
Geographic Code:1U4ND
Date:Apr 1, 2005
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