Fibronectin fragments mediate matrix metalloproteinase upregulation and cartilage damage through proline rich tyrosine kinase 2, c-src, NF-kappaB and protein kinase Cdelta.
OBJECTIVE
Since fibronectin fragments (Fn-fs) enhance cartilage damage through integrins, the objective was to investigate the role of integrin linked kinases, focal adhesion kinase (FAK) and a soluble form of FAK, proline rich tyrosine kinase 2 (Pyk2) and cellular src kinase (c-src) and the transcription factor, nuclear factor kappaB (NF-kappaB) in cartilage damage.


METHODS
Bovine chondrocytes were cultured with various concentrations of three different Fn-fs, an amino-terminal 29 kDa, a gelatin binding 50 kDa and a central 140-kDa Fn-fs, each with progressively weaker cartilage damaging activity, or with native fibronectin (Fn), and lysates probed for activation of the selected kinases. Confocal microscopy was used to visualize intracellular location of activated kinases and NF-kappaB. Various kinase inhibitors were tested for their effects on Fn-f mediated upregulation of matrix metalloproteinase (MMP)-3 and -13 and cartilage proteoglycan (PG) depletion.


RESULTS
The Fn-fs kinetically enhanced phosphorylation of FAK but did not show a clear dose-response effect. The 29-kDa and 50-kDa Fn-fs enhanced phosphorylation of Pyk2, c-src and NF-kappaB to a much greater extent than the 140-kDa Fn-f and native Fn and did so as a function of dose. The 29-kDa Fn-f enhanced the phosphorylation of nuclear Pyk2 as compared with no treatment or native Fn. Inhibitors of Pyk2, c-src, NF-kappaB and protein kinase Cdelta (PKCdelta) decreased MMP upregulation and decreased Fn-f mediated damage to cartilage.


CONCLUSIONS
These studies enhance our knowledge of crucial factors in Fn-f mediated signaling in MMP upregulation and cartilage damage and because of the potential physiologic relevance of Fn-fs, provide a better knowledge of cartilage degeneration in general.
Ding
L
L
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Box 9037, Grand Forks, ND 58202, USA.
Guo
D
D
Homandberg
G A
GA
D000818:Animals; D002356:Cartilage / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism / Q000494:pharmacology*; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism; D015854:Up-Regulation
D005353:Fibronectins; D016328:NF-kappa B; D011509:Proteoglycans; D011494:Protein Kinases; D051418:Focal Adhesion Kinase 2; D051745:Protein Kinase C-delta; D020782:Matrix Metalloproteinases