Fibronectin fragment activation of proline-rich tyrosine kinase PYK2 mediates integrin signals regulating collagenase-3 expression by human chondrocytes through a protein kinase C-dependent pathway.
Fibronectin fragments (FN-f), including the 110-kDa fragment that binds the alpha5beta1 integrin, stimulate collagenase-3 (MMP-13) production and cartilage destruction. In the present study, treatment of chondrocytes with the 110-kDa FN-f or an activating antibody to the alpha5beta1 integrin was found to increase tyrosine autophosphorylation (Tyr-402) of the proline-rich tyrosine kinase-2 (PYK2) without significant change in autophosphorylation (Tyr-397) of focal adhesion kinase (FAK). The tyrosine kinase inhibitor tyrphostin A9, shown previously to block a PYK2-dependent pathway, blocked the FN-f-stimulated increase in MMP-13, whereas tyrphostin A25 did not. FN-f-stimulated PYK2 phosphorylation and MMP-13 production was also blocked by reducing intracellular calcium levels. Adenovirally mediated overexpression of wild type but not mutant PYK2 resulted in increased MMP-13 production. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate stimulated PYK2 phosphorylation and MMP-13 production. MMP-13 expression stimulated by either phorbol 12-myristate 13-acetate or FN-f was blocked by PKC inhibitors including the PKCdelta inhibitor rottlerin. Furthermore, PKCdelta translocation from cytosol to membrane was noted within 5 min of stimulation with FN-f. Immortalized human chondrocytes, transiently transfected with MMP-13 promoter-luciferase reporter constructs, showed increased promoter activity after FN-f treatment that was inhibited by co-transfection with either of two dominant negative mutants of PYK2 (Y402F and K457A). No inhibition was seen after cotransfection with wild type PYK2, a dominant negative of FAK (FRNK) or empty vector plasmid. FN-f-stimulated MMP-13 promoter activity was also inhibited by chemical inhibitors of ERK, JNK, and p38 mitogen-activated protein (MAP) kinases or by co-transfection of dominant negative MAP kinase mutant constructs. These studies have identified a novel pathway for the MAP kinase regulation of MMP-13 production which involves FN-f stimulation of the alpha5beta1 integrin and activation of the nonreceptor tyrosine kinase PYK2 by PKC, most likely PKCdelta
Department of Internal Medicine, Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA. rloeser@rush.edu
Richard F
RF
Loeser
Christopher B
CB
Forsyth
Allen M
AM
Samarel
Hee-Jeong
HJ
Im
D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D017364:Collagenases / Q000096:biosynthesis*; D004789:Enzyme Activation; D005353:Fibronectins / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism; D053509:Matrix Metalloproteinase 13; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
D005353:Fibronectins; D039081:Integrin alpha5beta1; D010446:Peptide Fragments; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D011493:Protein Kinase C; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13