Abstract
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
Highlights
Matrix metalloproteinases (MMPs)1 are expressed by a number of different cell types and play a key role in diverse processes ranging from morphogenesis to tumor invasion to tissue remodeling
The 110-kDa Fibronectin Fragment and Antibodies to the ␣51 Integrin Stimulate proline-rich tyrosine kinase-2 (PYK2) Phosphorylation—In a previous study we found that treatment of chondrocytes with Fibronectin fragments (FN-f) stimulated expression of MMP-13 which was associated with mitogen-activated protein (MAP) kinase activation [12]
Recent studies have indicated that in arthritic cartilage overproduction of collagenases, in particular MMP-13 and MMP-1, by chondrocytes plays a central role in collagen degradation [4, 5, 34]
Summary
Matrix metalloproteinases (MMPs) are expressed by a number of different cell types and play a key role in diverse processes ranging from morphogenesis to tumor invasion to tissue remodeling (reviewed in Ref. 1) Because of their potent ability to degrade the extracellular matrix and the undesirable consequences of excess matrix degradation, the production and activation of MMPs must be tightly regulated. The increase in MMP-13 was accompanied by an increase in expression of IL-1, IL-1 was not required for the initial FN-f stimulation of MMP-13 expression These studies suggest that FN-f stimulation of integrin signaling could play an important role in mediating cartilage matrix degradation through stimulation of MMP and cytokine production. A role for FAK has been demonstrated for MMP secretion in response to concanavalin A [24] and hyaluronan [25], to our knowledge the possibility that activation of either FAK or PYK2 might be required for integrin signaling which regulates MMP expression has not been studied
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
