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

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

Fig. 1. Phosphorylation of PYK2 in chondrocytes treated with fibronectin fragments or an α₅β₁ integrin antibody

Human articular chondrocyte cultures were made serum free and then stimulated with 1 μm of the 110-kDa FN-f (A) or 25 μg/ml of an activating antibody to the α₅β₁ integrin (B). Cell lysates were prepared at the indicated time points and used for immunoblotting with antibodies to the autophosphorylation sites of PYK (Tyr-402, p-PYK2) and FAK (Tyr-397, p-FAK). C, mean band intensity from immunoblots of cells treated with FN-f was calculated as % of time 0 controls for p-PYK2 (filled bars) and p-FAK (open bars).

Fig. 2. PKC inhibition blocks fibronectin fragment stimulation of PYK2 phosphorylation and MMP-13 expression

A, human articular chondrocytes were treated for 30 min with control media or media containing 1 μm FN-f, 10 ng/ml IL-β, 50 nm PMA, or FN-f after pretreatment with 4 μm rottlerin or 10 μm BIM. Cell lysates were immunoblotted with antibodies to phosphorylated PYK2 (p-PYK2). B, conditioned media were collected from human articular chondrocytes treated for 24 h with FN-f with or without the indicated concentrations of BIM or rottlerin. Media samples were concentrated and equal amounts used for immunoblotting with antibodies to MMP-13. C and D, mean band intensity results for the experiments shown in A and B, respectively, expressed as % of unstimulated controls.

Fig. 3. Chondrocyte PKC activation in response to fibronectin fragments

Human articular chondrocytes were stimulated for the indicated periods with 1 μm FN-f. Cytosol and membrane fractions were prepared, and samples equalized for total protein were immunoblotted with antibodies to the specific PKC isoforms.

Fig. 4. Inhibition of intracellular calcium signaling blocks fibronectin fragment stimulation of PYK2 phosphorylation and MMP-13 expression

Human articular chondrocytes were pretreated for 30 min with control media or either 10 μm BAPTA-AM or 20 μm nifedipine and then stimulated with 1 μm FN-f. After 30 min, cell lysates were prepared and used for immunoblotting with antibodies to phosphorylated PYK2 (p-PYK2). Parallel cultures were allowed to incubate with FN-f and inhibitors for 24 h, and conditioned media were collected. Samples of equal volume were immunoblotted with antibodies to MMP-13.

Fig. 5. Tyrosine kinase inhibition with tyrphostin A9 (AG-17) blocks fibronectin fragment stimulation of MMP-13 synthesis

Human articular chondrocytes were pretreated for 30 min with the increasing concentrations of tyrphostin A9 (AG-17) (A) or tyrphostin A25 (AG-82) (B) and then treated overnight with 1 μm FN-f or control media without FN-f. Conditioned media were used for immunoblotting with antibodies to MMP-13.

Fig. 6. Inhibition of fibronectin fragment induced JNK phosphorylation by tyrphostin A9 (AG-17) and rottlerin

Human articular chondrocytes were pretreated for 30 min with the indicated inhibitors: 4 μm rottlerin, 10 μm tyrphostin A9 (AG-17), or 10 μm tyrphostin A25 (AG-82). The cells were then treated with 1 μm FN-f or control media without FN-f or inhibitors for 30 min at which time cell lysates were prepared. Chondrocyte lysates were immunoblotted with phosphospecific (phospho-) and non-phosphospecific (total) antibodies to JNK, p38, and ERK.

Fig. 7. Adenoviral overexpression of PYK2 stimulates chondrocyte MMP-13 production

Primary chondrocytes were treated with adenovirus containing a GFP expression construct (GFP) as control or GFP linked to wild type PYK2 (wtPYK2), a kinase dead PYK2 (kd-PYK2), or Y402F-PYK2. A, cell lysates were immunoblotted with phosphospecific and non-phosphospecific (total) antibodies to PYK2. A sample of lysate from an untreated control culture was included (cntl). B, media samples from duplicate wells of adenoviral treated cells were analyzed for MMP-13 by immunoblotting. The positive control (+cntl) was from cells treated with FN-f without adenoviral treatment, whereas none of the adenoviral treated cultures had received FN-f.

Fig. 8. MAP kinase inhibition blocks fibronectin fragment stimulation of MMP-13 and MMP-1 promoter activity

A, immortalized human chondrocytes (C-28I2 cells) were transiently transfected with −1600, −736, −370, −186 MMP-13, and −562 MMP-1 promoter-luciferase reporter constructs or the promoterless luciferase vector (basic) as control. After 24 h in complete media, the cells were changed to serum-free conditions for 20–24 h and then treated with 1 μm FN-f with or without MAP kinase inhibitors (30 μm PD98059, 20 μm SP600125, or 10 μm SB203580). After 24 h of incubation, cell lysates were prepared and used for luciferase assays. B, C-28I2 cells were co-transfected with the MMP promoter-reporter constructs and dominant negative (DN) mutant constructs for ERK-1, ERK-2, JNK, or p38 as indicated. Cells were incubated for 24 h in complete media, made serum-free for 24 h, and then treated with 1 μm FN-f for 24 h followed by analysis of luciferase activity.

Fig. 9. Inhibition of PYK2 blocks fibronectin fragment stimulation of MMP-13 and MMP-1 promoter activity

Immortalized human chondrocytes were co-transfected with the MMP promoter-reporter constructs described in FIG. 8 and constructs for wild type PYK2, dominant negative PYK2 mutants (PYK2–402 and -457), dominant negative FAK (FRNK), or an empty control vector. Tyrosine kinase inhibition by 10 μm tyrphostin A9 (AG-17) was also tested. Following 24 h of incubation in complete media and 24 h incubation in serum-free media, the cultures were treated with 1 μm FN-f as indicated. After 24 h of treatment, cell lysates were prepared and analyzed by luciferase assay.

Fig. 10. Inhibition of PYK2 blocks PMA-stimulated MMP-13 and MMP-1 promoter activity and PKC inhibition blocks fibronectin fragment stimulated activity

A, immortalized human chondrocytes were transiently transfected with MMP-13 and MMP-1 promoter-reporter constructs and in some cultures co-transfected with the PYK2 and FAK constructs as described in Fig. 9. The cultures were stimulated with 50 nM PMA with or without 5 μm BIM or 10 μm tyrphostin A9 (AG-17). After 24 h, cell lysates were prepared and used to measure luciferase activity. B, chondrocytes transiently transfected with the promoter-reporter constructs were treated with the FN-f in the absence or presence of 5 μm BIM.