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     

Focal adhesion kinase and mitogen-activated protein kinases are involved in chondrocyte activation by the 29-kDa amino-terminal fibronectin fragment.

The 29-kDa amino-terminal fibronectin fragment (FN-f) has a potent chondrolytic effect and is thought to be involved in cartilage degradation in arthritis. However, little is known about signal transduction pathways that are activated by FN-f. Here we demonstrated that FN-f induced nitric oxide (NO) production from human articular chondrocytes. Expression of inducible nitric-oxide synthase (iNOS) mRNA and NO production were observed at 6 and 48 h after FN-f treatment, respectively. Interleukin-1beta (IL-1beta) mRNA up-regulation was stimulated by FN-f in human chondrocytes. To address the possibility that FN-f-induced NO release is mediated by IL-1beta production, the effect of IL-1 receptor antagonist (IL-1ra) was determined. IL-1ra partially inhibited FN-f-induced NO release although it almost completely inhibited IL-1beta-induced NO release. Tyrosine phosphorylation of focal adhesion kinase was induced transiently by FN-f treatment. Blocking antibodies to alpha(5) or beta(1) integrin and Arg-Gly-Asp-containing peptides did not inhibit FN-f-induced NO production. PP2, a Src family kinase inhibitor, or cytochalasin D, which selectively disrupts the network of actin filaments, inhibited both FAK phosphorylation and NO production induced by FN-f, but the phosphatidylinositol 3-kinase inhibitor wortmannin had no effect. Analysis of mitogen-activated protein kinases (MAPK) showed activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase, and p38 MAPK. High concentrations of SB203580, which inhibit both JNK and p38 MAPK, and PD98059 a selective inhibitor of MEK1/2 that blocks ERK activation, inhibited FN-f induced NO production. These data suggest that focal adhesion kinase and MAPK mediate FN-f induced activation of human articular chondrocytes.     

MnTBAP, a synthetic metalloporphyrin, inhibits production of tumor necrosis factor-alpha in lipopolysaccharide-stimulated RAW 264.7 macrophages cells via inhibiting oxidative stress-mediating p38 and SAPK/JNK signaling

Antioxidants are able to inhibit inflammatory gene expression in response to lipopolysaccharide via down-regulating generation of intracellular reactive oxygen species (ROS) as second messengers. The effect of manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a synthetic metalloporphyrin with antioxidant activity, on tumor necrosis factor (TNF)-alpha production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells was examined. MnTBAP prevented the generation of intracellular ROS in lipopolysaccharide-stimulated RAW 264.7 cells and further inhibited lipopolysaccharide-induced TNF-alpha production. MnTBAP exclusively prevented the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK/JNK) whereas it did not affect the phosphorylation and activation of nuclear factor-kappaB and extracellular signal regulated kinase 1/2. MnTBAP was suggested to inhibit lipopolysaccharide-induced TNF-alpha production by the prevention of intracellular ROS generation and subsequent inactivation of p38 MAPK and SAPK/JNK.     

Endothelin-1 in osteoarthritic chondrocytes triggers nitric oxide production and upregulates collagenase production

The mechanism of endothelin-1 (ET-1)-induced nitric oxide (NO) production, MMP-1 production and MMP-13 production was investigated in human osteoarthritis chondrocytes. The cells were isolated from human articular cartilage obtained at surgery and were cultured in the absence or presence of ET-1 with or without inhibitors of protein kinase or LY83583 (an inhibitor of soluble guanylate cyclase and of cGMP). MMP-1, MMP-13 and NO levels were then measured by ELISA and Griess reaction, respectively. Additionally, inducible nitric oxide synthase (iNOS) and phosphorylated forms of p38 mitogen-activated protein kinase, p44/42, stress-activated protein kinase/Jun-N-terminal kinase and serine-threonine Akt kinase were determined by western blot. Results show that ET-1 greatly increased MMP-1 and MMP-13 production, iNOS expression and NO release. LY83583 decreased the production of both metalloproteases below basal levels, whereas the inhibitor of p38 kinase, SB202190, suppressed ET-1-stimulated production only. Similarly, the ET-1-induced NO production was partially suppressed by the p38 kinase inhibitor and was completely suppressed by the protein kinase A kinase inhibitor KT5720 and by LY83583, suggesting the involvement of these enzymes in relevant ET-1 signalling pathways. In human osteoarthritis chondrocytes, ET-1 controls the production of MMP-1 and MMP-13. ET-1 also induces NO release via iNOS induction. ET-1 and NO should thus become important target molecules for future therapies aimed at stopping cartilage destruction.     

Diphenyleneiodonium inhibits NF-kappaB activation and iNOS expression induced by IL-1beta: involvement of reactive oxygen species

AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS.     

Desferoxamine and ethyl-3,4-dihydroxybenzoate protect myocardium by activating NOS and generating mitochondrial ROS

Protection from a prolyl hydroxylase domain-containing enzyme (PHD) inhibitor, desferoxamine (DFO), was recently reported to be dependent on production of reactive oxygen species (ROS). Ischemic preconditioning triggers the protected state by stimulating nitric oxide (NO) production to open mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels, generating ROS required for protection. We tested whether DFO and a second PHD inhibitor, ethyl-3,4-dihydroxybenzoate (EDHB), might have similar mechanisms. EDHB and DFO increased ROS generation by 50-75% (P < 0.001) in isolated rabbit cardiomyocytes. This increase after EDHB exposure was blocked by N(omega)-nitro-L-arginine methyl ester (L-NAME), an NO synthase (NOS) inhibitor; ODQ, a guanylyl cyclase antagonist; and Rp-8-bromoguanosine-3',5'-cyclic monophosphorothioate Rp isomer, a PKG blocker, thus implicating the NO pathway in EDHB's signaling. Glibenclamide, a nonselective K(ATP) channel blocker, or 5-hydroxydecanoate, a selective mitoK(ATP) channel antagonist, also prevented EDHB's ROS production, as did blockade of mitochondrial electron transport with myxothiazol. NOS is activated by Akt. However, neither wortmannin, an inhibitor of phosphatidylinositol-3-kinase, nor Akt inhibitor blocked EDHB-induced ROS generation, indicating that EDHB initiates signaling downstream of Akt. DFO also increased ROS production, and this effect was blocked by ODQ, 5-hydroxydecanoate, and N-(2-mercaptopropionyl)glycine, an ROS scavenger. DFO increased cardiomyocyte production of nitrite, a metabolite of NO, and this effect was blocked by an inhibitor of NOS. DFO also spared ischemic myocardium in intact hearts. This infarct-sparing effect was blocked by ODQ, L-NAME, and N-(2-mercaptopropionyl)glycine. Hence, DFO and EDHB stimulate NO-dependent activation of PKG to open mitoK(ATP) channels and produce ROS, which act as second messengers to trigger entrance into the preconditioned state.     

4-Hydroxynonenal enhances MMP-2 production in vascular smooth muscle cells via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways

4-Hydroxynonenal (HNE) accumulates at atherosclerotic lesions, but its role in the progression of atherosclerosis is not clear. Considering the role of matrix metalloproteinases (MMP) in plaque destabilization, we investigated the mechanism by which HNE induces MMP production in vascular smooth muscle cells (VSMC). VSMC stimulated by HNE (1.0 microM) produced enzymatically active MMP-2 with an increased promoter activity, which was abolished by mutation of the NF-kappaB binding site in the promoter region. The increased NF-kappaB activity with subsequent MMP-2 production by HNE was significantly attenuated by transfection with Akt siRNA as well as by pretreatment with the PI3K/Akt inhibitors LY294002 (10 microM) and SH-5 (1.0 microM). The phosphorylation of Akt occurred as early as 5 min in VSMC exposed to HNE and was markedly attenuated by inhibition of mitochondrial reactive oxygen species (ROS). Furthermore, the impact of mitochondrial ROS on HNE-induced Akt phosphorylation with subsequent MMP-2 production was also demonstrated in mitochondrial function-deficient VSMC, as well as in cells transfected with manganese superoxide dismutase. Taken together, these results suggest that HNE enhances MMP-2 production in VSMC via mitochondrial ROS-mediated activation of the Akt/NF-kappaB signaling pathways.     

Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

The shear-induced intracellular signal transduction pathway invascular endothelial cells involves tyrosine phosphorylation andactivation of mitogen-activated protein (MAP) kinase, which may beresponsible for the sustained release of nitric oxide. MAP kinase isknown to be activated by reactive oxygen species (ROS), such asH₂O₂,in several cell types. ROS production in ligand-stimulatednonphagocytic cells appears to require the participation of aRas-related small GTP-binding protein, Rac1. We hypothesized that Rac1might serve as a mediator for the effect of shear stress on MAP kinaseactivation. Exposure of bovine aortic endothelial cells to laminarshear stress of 20 dyn/cm² for5-30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidantsN-acetylcysteine and pyrrolidinedithiocarbamate inhibited in a dose-dependent manner theshear-stimulated increase in total cytosolic and, specifically, MAPkinase tyrosine phosphorylation. Hence, the onset of shear stresscaused an enhanced generation of intracellular ROS, as evidenced by anoxidized protein detection kit, which were required for theshear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing adominant negative Rac1 gene product (N17rac1). We concluded that theGTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAPkinase via regulation of the flow-dependent redox changes inendothelial cells in physiological and pathological circumstances.     

Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes

Hyaluronan exerts a variety of biological effects on cells including changes in cell migration, proliferation, and matrix metabolism. However, the signaling pathways associated with the action of hyaluronan on cells have not been clearly defined. In some cells, signaling is induced by the loss of cell-hyaluronan interactions. The goal of this study was to use hyaluronan oligosaccharides as a molecular tool to explore the effects of changes in cell-hyaluronan interactions and determine the underlying molecular events that become activated. In this study, hyaluronan oligosaccharides induced the loss of extracellular matrix proteoglycan and collagen from cultured slices of normal adult human articular cartilage. This loss was coincident with an increased expression of matrix metalloproteinase (MMP)-13. MMP-13 expression was also induced in articular chondrocytes by hyaluronan (HA) hexasaccharides but not by HA tetrasaccharides nor high molecular weight hyaluronan. MMP-13 promoter-reporter constructs in CD44-null COS-7 cells revealed that both CD44-dependent and CD44-independent events mediate the induction of MMP-13 by hyaluronan oligosaccharides. Electromobility gel shift assays demonstrated the activation of chondrocyte NFkappaB by hyaluronan oligosaccharides. NFkappaB activation was also documented in C-28/I2 immortalized human chondrocytes by luciferase promoter assays and phosphorylation of IKK-alpha/beta. The link between activation of NFkappaB and MMP-13 induction by HA oligosaccharides was further confirmed through the use of the NFkappaB inhibitor helenalin. Inhibition of MAP kinases also demonstrated the involvement of p38 MAP kinase in the hyaluronan oligosaccharide induction of MMP-13. Our findings suggest that hyaluronan-CD44 interactions affect matrix metabolism via activation of NFkappaB and p38 MAP kinase.     

NF-kappa B mediates the stimulation of cytokine and chemokine expression by human articular chondrocytes in response to fibronectin fragments

Fibronectin fragments (FN-f) that bind to the alpha(5)beta(1) integrin stimulate chondrocyte-mediated cartilage destruction and could play an important role in the progression of arthritis. The objective of this study was to identify potential cytokine mediators of cartilage inflammation and destruction induced by FN-f and to investigate the mechanism of their stimulation. Human articular chondrocytes, isolated from normal ankle cartilage obtained from tissue donors, were treated with a 110-kDa FN-f in serum-free culture, and expression of various cytokine genes was analyzed by cDNA microarray and by a cytokine protein array. Compared with untreated control cultures, stimulation by FN-f resulted in a >2-fold increase in IL-6, IL-8, MCP-1, and growth-related oncogene beta (GRO-beta). Constitutive and FN-f-inducible expression of GRO-alpha and GRO-gamma were also noted by RT-PCR and confirmed by immunoblotting. Previous reports of IL-1beta expression induced by FN-f were also confirmed, while TNF expression was found to be very low. Inhibitor studies revealed that FN-f-induced stimulation of chondrocyte chemokine expression was dependent on NF-kappaB activity, but independent of IL-1 autocrine signaling. The ability of FN-f to stimulate chondrocyte expression of multiple proinflammatory cytokines and chemokines suggests that damage to the cartilage matrix is capable of inducing a proinflammatory state responsible for further progressive matrix destruction, which also includes the chemoattraction of inflammatory cells. Targeting the signaling pathways activated by FN-f may be an effective means of inhibiting production of multiple mediators of cartilage destruction.     

Role of mitogen-activated protein kinases and tyrosine kinases on IL-1-Induced NF-kappaB activation and iNOS expression in bovine articular chondrocytes.

Nitric oxide (NO), produced by the inducible isoform of the NO synthase (iNOS), plays an important role in the pathophysiology of arthritic diseases. This work aimed at elucidating the role of the mitogen-activated protein kinases (MAPK), p38MAPK and p42/44MAPK, and of protein tyrosine kinases (PTK) on interleukin-1beta (IL-1)-induced iNOS expression in bovine articular chondrocytes. The specific inhibitor of the p38MAPK, SB 203580, effectively inhibited IL-1-induced iNOS mRNA and protein synthesis, as well as NO production, while the specific inhibitor of the p42/44MAPK, PD 98059, had no effect. These responses to IL-1 were also inhibited by treatment of the cells with the tyrosine kinase inhibitors, genistein and tyrphostin B42, which also prevented IL-1-induced NF-kappaB activation. The p38MAPK inhibitor, SB 203580, had no effect on IL-1-induced NF-kappaB activation. Finally, the p42/44MAPK inhibitor, PD 98059, prevented IL-1-induced AP-1 activation in a concentration that did not inhibit iNOS expression. In conclusion, this study shows that (1) PTK are part of the signaling pathway that leads to IL-1-induced NF-kappaB activation and iNOS expression; (2) the p38MAPK cascade is required for IL-1-induced iNOS expression; (3) the p42/44MAPK and AP-1 are not involved in IL-1-induced iNOS expression; and (4) NF-kappaB and the p38MAPK lie on two distinct pathways that seem to be independently required for IL-1-induced iNOS expression. Hence, inhibition of any of these two signaling cascades is sufficient to prevent iNOS expression and the subsequent production of NO in articular chondrocytes.