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ABSTRACT: JNK-mediated cell signaling plays a critical role in matrix metalloproteinase (MMP) expression and joint destruction in rheumatoid arthritis (RA). Gadd45beta, which is an NF-kappaB-regulated gene, was recently identified as an endogenous negative regulator of the JNK pathway, since it could block the upstream kinase MKK-7. This study was carried out to evaluate whether low Gadd45beta expression in RA enhances JNK activation and overproduction of MMPs in RA, and whether Gadd45beta deficiency increases arthritis severity in passive K/BxN murine arthritis.
Activation of the NF-kappaB and JNK pathways and Gadd45beta expression were analyzed in human synovium and fibroblast-like synoviocytes (FLS) using quantitative polymerase chain reaction, immunoblotting, immunohistochemistry, electrophoretic mobility shift assay, and luciferase reporter constructs. Gadd45beta(-/-) and wild-type mice were evaluated in the K/BxN serum transfer model of inflammatory arthritis, and clinical signs of arthritis, osteoclast formation, and bone erosion were assessed.
Expression levels of the Gadd45beta gene and protein were unexpectedly low in human RA synovium despite abundant NF-kappaB activity. Forced Gadd45beta expression in human FLS attenuated tumor necrosis factor-induced signaling through the JNK pathway, reduced the activation of activator protein 1, and decreased the expression of MMP genes. Furthermore, Gadd45beta deficiency exacerbated K/BxN serum-induced arthritis in mice, dramatically increased signaling through the JNK pathway, elevated MMP3 and MMP13 gene expression in the mouse joints, and increased the synovial inflammation and number of osteoclasts.
Deficient Gadd45beta expression in RA can contribute to activation of JNK, exacerbate clinical arthritis, and augment joint destruction. This process can be mitigated by enhancing Gadd45beta expression or by inhibiting the activity of JNK or its upstream regulator, MKK-7.
Arthritis & Rheumatism 11/2009; 60(11):3229-40. · 7.87 Impact Factor
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ABSTRACT: c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-beta activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1beta-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1beta induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1beta-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1beta-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA.
Arthritis research & therapy 02/2007; 9(3):R57. · 4.27 Impact Factor
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ABSTRACT: JNK regulates matrix metalloproteinase (MMP) gene expression and joint destruction in rheumatoid arthritis (RA). Previous studies demonstrated that the 2 upstream MAPK kinases (MKK-4 and MKK-7) are phosphorylated in RA synovium and form a complex with JNK in fibroblast-like synoviocytes (FLS). However, the functional hierarchy of MKK-4 and MKK-7 in FLS has not been determined. We determined the relative contributions of these MKKs by evaluating the effect of MKK-4 and MKK-7 gene knockdown in cultured FLS.
FLS were transfected with MKK-4 and/or MKK-7 small interfering RNA, and protein levels were determined by immunoblotting. After stimulation with interleukin-1/beta (IL-1beta), tumor necrosis factor alpha(TNFalpha, or anisomycin, kinase function was determined by in vitro kinase assay. Activator protein 1 (AP-1) binding and transcriptional activity were determined by electrophoretic mobility shift assay and AP-1-luciferase promoter assay, respectively. MMP-3 expression was determined by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction.
IL-1beta-induced JNK phosphorylation was dependent on MKK-7 but not on MKK-4; however, anisomycin-activated JNK required both kinases. In vitro kinase assay demonstrated that IL-1beta-or TNFalpha induced JNK activity was only MKK-7 dependent, while anisomycin-activated JNK was both MKK-4 and MKK-7 dependent. IL-1beta-induced AP-1 binding activity and AP-1-driven gene expression were strictly MKK-7 dependent. Finally, MMP-3 production only required MKK-7, and there was no effect of MKK-4 deficiency.
These data indicate that only MKK-7 is required for JNK activation in FLS after cytokine stimulation; however, other forms of cellular stress utilize MKK-4. Thus, JNK function might be modulated by targeting MKK-7 to suppress cytokine-mediated FLS activation while leaving other stress responses intact.
Arthritis & Rheumatism 08/2006; 54(7):2127-35. · 7.87 Impact Factor
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ABSTRACT: Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-alpha (TNFalpha)-stimulated fibroblast-like synoviocytes (FLS) using the novel Syk inhibitor N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406). Using immunohistochemistry, Syk was detected in RA synovial tissue (ST), primarily in the synovial intimal lining. Western blot analysis demonstrated significantly greater amounts of phospho-Syk expression in RA ST compared with osteoarthritis ST. The kinase was expressed and functionally activated by TNFalpha in FLS and was blocked by R406. Western blot analysis demonstrated that Syk inhibition by R406 markedly suppressed TNFalpha-induced c-Jun N-terminal kinase (JNK) phosphorylation in FLS, with a modest decrease in extracellular signal-regulated kinase phosphorylation. Surprisingly, p38 activation was not affected by R406. The Syk inhibitor also decreased TNFalpha-induced mitogen-activated protein kinase kinase (MKK) 4 phosphorylation but not MKK3 and MKK6 phosphorylation, which is consistent with its selective sparing of p38. The connection between Syk and JNK was confirmed by demonstrating decreased phospho-c-Jun protein expression and complete inhibition of JNK function in R406-treated cells. R406 also suppressed downstream actions of JNK, as determined by activator protein 1 binding, as well as matrix metalloproteinase 3 gene expression. These data demonstrate that Syk activation plays an essential role in TNFalpha-induced cytokine and matrix metalloproteinase production in RA FLS, especially by suppressing activation of the JNK pathway.
Journal of Pharmacology and Experimental Therapeutics 06/2006; 317(2):571-8. · 3.83 Impact Factor
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ABSTRACT: p38 mitogen-activated protein kinase (MAPK) regulates cytokines in arthritis and is, in turn, regulated by MAPK kinase (MKK) 3 and MKK6. To modulate p38 function but potentially minimize toxicity, we evaluated the utility of targeting MKK3 by using MKK3(-/-) mice. These studies showed that TNF-alpha increased phosphorylation of p38 in WT cultured synoviocytes but that p38 activation, IL-1beta, and IL-6 expression were markedly lower in MKK3(-/-) synoviocytes. In contrast, IL-1beta or LPS-stimulated p38 phosphorylation and IL-6 production by MKK3(-/-) synoviocytes were normal. Detailed signaling studies showed that NF-kappaB also contributes to IL-6 production and that TNF-alpha-induced NF-kappaB activation is MKK3-dependent. In contrast, LPS-mediated activation of NF-kappaB does not require MKK3. To determine whether this dichotomy occurs in vivo, two inflammation models were studied. In K/BxN passive arthritis, the severity of arthritis was dramatically lower in MKK3(-/-) mice. Phospho-p38, phospho-MAPK activator protein kinase 2, IL-1beta, CXC ligand 1, IL-6, and matrix metalloproteinase (MMP) 3 levels in the joints of MKK3(-/-) mice were significantly lower than in controls. Exogenous IL-1beta administered during the first 4 days of the passive model restored arthritis to the same severity as in WT mice. In the second model, IL-6 production after systemic LPS administration was similar in WT and MKK3(-/-) mice. Therefore, selective MKK3 deficiency can suppress inflammatory arthritis and cytokine production while Toll-like receptor 4-mediated host defense remains intact.
Proceedings of the National Academy of Sciences 05/2006; 103(14):5484-9. · 9.68 Impact Factor
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ABSTRACT: The p38 MAPK signal transduction pathway is a key regulator of IL-1 and TNF-alpha production in rheumatoid arthritis. Previous studies demonstrated that upstream MAPK kinases (MKK3 and MKK6) that regulate p38 are activated in rheumatoid arthritis synovium. However, their functional relevance in fibroblast-like synoviocytes (FLS) has not been determined. To investigate the relative contribution of MKK3 and MKK6 to p38 activation, the effect of dominant-negative (DN) MKK3 and MKK6 constructs on cultured FLS was evaluated. Cultured FLS were stimulated with medium or IL-1beta, and immunoblotting was performed. In some experiments, cells were lysed and immunoprecipitated with anti-p38 Ab, followed by in vitro kinase assay with [gamma-(32)P]ATP and GST-activating transcription factor-2 as substrate. IL-1beta rapidly induced p38 phosphorylation in cells transfected with empty vector (pcDNA3.1), but was inhibited by 25% in cells expressing DN MKK3 or DN MKK6. Cotransfection with both DN plasmids decreased phospho-p38 by almost 75%. In vitro kinase assays on IL-1-stimulated FLS also showed that the combination of DN MKK3 and DN MKK6 markedly decreased kinase activity compared with empty vector or the individual DN plasmids. Furthermore, IL-1beta-induced IL-8, IL-6, and matrix metalloproteinase-3 protein production was significantly inhibited in DN MKK3/DN MKK6-transfected cells. The constructs had no effect on the respective mediator mRNA levels. These data demonstrate that MKK3 and MKK6 make individual contributions to p38 activation in FLS after cytokine stimulation, but that both must be blocked for maximum inhibition.
The Journal of Immunology 05/2005; 174(7):4301-6. · 5.79 Impact Factor
