Regulation of the JNK pathway by TGF-beta activated kinase 1 in rheumatoid arthritis synoviocytes

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-β 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-1β-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-1β induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1β-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1β-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.

In vivo RNAi-mediated silencing of TAK1 decreases inflammatory Th1 and Th17 cells through targeting of myeloid cells

“…As expected, TAK1 gene silencing was associated with a marked decrease in downstream inflammation-induced JNK and NF-B activation. In accordance with previously reported data for primary RA synovial fibroblasts, 37 we also show that TAK1 downregulation does not affect p38 activation in vivo, nor in vitro, as evidenced by a mouse monocytic cell line transfected with siTAK1 after TNF-␣ or LPS challenges. Although we cannot exclude that the direct silencing of TAK1 within other cell types, including T and B lymphocytes, as well as ECs (Figure 2 and supplemental Figure 3), might be responsible for the pronounced inhibition of TAK1 down-stream signaling pathways, an indirect effect through TAK1-silenced, MPS-derived cytokines is more likely to contribute to the decreased TAK1 activation observed in the whole spleen.…”

“…Indeed, in the context of degenerative joint disorders, such as RA, TAK1-mediated signal transduction has been reported to play an important role in the regulation of catabolic events and proinflammatory processes. 37 Interestingly, MEK kinase (MEKK) 1 and 2 and TAK1 are the most abundant MAP3Ks in inflamed rheumatoid joints, as well as in IL-1-induced fibroblast-like RA synoviocytes (RA-FLSs) in vitro. 38 Moreover, TLR2 and TLR4 that trigger TAK1 signaling have been shown to Relative expression levels of signaling mediators in arthritic mice were quantified as described in the Methods.…”

Phosphoinositide 3-Kinase δ Regulates Migration and Invasion of Synoviocytes in Rheumatoid Arthritis

“…FLS were used from passages 3 through 9, during which time they are a homogeneous population of cells (,1% CD11b positive, ,1% phagocytic, and ,1% FcgRII and FcgRIII receptor positive). The detailed characterization and phenotyping of FLS were previously described (15)(16)(17)(18)(19). FLS were cultured and used at 80% confluence.…”

MAPK Kinase Kinase-1 Is Essential for Cytokine-Induced c-Jun NH2-Terminal Kinase and Nuclear Factor-κB Activation in Human Pancreatic Islet Cells

“…A number of protein kinases have been identified as activators of IKK␤ and JNK in response to proinflammatory cytokines. For example, phosphorylation of JNK and/or IKK requires transforming growth factor-␤-activated kinase-1 (TAK1) (32) or MEKK-2 (33) in synoviocytes, mixed linage kinase 3 (MLK3) in T-cells (34), MEKK-1 in embryonic stem cells (35), NF-␤-inducing kinase (NIK) in HeLa cells (36), and apoptosis signal regulating kinase-1 (ASK1) in human umbilical vein endothelial cells (HUVECs) (37). However, the main intracellular activator(s) of JNK/IKK␤ in ␤-cells has, to our knowledge, hitherto not been identified.…”