ERK2 and JNK1 contribute to TNF-α-induced IL-8 expression in synovial fibroblasts

Abstract

Tumor necrosis factor α (TNF-α) induces the expression and secretion of interleukin 8 (IL-8), which contributes to synovitis in rheumatoid arthritis (RA). To elucidate the mechanism of the onset of RA, we used synovial fibroblasts without autoimmune inflammatory diseases and investigated MAPK signaling pathways in TNF-α-induced IL-8 expression. Synovial fibroblasts isolated from healthy dogs were characterized by flow cytometry, which were positive for the fibroblast markers CD29, CD44, and CD90 but negative for the hematopoietic cell markers CD14, CD34, CD45, and HLA-DR. TNF-α stimulated the secretion and mRNA expression of IL-8 in a time- and dose-dependent manner. ERK and JNK inhibitors attenuated TNF-α-induced IL-8 expression and secretion. TNF-α induced the phosphorylation of ERK1/2 and JNK1/2. TNF-α-induced IL-8 expression was attenuated both in ERK2- and JNK1-knockdown cells. TNF-α-induced ERK1/2 or JNK1/2 was observed in ERK2- or JNK1-knockdown cells, respectively, showing that there is no crosstalk between ERK2 and JNK1 pathways. These observations indicate that the individual activation of ERK2 and JNK1 pathways contributes to TNF-α-induced IL-8 expression in synovial fibroblasts, which appears to be involved in the progress in RA.

Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma

“…p38 and JNK are members of the MAPK family and activated by environmental stress, including ultraviolet exposure, oxidative stress, or inflammatory cytokines 36 . They have been suggested to induce COX2 expression in inflammatory processes, such as rheumatoid arthritis or Parkinson's disease, and in human cancer-promoting inflammation [37][38][39] . Our results suggested that the p38/JNK pathway also regulates PGE2 production in cUC cells in a manner different from that of the RAF/MEK/ERK pathway.…”

All-trans retinoic acid induces reprogramming of canine dedifferentiated cells into neuron-like cells

“…The cells were harvested using 0.25% trypsin-EDTA once they reached 90-95% confluence. The collected cells were suspended in CELLBANKER 1 plus medium at a density of 2 × 10 6 cells/500 μL, and 500 μL of the cell suspension was placed into each sterilized serum tube as previously described [28][29][30][31][32][33][34][35]. The tubes were then placed in a freezing vessel (BICELL) and cryopreserved at −80˚C.…”

“…Western blotting was performed as previously reported [28][29][30][31][32][33][34][35]. The cells were lyzed using lysis buffer containing 20 mM HEPES, 1 mM PMSF, 10 mM sodium fluoride, and a complete mini EDTA-free protease inhibitor cocktail at pH 7.4.…”

“…DFATs were characterized by flow cytometry analysis based on a previous report with slight modifications [26,31]. Cells were placed in 5-mL round-bottom tubes at 3 × 10 5 cells/tube with PBS containing 0.5% FBS, and incubated with antibodies (FITC-conjugated mouse monoclonal antibodies against human CD14, human CD29, human CD90, and human HLA-DR, FITC-conjugated rat monoclonal antibody against canine CD45, and PE-conjugated mouse monoclonal antibodies against canine CD34 and canine CD44) at 4˚C for 30 min.…”

“…Cells were transfected using Opti-MEM containing 5 μL/mL Lipofectamine 2000 and 50 nM JNK1, JNK2, JNK3, or scramble siRNA for 6 h [29][30][31][32][33][34][35]. The siRNA sequences are provided in Table 2.…”