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

<div><p>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.</p></div

TNF-α-induced IL-8 protein secretion and mRNA expression in synovial fibroblasts.

<p>IL-8 protein secretion (A) and IL-8 mRNA expression (C) increased in a time-dependent manner in synovial fibroblasts incubated with (closed circle) or without (open circle) 100 ng/mL TNF-α, for the indicated time periods. IL-8 protein secretion (B) and mRNA expression (D) showed TNF-α dose-dependent stimulation. After pretreatment with the ERK1/2 inhibitor FR180204 (25 μM), the JNK1/2 inhibitor SP600125 (10 μM), and the p38 inhibitors SB239063 (20 μM) and SKF86002 (10 μM) for 1 h, synovial fibroblasts were stimulated with TNF-α (50 ng/mL) for 6 h. ERK1/2 and JNK1/2 inhibitors significantly attenuated TNF-α-induced IL-8 mRNA expression, whereas p38 inhibitors had no effect (E). ERK1/2 and JNK1/2 inhibitors also inhibited TNF-α-induced IL-8 protein secretion (F). Results are presented as mean ± SE from three independent experiments. Synovial fibroblasts isolated from three male beagle dogs were used, and each experiment was performed with cells derived from a single donor. *<i>P</i> < 0.05, compared with 0 h (A, C) or 0 pM (B, D).</p

FGFR, PI3K, and Akt inhibitors attenuate bFGF-induced Akt phosphorylation.

<p>After pretreatment with the FGFR inhibitor SU5402 (25 μM), the PI3K inhibitor LY294002 (50 μM), and the Akt inhibitor MK2206 (1 μM) for 1 h, BMSCs were incubated with bFGF (100 ng/ml) for 10 min. Phosphorylation of Akt was examined by western blotting. The inhibitors of FGFR, PI3K, and Akt completely suppressed bFGF-induced Akt phosphorylation (arrowheads).</p

bFGF stimulates Akt phosphorylation in canine BMSCs.

<p>Western blotting for detection of phosphorylated Akt (p-Akt) in BMSCs treated with bFGF (100 ng/ml) for the indicated times (upper panel). Relative density of p-Akt compared with the results at 0 time (lower panel). Relative density of total Akt (t-Akt) compared with the results at 0 time (lower panel). bFGF stimulated the phosphorylation of Akt in a time-dependent manner. Results are presented as means ± SE. n = 3. *<i>p</i> < 0.05.</p

Characterization of synovial fibroblasts by flow cytometry.

<p>Synovial fibroblasts were isolated from three male beagle dogs. Solid and open histograms show non-specific and specific staining for the indicated marker, respectively. Cells were strongly positive for the fibroblast markers CD29, CD44, and CD90. In contrast, most of the cells were negative for the hematopoietic cell markers CD14, CD34, CD45, and HLA-DR. Results are representative in three independent experiments.</p

Schematic diagram of the contribution of ERK2 and JNK1 activation to TNF-α-induced IL-8 expression in synovial fibroblasts.

<p>In TNF-α-stimulated synovial fibroblasts, ERK2 and JNK1 cooperatively lead to the activation of IL-8 expression, without ERK1 and JNK2.</p

Parallel activation of ERK2 and JNK1 contributes to TNF-α-induced IL-8 expression.

<p>(A) Expression of phosphorylated JNK1 (p-JNK1), total JNK1 (t-JNK1), phosphorylated ERK2 (p-ERK2), total ERK2 (t-ERK2), and β-actin were detected by western blotting in cells transfected with ERK2 siRNA and subsequently stimulated with or without TNF-α (50 ng/mL) for 15 min (upper panel). β-actin was used as an internal standard. Relative densities of TNF-α-induced p-JNK1, p-ERK2, and t-ERK2 compared with those in the absence of TNF-α is shown in the lower panels. ERK2 siRNA transfection attenuated ERK2 protein expression and its phosphorylation, whereas no effect was observed on TNF-α-induced JNK1 phosphorylation. (B) Expression of phosphorylated ERK2 (p-ERK2), total ERK2 (t-ERK2), phosphorylated JNK1 (p-JNK1), total JNK1 (t-JNK1), and β-actin were detected by western blotting in cells transfected with JNK1 siRNA and subsequently stimulated with or without TNF-α (50 ng/mL) for 15 min (upper panel). β-actin was used as an internal standard. Relative densities of TNF-α-induced p-ERK2, p-JNK1, and t-JNK1 compared with those in the absence of TNF-α, are given in the lower panels. JNK1 siRNA transfection decreased the phosphorylation of JNK1, whereas no effect was observed on TNF-α-induced ERK2 phosphorylation. Results are presented as mean ± SE from three independent experiments. Synovial fibroblasts isolated from three male beagle dogs were used, and each experiment was performed with cells derived from a single donor. *<i>P</i> < 0.05.</p

Expression of various FGFR subtypes and binding of bFGF to these subtypes in canine BMSCs.

<p>A, mRNA expression of four different subtypes of FGFR determined by RT-PCR using total RNA extracted from canine BMSCs. PCR products for <i>FGFR-1</i> and <i>FGFR-2</i> were detected to be 120 and 193 bp, respectively. <i>TBP</i> control was used as an internal standard for the PCR analysis. B, Detection of FGFR-1 and FGFR-2 proteins (arrowheads) by western blotting using anti-FGFR-1 and anti-FGFR-2 antibodies. Canine brain extracts served as positive controls. C, Canine BMSCs were incubated with bFGF (100 ng/ml) for 2 min and cross-linked with DTSSP (1 mM) at 4°C for 2 h. Whole-cell proteins were extracted and immunoprecipitated with anti-bFGF antibody. Finally, the precipitated bFGF/FGFR complex was treated in the presence or absence DTT (200 mM) and detected by western blotting with anti-FGFR-1 or anti-FGFR-2 antibody. FGFR-2 protein was clearly detected after CLIP in the absence of DTT as indicated by an arrowhead.</p

Contribution of ERK isoforms to TNF-α-induced IL-8 expression.

<p>(A) The levels of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) were detected by western blotting in cells treated with TNF-α (50 ng/mL) for 0–120 min (upper panel). Time-dependent change of relative densities of p-ERK1/2 compared with those at time 0 is shown (lower panel). (B) In cells pretreated with or without the ERK1/2 inhibitor FR180204 (25 μM) for 1 h and subsequently stimulated with or without TNF-α (50 ng/mL) for 5 min, TNF-α-induced ERK1/2 phosphorylation was clearly attenuated (upper panel). Relative density of attenuation of TNF-α-induced p-ERK compared with that in the absence of TNF-α is shown (lower panel). (C) ERK1 and ERK2 protein expression was significantly decreased in cells transfected with the respective siRNAs but not in scramble siRNA-transfected cells (upper panel). β-actin was used as an internal standard. Relative density of ERK1 and 2 in the respective siRNA-transfected cells compared with that in scramble siRNA-transfected cells is shown in lower left and lower right panels, respectively. (D) TNF-α-induced IL-8 mRNA expression was attenuated in cells transfected with ERK2 siRNA but not in those transfected with ERK1 or scramble siRNA. Cells transfected with ERK1, ERK2, or scramble siRNA were stimulated with or without TNF-α (50 ng/mL), for 6 h. Results are presented as mean ± SE from three independent experiments. Synovial fibroblasts isolated from three male beagle dogs were used, and each experiment was performed with cells derived from a single donor. *<i>P</i> < 0.05.</p

siRNA sequence for canine FGFR-2.

<p>siRNA sequence for canine FGFR-2.</p