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

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

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

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

<p>(A) Expression of phosphorylated JNK1/2 (p-JNK1/2) and total JNK1/2 (t-JNK1/2) was detected by western blotting in cells treated with TNF-α (50 ng/mL) for 0–120 min (upper panel). Time-dependent change of relative density of p-JNK compared with that at time 0 is described (lower panel). (B) TNF-α-induced JNK1/2 phosphorylation was clearly attenuated in cells pretreated with the JNK1/2 inhibitor SP600125 (10 μM) for 1 h, and subsequently stimulated with TNF-α (50 ng/mL) for 15 min (upper panel). Relative density of the attenuation of TNF-α-induced p-JNK compared with that in the absence of TNF-α is shown (lower panel). (C) JNK1 and JNK2 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 densities of JNK1 and 2 protein expression 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 JNK1 siRNA but not in those transfected with JNK2 or scramble siRNA. The cells transfected with JNK1, JNK2, 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

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 and Function of Interleukin-1β-Induced Neutrophil Gelatinase-Associated Lipocalin in Renal Tubular Cells

<div><p>Acute kidney injury (AKI) is characterized by a sudden loss of renal function. Early recognition of AKI, especially in critically ill patients, is essential for adequate therapy. Currently, neutrophil gelatinase-associated lipocalin (NGAL) is considered to be an effective biomarker of AKI; however, the regulation of its expression and function in renal tubular cells remains unclear. In this study, we investigated the regulation of the expression and function of NGAL in IL-1β-treated Madin–Darby canine kidney (MDCK) cells as a model of renal tubular cells. IL-1β induced a disturbance in the localization of E-cadherin and zonaoccludin-1 (ZO-1). The transepithelial electrical resistance (TER) also decreased 5 days after IL-1β treatment. IL-1β induced NGAL mRNA expression and protein secretion in a time- and dose-dependent manner, which occurred faster than the decrease in TER. In the presence of ERK1/2 and p38 inhibitors, IL-1β-induced NGAL mRNA expression and protein secretion were significantly attenuated. In the presence of recombinant NGAL, IL-1β-induced disturbance in the localization of E-cadherin and ZO-1 was attenuated, and the decrease in TER was partially maintained. These results suggest that NGAL can be used as a biomarker for AKI and that it functions as a protector from AKI.</p></div

Expression and Function of Interleukin-1β-Induced Neutrophil Gelatinase-Associated Lipocalin in Renal Tubular Cells - Fig 4

<p>p38 and ERK1/2 inhibitors attenuated IL-1β-induced NGAL mRNA expression (A) and protein secretion (B) in MDCK cells. After pre-treatment with or without the ERK1/2 inhibitor FR180204 (25 μM), p38 inhibitor SB239063 (20 μM), JNK inhibitor SP600125 (10 μM), MEK inhibitor U0126 (20 μM), IKKα/β inhibitor BAY-117082 (10 μM), or IKKβ inhibitor TPCA-1 (10 μM) for 1 h, the cells were incubated with (black column) or without (white column) IL-1β (50 pM) for 48 h. Results are presented as mean ± SE from three independent experiments. *<i>P</i> < 0.05.</p

Recombinant NGAL prevented IL-1β-induced disruption in the localization of adherence and tight junction proteins in MDCK cells.

<p>After treatment with or without IL-1β (50 pM) in culture medium containing 10% or 2% FBS in the presence or absence of 50 ng/ml recombinant NGAL for 7 days, the cells were labeled with fluorescent dye for nuclear staining (blue, nuclei) and antibodies against E-cadherin (green) and ZO-1 (red). The IL-1β-treated cells displayed a loss of membrane localization of E-cadherin and ZO-1. Spotty cytoplasmic localization patterns of E-cadherin and ZO-1 (arrows) were observed in the IL-1β-treated cells. However, in the presence of recombinant NGAL, IL-1β-induced loss of membrane localization and spotty cytoplasmic localization of E-cadherin and ZO-1 were attenuated.</p