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

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

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

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

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

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

A Comparative Effectiveness Study of Renal Parameters Between Imidapril and Amlodipine in Patients with Hypertension: A Retrospective Cohort Study

<p><b>Article full text</b></p> <p><br></p> <p>The full text of this article can be found here<b>. </b><a href="https://link.springer.com/article/10.1007/s40119-016-0080-4">https://link.springer.com/article/10.1007/s40119-016-0080-4</a></p><p></p> <p><br></p> <p><b>Provide enhanced content for this article</b></p> <p><br></p> <p>If you are an author of this publication and would like to provide additional enhanced content for your article then please contact <a href="http://www.medengine.com/Redeem/”mailto:[email protected]”"><b>[email protected]</b></a>.</p> <p><br></p> <p>The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.</p> <p><br></p> <p>Other enhanced features include, but are not limited to:</p> <p><br></p> <p>• Slide decks</p> <p>• Videos and animations</p> <p>• Audio abstracts</p> <p>• Audio slides</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

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