Phosphorylation of STAT proteins by recombinant human IL-6 in immortalized human chondrocyte cell lines, T/C28a2 and C28/I2

Abstract

Two immortalized human juvenile chondrocyte cell lines, T/C28a2 and C28/I2, were employed to determine the extent to which recombinant human (rh) IL-6, a known cytokine activator of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway in many cell types, caused STAT proteins to be phosphorylated. The results showed that STAT3 was constitutively phosphorylated in the absence of rhIL-6 in T/C28a2 chondrocytes. However, C28/I2 chondrocytes treated with rhIL-6 caused STAT1, STAT3, and STAT5 to be phosphorylated without altering total unphosphorylated STAT proteins. STAT3 phosphorylation in response to rhIL-6 in T/C28a and C28/I2 chondrocytes was efficiently blocked by the JAK3-selective inhibitor WHI-P131 (Janex-1) and by soluble IL-6 receptor-α (sIL-6R). However, the combination of rhIL-6 and ruxolitinib, a JAK1/JAK2-selective inhibitor, was a less effective inhibitor of STAT protein activation. These findings showed that rhIL-6 activated STAT proteins in the C28/I2 chondrocyte cell line. STAT protein phosphorylation could be blocked by a JAK3-selective inhibitor or by the combination of rhIL-6 and sIL-6R.

Matrix Metalloproteinase-9 Production by Immortalized Human Chondrocyte Lines

“…We also showed western blots at Immunology summit-2015 demonstrating that STAT1 was constitutively phosphorylated in the T/C28a2 chondrocyte line [ 16 ]. However, C-28/I2 chondrocytes incubated with rhIL-6 (50 ng/ml) resulted in the phosphorylation of STAT3 without changing total STAT3 [ 15 ]. Moreover, the combination of rhIL-6 and the pan-JAK-small molecule inhibitor.…”

“…U0126, a small molecule inhibitor of MEK1/2, an upstream protein kinase required for the phosphorylation of ERK1/2 was purchased from Cell Signaling Technology. The Signal Transducer and Activator of Transcription-3 (U-STAT3) antibodies were purchased from R&D Systems and the β-actin antibody from Cell Signaling Technology [ 15 ]. An antibody which interacts with human neutrophil gelatinase-associated lipocalin (NGAL) was purchased from Pierce Biotechnology [ 14 ].…”

“…The presence of NGAL was also determined by ICC [ 14 ]. In addition, the experimental details for analyzing MMP-9 production as well as for the detection of STAT proteins by western blotting were performed as described in 2 papers published following the Keynote Presentation at Immunology Summit-2015 [ 14 , 15 ].…”

Pharmacologic Interventions for Preventing Chondrocyte Apoptosis in Rheumatoid Arthritis and Osteoarthritis

“…Analyses of synovial fluids and sera from RA and OA patients with active disease showed that these samples contained significantly elevated levels of various pro-inflammatory cytokines and growth factors when compared to a control group [52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68]. Of note, incubation of rat [69], non-arthritic or human chondrocytes from OA cartilage [70][71][72][73][74][75][76] or immortalized lines of human chondrocytes [77] with physiological levels of these cytokines, growth factors (e.g., VEGF and FGF) or additional soluble mediators (e.g., nitric oxide) were shown to induce apoptosis, which was accompanied by activation of SAPK/MAPK, JAK/STAT or PI3K/Akt/mTor signaling in these cells [78][79][80][81]. In addition, mediators of inflammation, including prostaglandin E 2 and neuropeptides (e.g., Substance P), are also implicated in perpetuating chronic inflammation [38].…”

“…The results of experimental studies cited in Refs. [70,72,77,80] were supported in part by grants from the National Institutes of Health, Takeda Pharmaceuticals of North America and Genentech/Roche Group.…”