Signal Transduction Pathways Involved in Rheumatoid Arthritis Synovial Fibroblast Interleukin-18-induced Vascular Cell Adhesion Molecule-1 Expression

Interleukin‐18 induces angiogenic factors in rheumatoid arthritis synovial tissue fibroblasts via distinct signaling pathways

“…Similarly, Src and PKC␣ I / ␤ II were not important for SDF-1␣/CXCL12 production (data not shown). We did not find toxic effects of chemical inhibitors or decreased viability of fibroblasts with the 10-M concentration of inhibitors used (25,26,29,30,32).…”

“…Our group has previously shown that IL-18 up-regulates CXC chemokines such as IL-8/CXCL8, growth-regulated oncogene ␣ (GRO␣)/CXCL1, and epithelial neutrophil-activating peptide 78 (ENA-78)/CXCL5 in RA ST fibroblasts (25,26). In this study, we demonstrated that IL-18 increases the production of SDF-1␣/CXCL12, MCP-1/ CCL2, and VEGF via overlapping but distinct signaling pathways.…”

“…All inhibitors were purchased from Calbiochem (San Diego, CA). Cells were pretreated for 1 hour with 10 M of each inhibitor before stimulation with IL-18 (25 nM), as described previously (25)(26)(27). TNF␣ (1.15 nM; Upjohn, Kalamazoo, MI) and phosphate buffered saline (PBS) were used as positive and negative controls, respectively.…”

“…FBS was then reduced to 5% for 24 hours, and subsequently to 0% for 24 hours; FBS was tapered gradually because we have noted in the past when working with RA ST fibroblasts that the morphologic features of these cells change, and they become apoptotic, if medium with 10% FBS is immediately switched to 0% FBS. We starve RA ST fibroblasts for 24 hours to remove the effect of FBS, since there are many factors in serum that can stimulate these cells, leading to false-positive results (25,26,29). RA ST fibroblasts were stimulated for 24 hours with IL-18 (25 nM).…”

“…Cell lysates in Laemmli sample buffer were boiled for 5 minutes, and samples containing equal amounts of protein (15 g total protein) were subjected to sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis followed by Western blot analysis (26). Membranes were probed with specific rabbit polyclonal antibodies to phosphorylated JNK, p38 MAPK, PKC␦, p65 NF B, or ATF-2 (all from Cell Signaling Technology, Beverly, MA).…”