bafilomycin-a1 and Osteoarthritis

To investigate the role of autophagy in the regulation of cell death in rheumatoid arthritis synovial fibroblasts (RASFs).. RASFs and osteoarthritis synovial fibroblasts (OASFs) were treated with thapsigargin (TG), an inducer of endoplasmic reticulum (ER) stress, and MG132, a proteasome inhibitor. Then, 3-methyladenine was used as an autophagy inhibitor and bafilomycin A1 as a lysosome inhibitor. Polyubiquitinated proteins, p62, and autophagy induction were evaluated by immunoblotting, immunofluorescence microscopy, and immunohistochemistry, respectively. OASFs were transfected with small interfering RNA targeting autophagy-linked FYVE protein (ALFY). Cell death was evaluated by flow cytometry and a caspase 3 activity assay.. In RASFs, the induction of autophagy by TG and MG132 was increased compared to that in OASFs. Whereas autophagy promoted a caspase 3-independent induction of cell death under ER stress, autophagy had a protective role in apoptosis induced by proteasome inhibition. Treatment of RASFs with 3-methyladenine blocked TG-induced cell death. ER stress induced a strong accumulation of p62-positive polyubiquitinated protein aggregates, accompanied by the formation of large vacuoles in RASFs but not OASFs. Furthermore, TG-induced p62 protein expression was increased, whereas TG-induced ALFY expression was reduced, in RASFs compared to OASFs. ALFY knockdown promoted the accumulation of p62, the formation of polyubiquitinated protein aggregates, and cell death.. Our data provide the first evidence of a dual role of autophagy in the regulation of death pathways in RASFs. A reduced expression of ALFY and the formation of p62-positive polyubiquitinated protein aggregates promote cell death in RASFs under severe ER stress.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Arthritis, Rheumatoid; Autophagy; Autophagy-Related Proteins; Caspase 3; Cell Death; Cells, Cultured; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Fibroblasts; Immunoblotting; Immunohistochemistry; Lysosomes; Macrolides; Membrane Proteins; Microscopy, Fluorescence; Osteoarthritis; Polyubiquitin; Sequestosome-1 Protein; Synovial Membrane; Transcription Factors

To determine the mechanism of matrix metalloproteinase (MMP)-13 upregulation in osteoarthritic synovial fibroblasts (OASF) in response to stimulation with basic calcium phosphate (BCP) crystals and to investigate the effect of prostaglandin (PG)E2 on BCP crystal-stimulated MMP expression.. Primary OASF were stimulated with BCP crystals; mRNA expression was measured by real-time reverse transcription-polymerase chain reaction and protein levels were assessed by Western blotting.. BCP crystals upregulated MMP-13 mRNA expression over 20-fold and increased MMP-13 protein production in OASF. BCP crystal-stimulated MMP-13 mRNA expression was blocked by inhibition of the extracellular regulated kinase (ERK1/2) and p38 mitogen activated protein kinase (MAPK) pathways and inhibition of the activation of nuclear factor kappaB. Addition of exogenous PGE2 downregulated BCP crystal-stimulated MMP-13 expression. In contrast, PGE2 upregulated, and had no effect, on BCP crystal stimulated MMP-3 and MMP-1 mRNA expression, respectively. These effects of PGE2 were diminished by L-161,982, a selective EP4 receptor antagonist, and mimicked by CAY10399, a selective EP2 receptor agonist, and forskolin, an adenylate cyclase activator.. These data suggest that BCP crystal induction of MMP-13 expression may involve the ERK1/2 and p38 MAPK pathways and activation of nuclear factor kappaB; this upregulation of MMP-13 may contribute to the accelerated cartilage breakdown in BCP crystal-associated osteoarthritis. PGE2 had contrasting effects on BCP crystal-stimulated MMP-3 and MMP-13 mRNA expression, mediated in an EP2/EP4/cAMP-dependent manner, suggesting that PGE2 may have beneficial as well as deleterious effects in BCP crystal-associated osteoarthritis.

Topics: Calcium Phosphates; Cells, Cultured; Crystallization; Cycloheximide; Dinoprostone; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression Regulation, Enzymologic; Humans; Macrolides; Matrix Metalloproteinase 13; NF-kappa B; Osteoarthritis; p38 Mitogen-Activated Protein Kinases; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP2 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Synovial Membrane; Up-Regulation