thapsigargin and Osteoarthritis

Excessive extracellular matrix degradation and chondrocyte apoptosis are the pathological features of osteoarthritis (OA). The ability of flavonoid compounds isolated from Chinese hawthorn leaves to exert protective effects on several diseases, via inhibition of oxidative stress and inflammation, has been demonstrated in several studies. This study explored the effects of vitexin on chondrocytes, and the underlying mechanisms thereof. Vitexin, an active ingredient in hawthorn leaf extracts, was shown to exert protective effects on chondrocytes, by inhibiting the expression of GRP78 and PDI, and an apoptotic protein (CHOP) induced by interleukin-1β. It also modulated thapsigargin-induced upregulation of GRP78 and PDI and subsequently an apoptotic protein (CHOP). Among rat chondrocytes, both the ER stress-activated nuclear factor kappa B (NF-κB) pathway and the induced expression of inflammatory cytokines (IL-6 and TNF-α) were significantly inhibited by vitexin. Finally, vitexin attenuated the progression of OA in vivo in rats. Taken together, all data demonstrate the relationship of ER stress and inflammation in the progression of OA, the ability of vitexin to protect chondrocytes and thus its therapeutic potential in patients with OA.

Topics: Animals; Apigenin; Apoptosis; Cartilage; Caspase 3; Cell Survival; Chondrocytes; Disease Models, Animal; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Inflammation; Interleukin-1beta; Male; NF-kappa B; Osteoarthritis; Rats; Rats, Sprague-Dawley; Sincalide; Thapsigargin; Transcription Factor CHOP; Tumor Necrosis Factor-alpha; Up-Regulation

To investigate the role of protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in the pathogenesis of rheumatoid arthritis (RA).. Synovial tissue samples from patients with RA and patients with osteoarthritis (OA) were stained for PTPN2. Synovial fibroblasts were stimulated with tumor necrosis factor (TNF) and interleukin-1β (IL-1β), lipopolysaccharide (LPS), TRAIL, or thapsigargin. The expression of PTPN2 in synovial fibroblasts and peripheral blood mononuclear cells (PBMCs) was analyzed by real-time polymerase chain reaction and Western blotting. Cell death, the release of IL-6 and IL-8, and the induction of autophagy were analyzed after PTPN2 silencing. Methylated DNA immunoprecipitation analysis was used to evaluate DNA methylation-regulated gene expression of PTPN2.. PTPN2 was significantly overexpressed in synovial tissue samples from RA patients compared to OA patients. Patients receiving anti-TNF therapy showed significantly reduced staining for PTPN2 compared with patients treated with nonbiologic agents. PTPN2 expression was higher in RA synovial fibroblasts (RASFs) than in OASFs. This differential expression was not regulated by DNA methylation. PTPN2 was further up-regulated after stimulation with TNF, TNF combined with IL-1β, or LPS. There was no significant difference in basal PTPN2 expression in PBMCs from patients with RA, ankylosing spondylitis, or systemic lupus erythematosus or healthy controls. Most interestingly, PTPN2 silencing in RASFs significantly increased the production of the inflammatory cytokine IL-6 but did not affect levels of IL-8. Moreover, functional analysis showed that high PTPN2 levels contributed to the increased apoptosis resistance of RASFs and increased autophagy.. This is the first study of PTPN2 in RASFs showing that PTPN2 regulates IL-6 production, cell death, and autophagy. Our findings indicate that PTPN2 is linked to the pathogenesis of RA via synovial fibroblasts.

Topics: Aged; Apoptosis; Arthritis, Rheumatoid; Autophagy; Biological Products; Cells, Cultured; Female; Fibroblasts; Humans; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Male; Middle Aged; Osteoarthritis; Protein Tyrosine Phosphatase, Non-Receptor Type 2; Synovial Membrane; Thapsigargin; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Up-Regulation

Nuclear protein 1 (Nupr1) is a stress-inducible protein that is involved in gene transcription. The present study was undertaken to determine whether chondrocytes express Nupr1 and whether Nupr1 regulates matrix metalloproteinase 13 (MMP-13) expression.. Paraffin-embedded cartilage sections from normal human and osteoarthritic (OA) cartilage were immunostained using anti-Nupr1 antibody. To measure Nupr1 expression, total RNA was isolated from joint tissue obtained 8 weeks after surgery from young (12-week-old) and older (12-month-old) mice that underwent destabilization of the medial meniscus (DMM) to induce OA. Human chondrocytes were stimulated with 1-10 ng/ml interleukin-1β (IL-1β), 25 μM tert-butyl-hydroperoxide (tBHP), or 2 μM thapsigargin, and Nupr1 expression was analyzed by quantitative polymerase chain reaction. In addition, chondrocytes were transfected with small interfering RNA to knock down Nupr1 expression and then stimulated overnight with IL-1β. After incubation, the conditioned medium was collected and MMP levels measured.. Increased Nupr1 immunostaining was noted in human OA cartilage compared to normal cartilage. Expression was also increased in joint tissue from 12-month-old mice that underwent DMM surgery compared to sham-operated controls. Stimulation of chondrocytes with IL-1β induced a 2-fold increase in Nupr1 messenger RNA (mRNA) within 1 hour, with the increase peaking to 4-fold at 6 hours. Treatment of chondrocytes with tBHP to induce oxidative stress increased Nupr1 mRNA expression by >2-fold; treatment with thapsigargin to induce endoplasmic reticulum stress did not produce a similar effect. Knockdown of Nupr1 inhibited IL-1β-mediated induction of MMP-13.. Nupr1 is expressed in cartilage, and its levels are increased in OA. Nupr1 expression is required for IL-1β-mediated expression of MMP-13. These findings provide evidence of a novel pathway for regulation of IL-1β-mediated production of MMPs in chondrocytes.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cartilage, Articular; Cells, Cultured; Chondrocytes; Disease Models, Animal; DNA-Binding Proteins; Humans; Interleukin-1beta; Matrix Metalloproteinase 13; Mice; Neoplasm Proteins; Osteoarthritis; Oxidative Stress; tert-Butylhydroperoxide; Thapsigargin

Synovial fibroblasts from rheumatoid arthritis show resistance to apoptotic stimuli, indicating they may be difficult to treat. To clearly understand these mechanisms of resistance, rheumatoid and osteoarthritis synovial fibroblasts (RASF and OASF) were exposed to endoplasmic reticulum (ER) stress such as thapsigargin, Ca2+-ATPase inhibitor.. Fibroblasts were assessed microscopically for cell viability by trypan blue exclusion and for autophagic cells by LC-3II formation. Caspase-3 activity was measured as aminomethyl-coumarin (AMC) liberated from AC-DEVD-AMC. Immunoblotting was performed to compare protein expression in OASF and RASF.. ER stress caused cell death in OASF but not in RASF. Thapsigargin, a Ca2+-ATPase inhibitor, did not change the expression of GRP78, an ER chaperone in OASF and RASF, but induced another ER stress protein, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) differently, showing high levels in OASF and low levels in RASF. Thapsigargin increased the autophagy response in RASF, with autophagosome formation, beclin expression, and LC3-II conversion. Transfection with beclin siRNA inhibited autophagy and increased the susceptibility to ER stress-induced cell death. On the other hand, CHOP siRNA increased autophagy and improved cell survival, especially in RASF, indicating that CHOP is involved in regulation of autophagy and cell death, but that low expression of CHOP protects RASF from apoptosis.. Autophagy induction and CHOP under-expression increases cell resistance against ER stress-induced cell death in fibroblasts from rheumatoid arthritis patients.

Topics: Arthritis, Rheumatoid; Autophagy; Blotting, Western; Caspase 3; Cell Survival; Down-Regulation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Fibroblasts; Gene Expression; Humans; Osteoarthritis; RNA, Small Interfering; Synovial Membrane; Thapsigargin; Transcription Factor CHOP; Transfection

To determine whether interleukin-1 (IL-1) initiates transient changes in the intracellular concentration of [Ca2+]i and the organization of filamentous actin (F-actin) in articular chondrocytes.. Articular chondrocytes within cartilage explants and enzymatically isolated chondrocytes were loaded with Ca(2+)-sensitive fluorescence indicators, and [Ca2+]i was measured using confocal fluorescence ratio imaging during exposure to 10 ng/ml IL-1alpha. Inhibitors of Ca2+ mobilization (Ca(2+)-free medium, thapsigargin [inhibitor of Ca-ATPases], U73122 [inhibitor of phospholipase C], and pertussis toxin [inhibitor of G proteins]) were used to determine the mechanisms of increased [Ca2+]i. Cellular F-actin was quantified using fluorescently labeled phalloidin. Toxin B was used to determine the role of the Rho family of small GTPases in F-actin reorganization.. In isolated cells on glass and in in situ chondrocytes within explants, exposure to IL-1 induced a transient peak in [Ca2+]i that was generally followed by a series of decaying oscillations. Thapsigargin, U73122, and pertussis toxin inhibited the percentage of cells responding to IL-1. IL-1 increased F-actin content in chondrocytes in a manner that was inhibited by toxin B.. Both isolated and in situ chondrocytes respond to IL-1 with transient increases in [Ca2+]i via intracellular Ca2+ release mediated by the phospholipase C and inositol trisphosphate pathways. The influx of Ca2+ from the extracellular space and the activation of G protein-coupled receptors also appear to contribute to these mechanisms. These findings suggest that Ca2+ mobilization may be one of the first signaling events in the response of chondrocytes to IL-1.

Topics: Actins; Animals; Bacterial Proteins; Bacterial Toxins; Calcium Signaling; Calcium-Transporting ATPases; Cartilage, Articular; Cells, Cultured; Chondrocytes; Estrenes; Female; GTP Phosphohydrolases; Interleukin-1; Osteoarthritis; Pertussis Toxin; Pyrrolidinones; Swine; Thapsigargin; Type C Phospholipases