thapsigargin and Arthritis--Rheumatoid

Fibroblast-like synoviocytes (FLSs) play a major role in the pathogenesis of rheumatoid arthritis (RA). Endoplasmic reticulum (ER) stress and dysregulation of unfolded protein response are involved in the resistance to apoptosis of FLSs in RA (RA-FLSs). MicroRNA (MiR)-211 plays an important role in controlling ER stress and apoptotic genes in a PKR-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-dependent manner. We investigated the effect of miR-211-5p overexpression on ER stress and apoptotic genes in RA-FLSs. FLSs were isolated from synovial tissues of trauma (n=10) and RA (n=10) patients. MiR-211-5p and mRNA expression of the selected genes involved in the PERK pathway and apoptosis regulation were measured in RA, trauma, and thapsigargin (Tg)-treated RA-FLSs. Afterward, Tg-treated RA-FLSs following miR-211-5p overexpression were evaluated for miR-211-5p and mRNA levels of the study genes. The expression of miR-211-5p, PERK, BAX, and BCL2 showed no differences between RA and trauma. However, the expression of ATF4 and BCL-XL showed a significant increase in trauma. In addition, the levels of C/EBP homologous protein (CHOP) and MCL1 indicated a significant increase in RA-FLSs. Tg treatment significantly increased the expression of PERK, ATF4, and CHOP in RA-FLSs with no effect on miR-211-5p, BAX, BCL2, BCL-XL, and MCL1. Furthermore, Tg treatment following miR-211-5p overexpression in RA-FLSs showed a significant increase in levels of miR-211-5p with no changes in apoptotic genes. MiR-211-5p overexpression in stimulated RA-FLSs did not alter the levels of selected genes involved in apoptosis regulation. However, more investigations are necessary to determine the ER stress role in apoptosis regulation in RA-FLSs.

Topics: Activating Transcription Factor 4; Apoptosis; Arthritis, Rheumatoid; bcl-2-Associated X Protein; Cell Proliferation; Cells, Cultured; Endoplasmic Reticulum Stress; Fibroblasts; Humans; MicroRNAs; Myeloid Cell Leukemia Sequence 1 Protein; RNA, Messenger; Synoviocytes; Thapsigargin

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

A series of experiments have been carried out to investigate the effects of different concentrations of thapsigargin (0, 0.001, 0.1, and 1 μM) on the proliferation and survival of human rheumatoid arthritis synovial cells (MH7A). The results showed that thapsigargin can block the cell proliferation in human rheumatoid arthritis synovial cells in a time- and dose-dependent manner. Results of Hoechst staining suggested that thapsigargin may induce cell apoptosis in MH7A cells in a time- and dose-dependent manner, and the percentages of cell death reached 44.6% at thapsigargin concentration of 1 μM treated for 4 days compared to the control. The protein and mRNA levels of cyclin D1 decreased gradually with the increasing of thapsigargin concentration and treatment times. Moreover, the protein levels of mTORC1 downstream indicators pS6K and p4EBP-1 were reduced by thapsigargin treatment at different concentrations and times, which should be responsible for the reduced cyclin D1 expressions. Our results revealed that thapsigargin may effectively impair the cell proliferation and survival of MH7A cells. The present findings will help to understand the molecular mechanism of fibroblast-like synoviocytes proliferations and suggest that thapsigargin is of potential for the clinical treatment of rheumatoid arthritis.

Topics: Arthritis, Rheumatoid; Cell Line; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Humans; Synovial Fluid; 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

Peripheral blood (PB) T cells from rheumatoid arthritis (RA) patients proliferate poorly to mitogen, a change that is related to decreased intracellular Ca2+ ([Ca2+]i) signaling after T cell receptor (TCR) stimulation. We hypothesized that this was, in part, due to the effect of mediators of inflammation and predicted that greater changes in [Ca2+]i signaling would be seen in synovial fluid (SF) T cells. We also examined the mechanisms underlying the altered [Ca2+]i signals.. Paired PB and SF T cells from patients with chronic inflammatory arthritis were stimulated with mitogen to assess the magnitude of the [Ca2+]i signal in cell populations by fluorometry, the pattern of the [Ca2+]i signal in individual cells in a single-cell ion-imaging system, and the spatial distribution of Ca2+ within intracellular organelles.. There was a significantly smaller [Ca2+]i signal after phytohemagglutinin protein stimulation of SF T cells (peak rise in [Ca2+]i signal PB versus SF 200 nM versus 180 nM; P < 0.05). In single SF T cells, a change in the pattern of the [Ca2+]i signal and a reduction in the number of responding cells was seen. These changes were a magnification of those seen in RA PB compared with control PB T cells. The contribution of Ca2+ release from intracellular stores to the final [Ca2+]i signal in PB and SF T cells was equal, but there was a significant increase in the Ca2+ remaining in the endoplasmic reticulum (ER) in SF T cells after TCR activation (PB versus SF 6 nM versus 19 nM; P < 0.05). Non-ER Ca2+ stores were not similarly affected.. We found abnormalities in the magnitude, pattern, and spatial distribution of [Ca2+]i signaling in T cells from SF of patients with chronic inflammatory arthritis. A reduction in the number of responding SF T cells may partly explain some of our observations. However, we propose that the observed redistribution of SF Ca2+ stores may underlie the altered [Ca2+]i signaling, thus making these cells hyporesponsive to mitogen. The inflammatory environment of the joint and the late stage of differentiation of SF T cells are both likely to contribute to these changes in [Ca2+]i signaling, resulting in aberrant T cell function and promotion of disease chronicity.

Topics: Arthritis, Rheumatoid; Calcium; Calcium Signaling; Chronic Disease; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Ionomycin; Ionophores; Organelles; Reference Values; Synovial Fluid; T-Lymphocytes; Thapsigargin

To pharmacologically and functionally characterize calcium-mobilizing purine receptors on adherent human rheumatoid synovial cells.. Fura-2-loaded synovial cells were screened for changes in cytosolic calcium concentration after the addition of purine receptor agonists. Release of interleukin-1 (IL-1) and prostaglandin E2 (PGE2) was assessed by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay, respectively. The effect of IL-1 prestimulation on purine-mediated PGE2 release was determined.. ATP (1-100 microM) and UTP (1-100 microM), but not 2-methylthio-ATP or adenosine, stimulated mobilization of calcium from intracellular stores in synovial cells. ATP and UTP stimulated a small, but significant, increase in PG release from resting synoviocytes and a dramatic increase in PG release from synoviocytes prestimulated with recombinant human IL-1alpha. Neither ATP nor UTP stimulated synoviocyte release of IL-1 as measured by specific ELISA. The effects of ATP and UTP on PG secretion were mimicked by phorbol 12-myristate 13-acetate and thapsigargin, and blocked by BAPTA buffering of cytosolic calcium.. Adherent human rheumatoid synovial cells mobilize intracellular calcium via a P2U-like purine receptor. P2U receptor agonists stimulate PGE2 release from synoviocytes, an effect that is greatly enhanced by IL-1alpha prestimulation and blocked by intracellular calcium buffering.

Topics: Adenosine Triphosphate; Adolescent; Adult; Aged; Arthritis, Rheumatoid; Buffers; Calcium; Dinoprostone; Drug Synergism; Humans; Interleukin-1; Middle Aged; Purinergic Agonists; Receptors, Purinergic; Synovial Membrane; Tetradecanoylphorbol Acetate; Thapsigargin; Uridine Triphosphate