geranylgeranyl-pyrophosphate and Arthritis--Rheumatoid

To study effects of drugs against rheumatoid arthritis (RA) synoviocytes or fibroblast like synoviocytes (FLS) are used. To overcome the drawbacks of using FLS, this study was conducted to show the validity of SW982 synovial cell line in RA study.. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Annexin V propidium iodide (PI) staining, mitochondrial membrane potential assay, Triton X-114 Phase partitioning, and immunolot for apoptosis signaling in SW982 human synovial cell line were performed.. Fluvastatin induced apoptosis in a dose- and time-dependent manner in TNFα -stimulated SW982 human synovial cells. A geranylgeranylpyrophosphate (GGPP) inhibitor, but not a farnesylpyrophosphate (FPP) inhibitor, induced apoptosis, and fluvastatin-induced apoptosis was associated with the translocation of isoprenylated RhoA and Rac1 proteins from the cell membrane to the cytosol. Fluvastatin-induced downstream apoptotic signals were associated with inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Accordingly, 89 kDa apoptotic cleavage fragment of poly (ADP-ribose) polymerase (PARP) was detected.. Collectively, our data indicate that fluvastatin induces apoptotic cell death in TNFα-stimulated SW982 human synovial cells through the inactivation of the geranylgerenylated membrane fraction of RhoA and Rac1 proteins and the subsequent inhibition of the PI3K/Akt signaling pathway. This finding shows the validity of SW982 cell line for RA study.

Topics: Apoptosis; Arthritis, Rheumatoid; Cell Line; Cell Survival; Fatty Acids, Monounsaturated; Fluvastatin; Humans; Indoles; Membrane Potential, Mitochondrial; Polyisoprenyl Phosphates; Signal Transduction; Synovial Fluid; Tetrazolium Salts; Thiazoles

To determine whether statins induce apoptosis in rheumatoid arthritis (RA) synoviocytes.. The effects of lipophilic and hydrophilic statins (fluvastatin and pravastatin, respectively) on the apoptosis of cultured RA synoviocytes were examined in vitro. Apoptosis was analyzed by flow cytometry after staining with JC-1 (to measure the mitochondrial transmembrane potential), active caspase 3, annexin V, and propidium iodide. Add-back experiments were conducted to determine which downstream products of the mevalonate pathway could suppress apoptosis. Modulation of various signaling pathways induced by statins, including protein prenylation, was also investigated.. Fluvastatin, but not pravastatin, induced apoptosis in RA synoviocytes in a concentration-dependent (1-10 microM) and time-dependent (48-96 hours) manner. Another lipophilic statin, pitavastatin, displayed almost the same effects as fluvastatin. In sharp contrast, lipophilic statins did not significantly increase apoptosis in synoviocytes from patients with osteoarthropathy. Apoptosis induced by fluvastatin was mitochondrial- and caspase 3-dependent and was abrogated by mevalonate and geranylgeranyl pyrophosphate, but not by farnesyl pyrophosphate. In addition, the geranylgeranyl transferase inhibitor GGTI-298 mimicked the effect of fluvastatin on RA synoviocytes. Treatment of RA synoviocytes with the RhoA kinase inhibitor Y-27632 caused apoptosis. Fluvastatin decreased the amount of RhoA protein in the membrane fraction, but increased the amount in the cytosolic fraction.. Fluvastatin induced apoptosis in RA synoviocytes through a mitochondrial- and caspase 3-dependent pathway and by the blockage of mevalonate pathways, particularly through the inhibition of protein geranylgeranylation and RhoA/RhoA kinase pathways. These findings suggest that lipophilic statins have potential as novel therapeutic agents for RA.

Topics: Apoptosis; Arthritis, Rheumatoid; Benzamides; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Mevalonic Acid; Polyisoprenyl Phosphates; Protein Prenylation; Synovial Membrane

Rheumatoid arthritis (RA) is a chronic inflammatory disease in which the synovial environment is characterized by intense immunological activity. Evidence suggests that statins modulate immune functions and may have a beneficial effect on patients with RA. We investigated whether simvastatin could inhibit the expression of interleukin 6 (IL-6) and IL-8 and cell proliferation induced by tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) obtained from RA patients undergoing joint replacement therapy.. RA FLS were cultured with or without 0.05-10 microM simvastatin for 12 h. Cytokine mRNA expression and secretion levels were detected using real-time PCR and ELISA, respectively. Cell proliferation of FLS induced by TNF-alpha was determined by MTT assay.. Real-time PCR analysis revealed that the levels of IL-6 and IL-8 mRNA expressed by FLS were reduced by simvastatin in a dose-dependent manner. Levels of IL-6 and IL-8 in FLS culture supernatants were decreased by simvastatin in a time-dependent and dose-dependent manner. MTT assay revealed that simvastatin could inhibit proliferation of FLS induced by TNF-alpha. These effects of simvastatin on IL-6 and IL-8 production and cell proliferation were reversed in the presence of mevalonic acid or geranylgeranyl-pyrophosphate, but not with farnesyl-pyrophosphate.. Our results suggest that the beneficial effect of simvastatin in RA patients may involve inhibition of IL-6 and IL-8 production, as well as reduction of cell proliferation.

Topics: Arthritis, Rheumatoid; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Drug Antagonism; Drug Combinations; Fibroblasts; Gene Expression; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Interleukin-6; Interleukin-8; Mevalonic Acid; Polyisoprenyl Phosphates; RNA, Messenger; Simvastatin; Synovial Membrane; Tumor Necrosis Factor-alpha

Statins, competitive inhibitors of hydroxymethylglutaryl-CoA reductase, have recently been shown to have a therapeutic effect in rheumatoid arthritis (RA). In RA, synovial fibroblasts in the synovial lining, are believed to be particularly important in the pathogenesis of disease because they recruit leukocytes into the synovium and secrete angiogenesis-promoting molecules and proteases that degrade extracellular matrix. In this study, we show a marked reduction in RA synovial fibroblast survival through the induction of apoptosis when the cells were cultured with statins. Simvastatin was more effective in RA synovial fibroblasts than atorvastatin, and both statins were more potent on tumor necrosis factor-alpha-induced cells. In contrast, in osteoarthritis synovial fibroblasts, neither the statin nor the activation state of the cell contributed to the efficacy of apoptosis induction. Viability of statin-treated cells could be rescued by geranylgeraniol but not by farnesol, suggesting a requirement for a geranylgeranylated protein for synovial fibroblast survival. Phase partitioning experiments confirmed that in the presence of statin, geranylgeranylated proteins are redistributed to the cytoplasm. siRNA experiments demonstrated a role for Rac1 in synovial fibroblast survival. Western blotting showed that the activated phosphorylated form of Akt, a protein previously implicated in RA synovial fibroblast survival, was decreased by about 75%. The results presented in this study lend further support to the importance of elevated pAkt levels to RA synovial fibroblast survival and suggest that statins might have a beneficial role in reducing the aberrant pAkt levels in patients with RA. The results may also partly explain the therapeutic effect of atorvastatin in patients with RA.

Topics: Apoptosis; Arthritis, Rheumatoid; Biological Transport; Cell Survival; Cells, Cultured; Cytoplasm; Diterpenes; Fibroblasts; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Membrane Proteins; Osteoarthritis; Polyisoprenyl Phosphates; Proto-Oncogene Proteins c-akt; rac1 GTP-Binding Protein; RNA, Small Interfering; Synovial Membrane; Tissue Distribution; Tumor Necrosis Factor-alpha