Effects of prostaglandin E2 on superoxide dismutase gene expression in chondrocyte

Abstract

Inflammation is part of the complex biological response of tissues characterized by a cascade of biochemical events that propagates the inflammatory response where Prostaglandin and Superoxide Dismutase seems to be an important part of the key. Although Osteoarthritis is known as a degenerative disease, secondary inflammation may play an important role in the tissular changes that occurs in this disease. Chondrocyte is able to synthetize and react to most of intermediate of inflammatory agents. Prostaglandin may be overproduced in 20 fold by chondrocytes. Reactive oxygen species (ROS) are implicated in cellular inflammatory response, and Superoxide dismutase are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide; they are important antioxidant and antiimmflamatory defense in nearly all cells. Purpose: The aims of this study was to further characterize the effects of Prostaglandin E2 on gene expression of Superoxide Dismutase (SOD) in bovine chondrocyte and ATDC-5 culture cells and determine their influence on inflammatory process in cartilage destruction. Methods: After establishing the best conditions for cell culture, proliferation, toxicity and transfection, second step; the expression of SOD promoter constructs was analyzed in Bovine Chondrocytes and ATDC-5 cell. Briefly, cells were collected by centrifugation, resuspended in serum-free DMEM media, and then transfected with ExGen 500, a cationic polymer transfection reagent according to the manufacturer´s recommendations (9 equivalents). Following transfection, cells were resuspended in media containing 5%FCS and plated in 12-well tissue culture dishes. Cells were treated, following transfection with SOD promoter, with differences concentrations of PgE2 x-9 to x-7, TNF protein as a positive control, ethanol and serum media as controls for 24h and then collected and lysed in luciferase lysis buffer. Luciferace assay was preformed according to the manufacture’s instructions with a luminometer. Results: These results indicate that Prostaglandin E2 has no direct effects on proliferations and toxicity in neither bovine chondrocyte nor ATDC-5, at this concentration and conditions of laboratory test. In contrast, Prostaglandin E2 is able to, directly stimulate peripheral receptors membranes, then, modulate intracellular signaling systems through transcription factors, transmitting this information to the nucleus with the consequent modulation of protein synthesis. In this case Superoxide Dismutase was almost completely inhibited by any concentration of PgE2 used, in compare to TNF control P < 0.001 and ethanol-medium controls P < 0.0001 in bovine chondrocyte which can result in an increased accumulation of free radicals at tissue levels and consequent degenerative changes by the presence of inflammations and oxidative stress Conclusion: During inflammatory process in Osteoarthritis, certain cells may secrete many factors that are thought to be deleterious to the articular cartilage and bone. Among these deleterious factor, a great deal of attention is focused on pro-inflammatory cytokines, matrix metalloproteases, arachidonic acid metabolites, free radicals and nitric oxide that mediate or are directly involved in these destructive processes. Superoxide Dismutase is part of a group of enzymes involved in a regulatory complex of inflammation. Low antioxidant levels are a risk factor for inflammation because they protect cartilage from ROS. Prostaglandin E2 is involved, activating pathway of inflammation in Osteoarthritis and we show the way PgE2 can switch the expression of genes dependent on the activation of the transcription factors inhibiting the production of SOD ULA-CDCHTA PROYECTO M-987-10-03-B