GLUCOCORTICOID SUPPRESSION OF COX-2 EXPRESSION IN HYPOTHALAMIC NEURONAL CELL LINE

Purpose: Glucocorticoids have long been administered as potent anti-inflammatory agents. They are secreted in response to activation of the hypothalamic-pituitary-adrenal (HPA) axis and act as regulators of the immune responses, preventing overshoot of inflammatory processes. Dysfunction of this regulatory feedback leads to inflammatory processes unchecked and has been implicated in various chronic inflammatory disorders such as rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis, all of which are associated with increased expression of pro-inflammatory genes. One pro-inflammatory gene regulated by glucocorticoids is cyclooxygenase-2 (cox-2), a key and rate-limiting enzyme in the production of prostaglandins (PGs). The mechanisms by which glucocorticoids suppress this enzyme's activity have been well studied outside of the central nervous system. However, there is limited knowledge on how glucocorticoids suppress cox-2 expression in the paraventricular nucleus of the hypothalamus (PVH). In the PVH, studies have shown that in response to inflammatory stimuli, cox-2 expression and prostaglandins levels increase which stimulate parvocellular neurons to release corticotropin-releasing hormone (CRH). CRH release ultimately results in inflammation-induced activation of the HPA axis. Uninhibited, cox-2 activity would lead to excessive levels of PGs and a hyperactive HPA response. The hypothesis for this study is, glucocorticoids suppress cox-2 expression by inhibiting Nuclear Factor-𝛋B(NF-kB) transcriptional activity in hypothalamic IVB neuronal cell line Methods: To characterize the model, a hypothalamic IVB cell line was analyzed for cox-2 expression following stimulation with Phorbol 12-Myristate 13-Acetate (PMA) for 30m, 60m, and 120m. A dose response curve was generated using 1µm, 0.3µm, 0.1µm, 30nm, 10nm, and 1nm of PMA. COX-2 mRNA expression was analyzed using Real time polymerase chain reaction (RT-PCR). COX-2 protein levels were probed by western blot. COX-2 immunoreactive cells were analyzed using Immunocytochemistry (ICC). Cells were treated with 10-7M dexamethasone and COX-2 mRNA, protein levels and immunoreactive cells were analyzed. Results: COX-2 mRNA, protein levels and immunoreactivity increased following PMA treatment of hypothalamic IVB neuronal cells. Dexamethasone (Dex) treatment decreased COX-2 mRNA, protein levels as well as COX-2ir cells Conclusions: PMA treatment induces cox-2 expression and dex treatment has an inhibitory effect on its expression.