Browsing by Subject "COX-2"
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Item GLUCOCORTICOID SUPPRESSION OF COX-2 EXPRESSION IN HYPOTHALAMIC NEURONAL CELL LINE(2013-04-12) Stacey, WinfredPurpose: 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.Item Mechanisms by which 17β-Estradiol (E2) suppress neuronal cox-2 expression(2015-12-01) Stacey, Winfred; Rosalie M. Uht; Rebecca L. Cunningham; Eric B. GonzalesData from animal models indicate that 17β-estradiol (E2) deprivation increases susceptibility to neurodegenerative diseases. E2 attenuates inflammatory response by suppressing expression of pro-inflammatory genes; however, the mechanisms by which E2 suppress neuronal pro-inflammatory genes are not well established. Histological analyses of postmortem human brains suggest that neuronal cyclooxygenase-2 (COX-2) is upregulated in early stages of Alzheimer’s disease (AD) and in Parkinson’s disease (PD). Given that COX-2 is selectively expressed in a subset of neurons in the hippocampus, cerebral cortex, and amygdala, we investigated mechanisms by which E2 could down-regulate cox-2 expression in a neuronal system. To characterize the effect of E2 on cox 2 in a neuronal system, we used the AR-5 and N27 rat neuronal cell line models. Our data indicate that E2 and ERβ agonist diarylpropionitrile (DPN) suppress COX-2 pre-mRNA and mRNA levels to the same extent in AR-5 but not in N27. Furthermore, PHTPP, a selective ERβ antagonist, reversed the effect of both E2 and DPN in AR-5. Because the cox-2 promoter lacks palindromic estrogen response elements (EREs), we targeted a proximal promoter region with a nuclear factor- ĸB (NF-ĸB) response element implicated in cox-2 regulation. E2 and DPN failed to increase ERβ occupancy at the cox-2 promoter. Rather, DPN decreased promoter occupancy of p65 NF-κB subunit and acetylation of histone 4 (Ac-H4). Treatment with the non-specific HDAC inhibitor Trichostatin A (TSA) counteracted DPN’s repressive effects on cox-2 expression. In keeping with the effect of TSA, E2 and DPN increase HDAC1 promoter occupancy; however recruitment of HDAC3 was unchanged. HDAC1 is known to form a complex with Swi-independent A (Sin3A); E2 and DPN increased Sin3A occupancy. The recruitment of HDAC1 seems to correlate with decreased acetylation of histone 4 (H4) and not histone 3 (H3). Furthermore E2 alone increased methylation status in the cox-2 proximal promoter. Taken together, these data suggest that E2 suppresses neuronal cox-2 expression through ERβ-mediated recruitment of HDAC1, Sin3A and a concomitant reduction of p65 and H4 levels. Here we conclude that E2 suppresses neuronal cox-2 expression through a mechanism that involves a combination of decreasing activator and increasing repressor recruitment to the cox-2 promoter.