The three independent experiments shown in Figure 1A were performed with different batches of LPS and BV 2 cells than the three independent Perifosine solubility experiments shown in Figure 1B. Another factor influencing the absolute response values is the composition of the culture medium. Performing the experiments shown in Figure 1B in medium with charcoal treated FBS resulted in almost two fold higher TNF mRNA expression levels, whereby the relative Inhibitors,Modulators,Libraries expression changes were comparable. As shown in Figure 1B preincubation Inhibitors,Modulators,Libraries of cells for 24 h with 25 nM cortico sterone, corresponding to low physiological glucocorti coid concentrations, resulted in a pronounced stimulation of TNF expression upon treatment with LPS. Compa rable effects, although somewhat less efficient, were observed when using 11 dehydrocorticosterone as sub strate.

This suggests a pro inflammatory effect of low en dogenous glucocorticoid concentrations and a role of the glucocorticoid activating enzyme 11B HSD1 in stimula ting inflammation. We measured IL 6 expression as an accepted read out to assess the sensitivity of exposure to LPS and subse Inhibitors,Modulators,Libraries quent activation of NF ��B. LPS induced IL 6 expres sion in a concentration dependent manner. Preincubation with 25 nM corticosterone potentiated IL 6 expression. A similar stimulation of IL 6 expression was observed upon preincubation with 50 nM 11 dehydrocorticosterone, an effect which was reversed by the two structurally distinct selective 11B HSD1 inhibitors BNW 16 and T0504. The im pact of LPS and glucocorticoids on IL 6 protein expres sion was assessed by ELISA.

Increased IL 6 protein was observed upon treatment of cells with LPS, whereby Inhibitors,Modulators,Libraries the effect on protein expression was more pronounced than the effect on mRNA levels, suggesting enhanced transla tion andor protein stability in addition to enhanced gene expression. Importantly, both preincubation with cortico sterone and 11 dehydrocorticosterone further increased IL 6 expression, whereby the effect of the latter was blocked by 11B HSD1 inhibitors. In the ab sence of LPS, incubation of BV 2 cells with 25 nM corticosterone and 50 nM 11 dehydrocorticosterone both enhanced IL 6 expression. As expected, the effect of 11 dehydrocorticosterone was abolished by 11B HSD1 inhibition.

MR and GR differentially modulate the IL 6 expression Since glucocorticoids are known as potent anti inflammatory drugs, we next determined the concentration dependence of IL 6 expression and compared the effects of 11 dehydrocorticosterone, Inhibitors,Modulators,Libraries corticosterone, and dexametha sone. The potent GR agonist dexamethasone suppressed IL 6 mRNA and protein Romidepsin solubility expression in a concentration dependent manner. Unlike the GR selective ligand dexamethasone, 11 dehydrocorticosterone showed a bi phasic response with peak stimulatory effects at about 50 nM and potent suppression at concentrations higher than 250 nM.