Inflammatory responses in many cell types are coordinately regulated by the opposing actions of NF-B and the glucocorticoid receptor (GR). The human glucocorticoid receptor (hGR) gene encodes two protein isoforms: a cytoplasmic alpha form (GR␣), which binds hormone, translocates to the nucleus, and regulates gene transcription, and a nuclear localized beta isoform (GR␤), which does not bind known ligands and attenuates GR␣ action. We report here the identification of a tumor necrosis factor (TNF)-responsive NF-B DNA binding site 5 to the hGR promoter that leads to a 1.5-fold increase in GR␣ mRNA and a 2.0-fold increase in GR␤ mRNA in HeLaS3 cells, which endogenously express both GR isoforms. However, TNF-␣ treatment disproportionately increased the steady-state levels of the GR␤ protein isoform over GR␣, making GR␤ the predominant endogenous receptor isoform. Similar results were observed following treatment of human CEMC7 lymphoid cells with TNF-␣ or IL-1. The increase in GR␤ protein expression correlated with the development of glucocorticoid resistance.

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Using microarray technology, we analyzed 12,000 genes for regulation by TNF-alpha and the synthetic glucocorticoid, dexamethasone, in the human lung epithelial cell line, A549. Only one gene was induced by both agents, the cellular inhibitor of apoptosis 2 (c-IAP2), which was induced 17-fold and 5-fold by TNF-alpha at 2 h and 24 h, respectively, and increased 14-fold and 9-fold by dexamethasone at 2 h and 24 h, respectively. The combination of the two agents together led to an additive increase (34-fold) at 2 h and a more than additive effect (36-fold) at 24 h. The human c-IAP2 promoter contains two nuclear factor (NF)-kappaB sites that have been shown to be required for transcriptional activation by TNF-alpha. To test whether glucocorticoids regulate the c-IAP2 gene at the level of the promoter, a reporter vector containing 947 bases upstream of the start site of transcription of the human c-IAP2 promoter was linked to luciferase [IAP(-947-+54)-LUC] and transfected into A549 cells. Dexamethasone and TNF-alpha each induced reporter activity, whereas the combination of the two agents led to greater induction of luciferase than either one alone. Truncation of the promoter region containing a putative glucocorticoid response element (GRE) at -515 [IAP(-395-+54)-LUC] or mutation of the GRE in the context of the natural promoter [IAP(-947-+54mutGRE)-LUC] resulted in a loss of dexamethasone-mediated induction of reporter activity. Although the functional NF-kappaB sites were retained in the truncated and mutant c-IAP2 promoter constructs, dexamethasone did not inhibit the TNF-alpha induction of luciferase activity, indicating that GR repression through the NF-kappaB sites did not occur. Regulation of the c-IAP2 gene is therefore unique, as GR and NF-kappaB signaling pathways are usually mutually antagonistic, not cooperative. Treatment of A549 cells with TNF-alpha and/or dexamethasone had no effect on cell death, but the two agents were able to inhibit interferon-gamma/anti-FAS antibody-mediated apoptosis. In human glioblastoma A172 cells, TNF-alpha and dexamethasone together elicited a greater than additive increase in c-IAP2 mRNA levels and also inhibited anti-FAS antibody-mediated A172 cell apoptosis. In contrast, in human CEM-C7 leukemic T cells, whereas TNF-alpha and dexamethasone treatment also led to an increase in c-IAP2 mRNA, the two agents were able to induce apoptosis on their own. However, TNF-alpha and dexamethasone were also able to blunt anti-FAS-induced apoptosis in the T cells. These data indicate that the induction of the antiapoptotic protein, c-IAP2, by glucocorticoids and TNF-alpha correlates with the ability of these agents to inhibit apoptosis in a variety of cell types.

1 We show that a portion of the TM2 domain regulates the sensitivity of beta subunit-containing rat neuronal nicotinic AChR to the ganglionic blocker mecamylamine, such that the substitution of 4 amino acids of the muscle beta subunit sequence into the neuronal beta4 sequence decreases the potency of mecamylamine by a factor of 200 and eliminates any long-term e ects of this drug on receptor function. 2 The same exchange of sequence that decreases inhibition by mecamylamine produces a comparable potentiation of long-term inhibition by nicotine. 3 Inhibition by mecamylamine is voltage-dependent, suggesting a direct interaction of mecamylamine with sequence elements within the membrane ®eld. We have previously shown that sensitivity to TMP (tetramethylpiperidine) inhibitors is controlled by the same sequence elements that determine mecamylamine sensitivity. However, inhibition by bis-TMP compounds is independent of voltage. 4 Our experiments did not show any in¯uence of voltage on the inhibition of chimeric receptors by nicotine, suggesting that the inhibitory e ects of nicotine are mediated by binding to a site outside the membrane's electric ®eld. 5 An analysis of point mutations indicates that the residues at the 6' position within the beta subunit TM2 domain may be important for determining the e ects of both mecamylamine and nicotine in a reciprocal manner. Single mutations at the 10' position are not su cient to produce e ects, but 6' 10' double mutants show more e ect than do the 6' single mutants.

Human nicotinic acetylcholine (ACh) receptor subtypes expressed in Xenopus oocytes were characterized in terms of their activation by the experimental agonist RJR-2403. Responses to RJR-2403 were compared with those evoked by ACh and nicotine. These agonists were also characterized in terms of whether application of the drugs had the effect of producing a residual inhibition that was manifest as a decrease in subsequent control responses to ACh measured 5 min after the washout of the drug. For the activation of ␣4␤2 receptors, RJR-2403 had an efficacy equivalent to that of ACh and was more potent than ACh. RJR-2403 was less efficacious than ACh for other human receptor subtypes, suggesting that it is a partial agonist for all these receptors. Nicotine activated peak currents in human ␣4␤2 and ␣3␤2 receptors that were 85 and 50% of the respective ACh maximum responses. Nicotine was an efficacious activator of human ␣7 receptors, with a potency similar to ACh, whereas RJR-2403 had very low potency and efficacy for these receptors. At concentrations of Ͻ1 mM, RJR-2403 did not produce any residual inhibition of subsequent ACh responses for any receptor subtype. In contrast, nicotine produced profound residual inhibition of human ␣4␤2, ␣3␤2, and ␣7 receptors with IC 50 values of 150, 200, and 150 M, respectively. Co-expression of the human ␣5 subunit with ␣3 and ␤2 subunits had the effect of producing protracted responses to ACh and increasing residual inhibition by ACh and nicotine but not RJR-2403. In conclusion, our results, presented in the context of the complex pharmacology of nicotine for both activating and inhibiting neuronal nicotinic receptor subtypes, suggest that RJR-2403 will be a potent and relatively selective activator of human ␣4␤2 receptors.

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