Gene regulation of α4β2 nicotinic receptors: microarray analysis of nicotine-induced receptor up-regulation and anti-inflammatory effects (original) (raw)
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Molecular Pharmacology, 2011
Despite evidence that smoking confers protection against neurological disorders, how and whether specific nicotinic receptor subtypes are involved is unknown. We reported previously that nicotine suppresses constitutive nuclear factor B (NF-B) activity and thereby proinflammatory cytokine (PIC) production in SHEP1 cells stably transfected with ␣42 nicotinic receptors. Here, we report the anti-inflammatory effects of nicotine pretreatment in lipopolysaccharide (LPS)-stimulated SHEP1 cells. Nicotine (100 -300 nM, concentrations found in smoker's blood) blocked LPS-induced NF-B translocation and production of PICs interleukin (IL)-1 and IL-6 but only partially blocked inhibitor of nuclear factor-B␣ (IB␣) phosphorylation. These effects were exclusively in cells transfected with ␣42 receptors but not in wild types. The cell-permeable calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid-acetoxymethyl ester, the adenylate cyclase stimulant for-skolin, and a specific protein kinase A (PKA) inhibitor PKI 14-22-amide failed to block the effect of nicotine on LPS-induced NF-B translocation and IB␣ phosphorylation. However, the effects of nicotine on NF-B activity were significantly blocked by the highly specific janus kinase 2 (JAK2) inhibitor ␣-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490) and the signal transducer and activator of transcription 3 (STAT3) inhibitor 2-hydroxy-4- [[[[(4-methylphenyl)sulfonyl]oxy]acetyl]amino]benzoic acid (NSC74859). These findings reveal a calcium-and cAMP-PKA-independent signaling cascade and suggest a role for JAK2-STAT3 transduction in ␣42-mediated attenuation of LPS-induced inflammation. Anti-inflammatory effects of nicotine may therefore be mediated through ␣42 receptors, the predominant high-affinity binding sites for nicotine in the central nervous system, in addition to the better-established ␣7 receptors.
PLoS ONE, 2014
Nicotine stimulation of a7 nicotinic acetylcholine receptor (a7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the a7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-kB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLRmediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to a7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-a production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-a response to an additional LPS challenge, a behavior reminiscent of the 'endotoxin tolerant' phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.
Function of Partially Duplicated Human α7 Nicotinic Receptor Subunit CHRFAM7A Gene
Journal of Biological Chemistry, 2010
The neuronal ␣7 nicotinic receptor subunit gene (CHRNA7) is partially duplicated in the human genome forming a hybrid gene (CHRFAM7A) with the novel FAM7A gene. The hybrid gene transcript, dup␣7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dup␣7 cDNA has been cloned and expressed in GH4C1 cells and Xenopus oocytes to study the pattern and functional role of the expressed protein. Our results reveal that dup␣7 transcript was natively translated in HL-60 cells and heterologously expressed in GH4C1 cells and oocytes. Injection of dup␣7 mRNA into oocytes failed to generate functional receptors, but when co-injected with ␣7 mRNA at ␣7/dup␣7 ratios of 5:1, 2:1, 1:1, 1:5, and 1:10, it reduced the nicotine-elicited ␣7 current generated in control oocytes (␣7 alone) by 26, 53, 75, 93, and 94%, respectively. This effect is mainly due to a reduction in the number of functional ␣7 receptors reaching the oocyte membrane, as deduced from ␣-bungarotoxin binding and fluorescent confocal assays. Two additional findings open the possibility that the dominant negative effect of dup␣7 on ␣7 receptor activity observed in vitro could be extrapolated to in vivo situations. (i) Compared with ␣7 mRNA, basal dup␣7 mRNA levels are substantial in human cerebral cortex and higher in macrophages. (ii) dup␣7 mRNA levels in macrophages are down-regulated by IL-1, LPS, and nicotine. Thus, dup␣7 could modulate ␣7 receptor-mediated synaptic transmission and cholinergic antiinflammatory response.
Clinical and Experimental Immunology, 2006
Summary Macrophages/monocytes and the proinflammatory mediators, such as tumour necrosis factor (TNF)-α, prostaglandin E2 (PGE2), macrophage inflammatory protein (MIP)-1α and MIP-1α, play a critical role in the progression of immunological disorders including rheumatoid arthritis, Behçet’s disease and Crohn’s disease. In addition, the nicotinic acetylcholine receptor-α7 (α7nAChR) subunit is an essential regulator of inflammation. In this study, we evaluated the expression of the α7nAChR subunit on human peripheral monocytes and the effect of nicotine on the production of these proinflammatory mediators by activated monocytes. Fluorescein isothiocyanate (FITC)-labelled α-bungarotoxin demonstrated the cell surface expression of the α7nAchR subunit. Pretreatment with low-dose nicotine caused inhibition of TNF-α, PGE2, MIP-1α and MIP-1α production, and mRNA expression of TNF-α, MIP-1α and MIP-1α and COX-2 in lipopolysaccharide (LPS)-activated monocytes. These suppressive effects of nico...
Molecular Pharmacology, 2010
Despite evidence that smoking confers protection against neurological disorders, how and whether specific nicotinic receptor subtypes are involved is unknown. We reported previously that nicotine suppresses constitutive nuclear factor B (NF-B) activity and thereby proinflammatory cytokine (PIC) production in SHEP1 cells stably transfected with ␣42 nicotinic receptors. Here, we report the anti-inflammatory effects of nicotine pretreatment in lipopolysaccharide (LPS)-stimulated SHEP1 cells. Nicotine (100 -300 nM, concentrations found in smoker's blood) blocked LPS-induced NF-B translocation and production of PICs interleukin (IL)-1 and IL-6 but only partially blocked inhibitor of nuclear factor-B␣ (IB␣) phosphorylation. These effects were exclusively in cells transfected with ␣42 receptors but not in wild types. The cell-permeable calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid-acetoxymethyl ester, the adenylate cyclase stimulant for-skolin, and a specific protein kinase A (PKA) inhibitor PKI 14-22-amide failed to block the effect of nicotine on LPS-induced NF-B translocation and IB␣ phosphorylation. However, the effects of nicotine on NF-B activity were significantly blocked by the highly specific janus kinase 2 (JAK2) inhibitor ␣-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG-490) and the signal transducer and activator of transcription 3 (STAT3) inhibitor 2-hydroxy-4- [[[[(4-methylphenyl)sulfonyl]oxy]acetyl]amino]benzoic acid (NSC74859). These findings reveal a calcium-and cAMP-PKA-independent signaling cascade and suggest a role for JAK2-STAT3 transduction in ␣42-mediated attenuation of LPS-induced inflammation. Anti-inflammatory effects of nicotine may therefore be mediated through ␣42 receptors, the predominant high-affinity binding sites for nicotine in the central nervous system, in addition to the better-established ␣7 receptors.
The Journal of Immunology, 2010
Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC 4 . Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a highaffinity receptor for IgE (Fc«RI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using a-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) a7, a9, and a10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for ‡8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A 2 activity and the PI3K/ERK/NF-kB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kB. The suppressive effect of nicotine on the late-phase response was blocked by the a7/a9-nAChR antagonists methyllycaconitine and a-bungarotoxin, as well as by small interfering RNA knockdown of a7-, a9-, or a10-nAChRs, suggesting a functional interaction between a7-, a9-, and a10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.
British journal of pharmacology, 2017
Genomic analysis has shown many variants in both CHRNA4 and CHRNB2, genes which encode the α4 and β2 subunits of nicotinic ACh receptors (nAChR) respectively. Some variants influence receptor expression, raising the possibility that CHRNA4 variants may affect response to tobacco use in humans. Chronic exposure to nicotine increases expression of nAChRs, particularly α4β2-nAChRs, in humans and laboratory animals. Here, we have evaluated whether the initial level of receptor expression affects the increase in expression. Mice differing in expression of α4 and/or β2 nAChR subunits were chronically treated with saline, 0.25, 1.0 or 4.0 mg·kg(-1) ·h(-1) nicotine. Brain preparations were analysed autoradiographically by [(125) I]-epibatidine binding, immunoprecipitation and Western blotting. Immunochemical studies confirmed that most of the [(3) H]-epibatidine binding corresponds to α4β2*-nAChR and that increases in binding correspond to increases in α4 and β2 proteins. Consistent with pr...
Up-regulation of Nicotinic Receptors by Nicotine Varies with Receptor Subtype
Journal of Biological Chemistry, 2008
α4β2 and α3-containing nicotinic receptors, α6containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that α6β2, like α3β2 and α4β2 receptors, formed high-affinity, epibatidine-binding complexes that are pentameric, trafficked to the cell surface and produced acetylcholine-evoked currents. Chronic nicotine exposure upregulated α6β2 receptors with differences in upregulation time course and concentration-dependence compared to α4β2 receptors, the predominant high-affinity nicotine binding site in brain. The α6β2 receptor upregulation required higher http://www.jbc.org/cgi/
Nicotine and acetylcholine lead to distinct modulation of gene regulation
Background: Sepsis is well known to lead to the activation of multiple pro-inflammatory markers, like MCP-1 (Monocyte chemotactic protein 1), TNF-alpha (Tumor necrosis factor alpha), while the underlying genetic changes still remain poorly studied.Methods: We used human umbilical vein endothelial cells to test the reactions to nicotine or acetylcholine/pyridostigmine administration in regards to MCP-1 levels, gene regulation and RNR expression. Results: Pyridostigmine and Acetylcholine (Ach) lead to a significant decrease of MCP-1 levels in TNF-alpha stimulated human umbilical vein endothelial cells, while nicotine had no effect. Interestingly nicotine and acetylcholine lead to different gene expression (nicotine up-regulates epidermal growth factor and down-regulates matrix metalloproteinase-8, while Ach/pyridostigmine up-regulates thioredoxin interacting protein and down-regulates insulin like growth factor 1). Furthermore RNA levels and gene activation were similar after nicotine...
Differential modulation of EAE by α9*‐ and β2*‐nicotinic acetylcholine receptors
Immunology & Cell Biology, 2013
Nicotine is a potent inhibitor of the immune response and is protective against experimental autoimmune encephalomyelitis (EAE). Initial studies suggested that the cholinergic system modulates inflammation via the α7‐nicotinic acetylcholine receptor (nAChR) subtype. We recently have shown that effector T cells and myeloid cells constitutively express mRNAs encoding nAChR α9 and β2 subunits and found evidence for immune system roles for non‐α7‐nAChRs. In the present study, we assessed the effects of nAChR α9 or β2 subunit gene deletion on EAE onset and severity, with or without nicotine treatment. We report again that disease onset is delayed and severity is attenuated in nicotine‐treated, wild‐type mice, an effect that also is observed in α9 subunit knock‐out (KO) mice irrespective of nicotine treatment. On the other hand, β2 KO mice fail to recover from peak measures of disease severity regardless of nicotine treatment, despite retaining sensitivity to nicotine's attenuation of...