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Function of partially duplicated human ?77 nicotinic receptor subunit CHRFAM7A gene: potential implications for the cholinergic anti-inflammatory response.

ABSTRACT: 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 anti-inflammatory response.

SUBMITTER: de Lucas-Cerrillo AM

PROVIDER: S-EPMC3013019 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Function of partially duplicated human α77 nicotinic receptor subunit CHRFAM7A gene: potential implications for the cholinergic anti-inflammatory response.

de Lucas-Cerrillo Ana M AM Maldifassi M Constanza MC Arnalich Francisco F Renart Jaime J Atienza Gema G Serantes Rocío R Cruces Jesús J Sánchez-Pacheco Aurora A Andrés-Mateos Eva E Montiel Carmen C

The Journal of biological chemistry 20101103 1

The neuronal α7 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, dupα7, has been identified in brain, immune cells, and the HL-60 cell line, although its translation and function are still unknown. In this study, dupα7 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 t ...[more]

PMID: 21047781

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Project description:ObjectiveMuscle acetylcholine receptors have five alpha subunits (α, β, δ, ε, or γ), and cholinergic receptor nicotinic gamma subunit (CHRNG) is the γ subunit. It may also play an essential role in biological processes, including cell differentiation, growth, and survival, while the role of CHRNG has not been studied in the literature. Therefore, the purpose of this study is to clarify the effect of CHRNG on the proliferation and differentiation of bovine preadipocytes.MethodsWe constructed a CHRNG overexpression adenovirus vector and successfully overexpressed it on bovine preadipocytes. The effects of CHRNG on bovine preadipocyte proliferation were detected by Edu assay, cell counting Kit-8 (CCK-8), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), Western blot and other techniques. We also performed oil red O, RT-qPCR, Western blot to explore its effect on the differentiation of preadipocytes.ResultsThe results of Edu proliferation experiments showed that the number of EDU-positive cells in the overexpression group was significantly less. CCK-8 experiments found that the optical density values of the cells in the overexpression group were lower than those of the control group, the mRNA levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin D2 (CCND2) decreased significantly after CHRNG gene overexpression, the mRNA levels of cyclin dependent kinase inhibitor 1A (CDKN1A) increased significantly, and the protein levels of PCNA, CCNB1, CCND2 decreased significantly. Overexpression of CHRNG inhibited the differentiation of bovine preadipocytes. The results of oil red O and triglyceride determination showed that the size and speed of lipid droplets accumulation in the overexpression group were significantly lower. The mRNA and protein levels of peroxisome proliferator activated receptor gamma (PPARγ), CCAAT enhancer binding protein alpha (CEBPα), fatty acid binding protein 4 (FABP4), fatty acid synthase (FASN) decreased significantly.ConclusionOverexpression of CHRNG in bovine preadipocytes inhibits the proliferation and differentiation of bovine preadipocytes.

Project description:BACKGROUND:The identification of variants in the nicotinic acetylcholine receptor (nAChR) subunit genes associated with smoking phenotypes are increasingly important for prevention and treatment of nicotine dependence. In the context of personalized medicine, the aims of this study were to evaluate whether cholinergic receptor nicotinic alpha 2 (CHRNA2), cholinergic receptor nicotinic alpha 3 (CHRNA3), cholinergic receptor nicotinic alpha 5 (CHRNA5) and cholinergic receptor nicotinic beta 3 (CHRNB3) polymorphisms were associated with nicotine dependence severity, and to investigate possible pharmacogenetics markers of smoking cessation treatment. METHODS:This study cohort enrolled 1049 smoking patients who received pharmacological treatment (varenicline, varenicline plus bupropion, bupropion plus/or nicotine replacement therapy). Smoking cessation success was considered for patients who completed 6 months of continuous abstinence. Fagerström test for nicotine dependence (FTND) and Issa situational smoking scores (Issa score) were analyzed for nicotine dependence. CHRNA2 (rs2472553), CHRNA3 (rs1051730), CHRNA5 (rs16969968 and rs2036527) and CHRNB3 (rs6474413) polymorphisms were genotyped by high resolution melting analysis. RESULTS:Females with GA and AA genotypes for CHRNA5 rs16969968 and rs2036527 polymorphisms had higher success rate in smoking cessation treatment: 44.0% and 56.3% (rs16969968), 41.5% and 56.5% (rs2036527), respectively, compared with carriers of the GG genotypes: 35.7% (rs16969968), 34.8% (rs2036527), (P?=?0.03, n?=?389; P?=?0.01, n?=?391). The GA or AA genotypes for the rs16969968 and rs2036527 were associated with higher odds ratio for success in women (OR?=?1.63; 95% CI?=?1.04 to 2.54; P?=?0.03 and OR?=?1.59, 95% CI?=?1.02 to 2.48; P?=?0.04; respectively). We did not find association of these polymorphisms with nicotine dependence related scores. Polymorphisms in the CHRNA2, CHRNA3 and CHRNB3 genes were not associated with the phenotypes studied. CONCLUSION:CHRNA5 rs16969968 and rs2036527 were associated with higher success rate in the smoking cessation treatment in women. These findings might contribute to advances in personalized medicine.

Dataset Information

Function of partially duplicated human ?77 nicotinic receptor subunit CHRFAM7A gene: potential implications for the cholinergic anti-inflammatory response.

Publications

Function of partially duplicated human α77 nicotinic receptor subunit CHRFAM7A gene: potential implications for the cholinergic anti-inflammatory response.

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