Overexpressed alpha7 nicotinic acetylcholine receptor inhibited proinflammatory cytokine release in NIH3T3 cells.
Alpha-7 nicotinic acetylcholine receptor (alpha7nAChR) played an important role during the process of inflammation, and was a potential therapeutic target for many diseases, including Alzheimer's disease, endotoxaemia, hypertension etc. However, it was difficult to express alpha7nAChR on commonly used host cells because of the lack of the chaperone protein RIC-3, which was indispensable to promote folding, assembly, and surface expression of alpha7nAChR. This work was designed to develop a cell line, which not only expressed alpha7nAChR highly and stably, but possessed biological activity. Full-length alpha7nAChR gene was extracted from rat peritoneal macrophages, recombinant plasmid pIRES2-EGFP-alpha7nAChR was constructed, and then transfected to the mouse fibroblast cell line NIH3T3, a cell line expressing RIC-3. Compared with the NIH3T3-pIRES2-EGFP, expression of alpha7nAChR was significantly higher in NIH3T3-pIRES2-EGFP-alpha7nAChR on both mRNA and protein levels. To evaluate the anti-inflammation activity of the over-expressed alpha7nAChR, TNF-alpha, IL-6, and IL-1beta release induced by lipopolysaccharide was determined. No changes were found between NIH3T3-pIRES2-EGFP and NIH3T3-pIRES2-EGFP-alpha7nAChR culture supernatant under LPS challenge, however under nicotine pre-stimulation, pro-inflammatory cytokine release was significantly attenuated in alpha7nAChR transfected cell line. Moreover, in this established cell line, PNU-282987, a selective alpha7nAChR agonist, exerted a stronger ability to reduce TNF-alpha release than nicotine. We concluded that a functional NIH3T3-pIRES2-EGFP-alpha7nAChR cell line was developed, which might be useful for agonist screen and biological research.
Li
Dong-Jie
DJ
School of Life Science and Technology, China Pharmaceutical University, Nanjing, Jiangsu 210009, China.
Tang
Qiu
Q
Shen
Fu-Ming
FM
Su
Ding-Feng
DF
Duan
Jun-Li
JL
Xi
Tao
T
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis*; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D018836:Inflammation Mediators / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis*; D064569:alpha7 Nicotinic Acetylcholine Receptor
D001549:Benzamides; D001643:Bridged Bicyclo Compounds; C579166:Chrna7 protein, mouse; C579165:Chrna7 protein, rat; D016207:Cytokines; D018836:Inflammation Mediators; D018722:Nicotinic Agonists; C498513:PNU-282987; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D009538:Nicotine