[Establishment of a cell model targeted to NFAT signal transduction pathway for preliminary screening of FK506-like immunosuppressants].
To screen NFAT antagonistic drugs and research signal transduction pathway related to NFAT. Four recombinant vectors were constructed. Each consists of three tandem copies of the human IL-2 distal NFAT-AP1 binding site in the context of the minimal IL-2 enhancer, either the sequence from -326 - +46 or the sequence from -89 - +46 (containing only the TATA box), driving a luciferase reporter gene or a destabilized enhanced green fluorescence protein (d2EGFP) reporter gene, respectively. Transient transfection of Jurkat cells was achieved by electroporation with 5 - 10 microg of the above plasmid and one pulse at 200V, 65ms. Plasmid pEFBos-mNFAT1 constitutively expressing murine full length NFAT1 protein was used for transient cotransfection. The results showed that neither of non-stimulation nor PMA or ionomycin stimulation alone could activate the reporter gene except PMA plus ionomycin costimulation. Furthermore, overexpressed murine NFAT1 augmented the activation of either IL-2 promoter or NFAT-AP1 enhancer drived reporter gene compared to the endogenous did. However, the reporter gene expression was nearly completely inhibited by pretreatment for 1h with FK506 at 5 microg/mL and then stimulation for 6-12h with PMA plus ionomycin in the presence of FK506. These findings indicated that such a transient Jurkat cell model offered a potential platform for preliminary screening of FK506 or CsA-like immunosuppressive agents.
Xiao
He
H
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
Qian
Lu
L
Qin
Wei-Song
WS
Li
Song
S
Shen
Bei-Fen
BF
Li
Yan
Y
D000818:Animals; D004353:Drug Evaluation, Preclinical; D004742:Enhancer Elements, Genetic / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007376:Interleukin-2 / Q000235:genetics; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D016559:Tacrolimus / Q000494:pharmacology*
D007166:Immunosuppressive Agents; D007376:Interleukin-2; D050778:NFATC Transcription Factors; C494007:Nfatc2 protein, mouse; D049452:Green Fluorescent Proteins; D008156:Luciferases; D016559:Tacrolimus