Establishment of a cell model targeted to NFAT signal transduction pathway for preliminary screening of FK506-like immunosuppressants.
- Author:
He XIAO
1
;
Lu QIAN
;
Wei-Song QIN
;
Song LI
;
Bei-Fen SHEN
;
Yan LI
Author Information
1. Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Drug Evaluation, Preclinical;
Enhancer Elements, Genetic;
genetics;
Green Fluorescent Proteins;
genetics;
Humans;
Immunosuppressive Agents;
pharmacology;
Interleukin-2;
genetics;
Jurkat Cells;
Luciferases;
genetics;
metabolism;
Mice;
Models, Biological;
NFATC Transcription Factors;
genetics;
metabolism;
Promoter Regions, Genetic;
Signal Transduction;
Tacrolimus;
pharmacology
- From:
Chinese Journal of Biotechnology
2005;21(5):759-765
- CountryChina
- Language:Chinese
-
Abstract:
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.
