Development of cell – based model for NFAT (Nuclear factor of activated T-cell) nuclear – cytoplasm translocation and its application for immunomodulator screening

Abstract

Nuclear factor of activated T-cells (NFATs) together with calcium ion play an important role in immune reactions. The NFAT family encodes four distinct classes of proteins including NFAT1 (NFATp), NFAT2 (NFATc), NFAT3 and NFAT4 (NFATx). The objective of this study is to develop and validate the standard procedure for investigation NFAT4 and NFAT3 nuclear-cytoplasm translocation for bioactive immuno-modulator screening.

NFAT4 (N) and NFAT3 (N) genes were isolated from the library, GFP-fusion recombined, pcDNA 3.1 vector-ligated and propagated in DH5α strain E.coli. Human Embryonic Kidney (HEK) cells and primary porcine skin fibroblasts were cultured in modified Eagle’s medium containing 5% fetal bovine serum and supplemented with 100U/ml penicillin and 100μg/ml streptomycin. The vectors containing GFP as a negative control, GFP-NFAT3(N) as a dominant negative control, and GFP-NFAT4(N) were transfected into HEK cells and primary porcine skin fibroblasts using Lipofectamine 2000 (Invitrogen). The transfected cells were cultured for 24 h allowing protein expression. The GFP and NFAT fusion proteins nuclear-cytoplasm translocation patterns were studied by adding 1μM of calcium ionophore (A23187) with/without 1 and 5μM cyclosporine A (CsA). The fluorescence images from GFP were captured and analyzed with an inverted fluorescent microscope (Carl Zeiss). The kinetic of A23187 (stimulation) and CsA (inhibition) were intensively studied. Furthermore, this cell-base model was applied for determining the NFAT pathway inhibitory effect of curcumin (Curcuma longa) and andrographolide (Andrographis paniculata) extracts. The previous studies reported that both extracts could inhibit interleukin 2 production induced by T-cell activation.

The fluorescent signal from the GFP transfected cells is diffused throughout the cells both with and without cytosolic calcium induction by A23187, whereas the GFP-NFAT4(N) transfected cells significantly showed the translocation of the signal from cytoplasm to nucleus. The GFP-NFAT4(N) nuclear translocation, however, was significantly inhibited by 1 and 5μM CsA. As a dominant negative, the fluorescent signal from the GFP-NFAT3(N) transfected cells remained predominantly in the cytosol even after A23187 stimulation. NFAT nuclear translocation kinetic studies of A23187 and CsA were performed. The results suggested that the stimulation of NFAT nuclear translocation by A23187 was transient and reversible, whereas the inhibitory effect of NFAT nuclear translocation by CsA was last longer than 24 hours and irreversible. Using competitive kinetic study, the results confirmed that A23187 and CsA did not compete at the same effective site of calcineurin, and CsA was the non-competitive irreversible inhibitor for calcineurin enzyme. Our study confirmed that both curcumin and anndrographolide extracts cold not inhibit or stimulate NFAT nuclear translocation, indicating both substances may exert their pharmacologic action by other pathways, but not the NFAT pathway, such as c-jun transcription factor and NF- ϰB activation.

By induction of intracellular calcium concentrations in HEK cells and primary porcine skin fibroblasts transfected with either NFAT4 (N) or NFAT3 (N), demonstrated a specific patterns of cytoplasm-nuclear NFATs-protein translocation. Potentially, the findings from this study can be applied for the development of a standard cell-based method for screening of bioactive immuno-modulators signaling through NFATs pathway.

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