NFAT but not NF-kappaB is critical for transcriptional induction of the prosurvival gene A1 after IgE receptor activation in mast cells

Abstract

FcepsilonRI-activation-induced survival of mast cells is dependent on the expression and function of the prosurvival protein A1. The expression of A1 in lymphocytes and monocytes has previously been described to be transcriptionally regulated by NF-kappaB. Here we demonstrate that the expression of A1 in mast cells is not dependent on NF-kappaB but that NFAT plays a crucial role. FcepsilonRI-induced A1 expression was not affected in mast cells overexpressing an IkappaB-alpha super-repressor or cells lacking NF-kappaB subunits RelA, c-Rel, or c-Rel plus NF-kappaB1 p50. In contrast, inhibition of calcineurin and NFAT by cyclosporin A abrogated the expression of A1 in mast cells on FcepsilonRI-activation but had no effect on lipopolysaccharide-induced expression of A1 in J774A.1 monocytic cells. Cyclosporin A also inhibited luciferase expression in an A1 promoter reporter assay. A putative NFAT binding site in the A1 promoter showed inducible protein binding after FcepsilonRI crosslinking or treatment with ionomycin as detected in a band shift assay or chromatin immunoprecipitation. The binding protein was identified as NFAT1. Finally, mast cells expressing constitutively active NFAT1 exhibit increased expression of A1 after FcepsilonRI-stimulation. These results indicate that, in FcepsilonRI stimulated mast cells, A1 is transcriptionally regulated by NFAT1 but not by NF-kappaB.

Figure 1

IgE receptor crosslinking induces mast cell survival and A1 expression. (A) Murine BMMCs, cultured under IL-3 deprivation, show enhanced survival when activated by IgE receptor crosslinking (IgER CL). (B) RNase protection assay (RPA) shows an increased expression of A1 in both BMMC and the mouse mast cell line C57 on IgER CL. Data are presented as mean plus or minus SEM of 3 independent experiments.

Figure 2

A1 induction is intact in NF-κB–deficient mast cells. (A,B) Reverse transcriptase PCR (RT-PCR) analysis of A1 expression in BMMCs deficient for 3 NF-κB subunits c-Rel (c-rel^−/−) or c-Rel plus NF-κB1 p50 (c-rel^−/−nfκb1^−/−) (A) or RelA (rela^−/−) (B) shows strong induction of A1 after IgER CL or ionomycin stimulation. (C) IgER CL increases A1 mRNA levels in C57 cells stably transfected with the nondegradable NF-κB super-repressor, IκB-α SR. Western blot analysis (WB) of cytosolic and nuclear extracts reveal intact IκB levels after IgER CL and an active IκB-α SR. The IκB-α SR is of human origin and runs at a slower mobility. (D) EMSA shows less protein-DNA complex of nuclear RelA and the oligonucleotide probe containing an RelA binding site, from C57 stable transfected with an active IκB-α SR compared with C57 cell transfected with an empty vector. Probing for GAPDH and ERK was used as loading controls in RT-PCR and Western blot analyses, respectively. RT-PCR experiments in panels A, B, and C were performed 3 times with similar results, whereas the Western blots and EMSA in panels C and D were performed twice and once, respectively.

Figure 3

A1 induction is abrogated by the calcineurin inhibitor cyclosporin A specifically in mast cells. BMMCs (A) and the mast cell line C57 (B) show increased expression of A1 mRNA when activated by IgER CL. The calcineurin inhibitor cyclosporin A abrogated the induction of A1. (C) The inhibitory effect of cyclosporin A on A1 induction was not observed in the macrophage cell line J774A.1 activated by LPS. Expression of mRNA was analyzed by RNase protection assays (RPA). Probing for L32 and Gapdh was used as a control. The data are from one (B) and 2 (A, C) independent experiments. Results have also been confirmed by RT-PCR analysis, which was repeated 3 times (data not shown).

Figure 4

An A1 promoter, lacking the NF-κB site, can still be activated by IgE receptor crosslinking or treatment with the calcium ionophore ionomycin. (A) Schematic view of the 2 vectors with luciferase as a reporter, containing upstream regions of A1. The −389 pA1 Luc does not contain the NF-κB binding site. (B) C57 cells transiently transfected with −1996 pA1 Luc or −389 pA1 Luc show a 3- to 5-fold increase in luciferase activity after IgE receptor crosslinking or treatment with ionomycin (1 μM). (C) The increased luciferase activity was abrogated by the addition of cyclosporin A. Data represent mean plus or minus SEM from 2 or 3 independent experiments.

Figure 5

An NFAT-containing protein complex binds to a putative NFAT site in the A1 proximal promoter. (A) Sequences of the wild-type (wt) and mutated (mut) oligonucleotide probes used in the EMSA with the putative binding site for NFAT indicated in bold and the 3 base pair mutation in italics. (B) EMSA on nuclear extracts from C57 cells activated by IgE receptor crosslinking or treatment with ionomycin (1 μM). Activation of C57 cells results in an increased binding of a nuclear protein complex to the oligonucleotide probe shown in panel A. This complex formation is abrogated by pretreatment with unlabeled competitive wt but not mutated oligonucleotide probe. Mutated oligonucleotide probe does not form binding complex at all. (C) The augmented complex binding from activated C57 is inhibited by pretreatment of the cells with cyclosporin A. (D) NFAT-specific antibodies (anti-NFAT = NFATc1; anti-NFAT1 = NFATc2) cause a band shift in the EMSA, indicating the presence of NFAT within the DNA-protein complex. Representative results from 2 independent experiments are shown.

Figure 6

NFAT1-containing protein complexes bind a promoter region of A1 on activation in mast cells but not the macrophage cell line J774A.1. (A) ChIP reveals an increase of NFAT1-containing protein complexes bound to the A1 promoter in C57 cells stimulated with ionomycin for 3 hours compared with unstimulated cells. The binding is inhibited by pretreatment (10 minutes) of the cells with cyclosporin A before stimulation with ionomycin. Chromatin fragments were prepared and immunoprecipitated with antibodies against NFAT1, control IgG (negative control), and RNA polymerase II (positive control). The immunoprecipitated DNA and input DNA were analyzed by PCR using specific primer pairs for A1 resulting in PCR products covering the sequence shown in Figure 5A, and for the promoter of IL-13. Shown is a representative of 3 independent experiments. (B) Activation of the macrophage cell line J774A.1 by LPS for 3 hours increases complex formation of the NF-κB subunit RelA and chromatin of the A1 promoter region. On the contrary, the NFAT1-specific antibody does not pull down any chromatin bound protein complexes in J774A.1 but in ionomycin-activated C57 cells.

Figure 7

Expression of constitutively active NFAT1 increases A1 mRNA levels in FcϵRI-stimulated mast cells. (A) RT-PCR analysis was used to determine the levels of A1 mRNA in C57 cells transiently transfected with vectors expressing constitutively active NFAT1, NFAT2, or empty vector. The cells were left untreated or were activated by FcϵRI crosslinking. Amplification of GAPDH was used as control. Overexpression of NFAT1, but not NFAT2, resulted in increased levels of A1 mRNA. Representative results from 2 independent experiments are shown. (B) Real-time quantitative PCR analysis of A1 mRNA shows increased levels of A1 expression in C57 cells expressing constitutively active NFAT1 compared with cells transfected with empty vector. Data are presented as mean plus or minus SEM from 3 independent experiments.