( New PathDetect reporting...)Cis-Reporting System Monitors Intracellular Calcium Mobilization

New PathDetect reporting system for studying NFAT activation

Li Xu Matthew Hsu Mary Buchanan Chao-Feng Zheng
Stratagene

Stratagenes new PathDetect cis-reporter plasmid, the pNFAT-Luc reporter plasmid, is designed for studying the activation of the nuclear factor of activated T-cell (NFAT) transcription factors. These interesting proteins, which play an essential role in inducible gene expression during immune responses, are activated by stimuli that increase free calcium levels in the cytoplasm. NFAT transcription factors are studied intensively as potential targets for newer and safer immunosuppressive drugs and for assessing cellular signal transduction pathways that are responsive to calcium mobilization.

Proteins that comprise the family of transcription factors known as nuclear factor of activated T cells play an essential role in inducible gene expression during immune responses. Although first identified in T cells, these proteins have also been detected in many other tissues and cell types. NFAT transcription factors are regulated by calcineurin, a calcium/calmodulin-dependent phosphatase. NFAT proteins, as substrates for calcineurin, mediate the effects of cyclosporin A (CsA) and FK506, two immunosuppresive drugs that revolutionized transplant surgery in 1983.¹

Members of the NFAT family are divided according to primary structure homology into four subgroups: NFAT1 (1A, 1B, and 1C), NFAT2 (NFATc.a and NFATc.b), NFAT3, and NFAT4 (4a, 4b, 4c, and NFATx). NFAT proteins vary in size from NFAT4a (708 amino acid residues) to NFATx (1075 amino acid residues). Two major regions of homology exist among these subgroups: the DNA-binding domain (dbd) and the NFAT homology region (NHR). The dbd is positioned within amino acid residues ~400 and ~700 in the known isoforms of NFAT and shows some similarity to the dbd of Rel-family transcription factors.¹ The NHR is about 300 amino acid residues in length and is located N-terminal to the dbd. Although the dbd sequences of different isoforms of NFAT proteins have distinct affinities, they bind to a consensus sequence of T(A)GGAAAAT(A/C).^1,2 This site is present in many genes involved in immune responses, including interleukin-2 (IL-2), IL-4, IL-5, granular macrophage colony-stimulating factor (GM-CSF), IFN-g, and IL-13. In many cases, for DNA binding and transactivation, NFAT proteins cooperate with AP-1, which binds c-Jun/fos dimers. Complexes formed from cooperative binding of NFAT proteins, AP-1, and DNA are significantly more stable than complexes of NFAT and DNA alone.^3,4

In resting cells, NFAT proteins are phosphorylated and reside in the cytoplasm. Stimuli that increase calcium mobilization result in the activation of NFAT proteins. The rapid dephosphorylation and translocation of NFAT proteins to the nucleus characterize activation; upon translocation, the dephosphorylated proteins show higher affinity for DNA. CsA or FK506 can block each step in the activation of NFAT proteins. Therefore, NFAT activation follows precisely the activation of calcineurin by increased free calcium levels in the cytoplasm.^5,6 Researchers from diverse fields are interested in the NFAT family of transcription factors, which are potential targets for newer and safer immunosuppressive drugs.¹ In addition, the activation of NFAT proteins involves various cellular signal transduction pathways, including calcium mobilization and MAP kinase pathways linked to T-cell receptors and Ras.¹ To assist researchers probing the activity of NFAT proteins, Stratagene has developed a new PathDetect cis-reporter plasmid, the pNFAT-Luc reporter plasmid, containing the NFAT binding site from the human IL-2 gene.^2,7-9 The NFAT cis-reporting system includes the transfection-ready pNFAT-Luc reporter plasmid and the pCIS-CK negative control plasmid.

Construction of the pNFAT-Luc Plasmid

The backbone of the 5749-base-pair pNFAT-Luc plasmid (Figure 1) is the pFR-Luc reporter plasmid of the PathDetect cis-reporting system. To this backbone, we replaced the GAL4 binding element with four direct repeats of the NFAT binding sequence (286 to 257) from the IL-2 gene promoter, the most studied and widely used NFAT binding sequence.^1,2,10,11 In addition to a polyadenylation signal downstream of the luciferase gene, the pNFAT-Luc plasmid contains another polyadenylation signal in front of the NFAT binding sites and TATA box. This design minimizes transcription read through from upstream sequences and background luciferase expression. For all reporter plasmids of the PathDetect cis-reporting systems, activation of the luciferase gene indicates interaction of uncharacterized gene products, extracellular stimuli, growth factors, or drug candidates with specific enhancer elements. When a plasmid expressing the gene of interest is cotransfected into mammalian cells along with a cis-reporter plasmid, luciferase activity indicates transcription activation.

Fig.1

Testing the pNFAT-Luc Plasmid in Jurkat Cells

Under natural cellular conditions, NFAT proteins are activated by antigen stimulation of the T-cell receptor.¹ Pharmacology studies have established that NFAT proteins can be activated by the protein kinase C activator phorbol ester (PMA) in combination with the calcium ionophore ionomycin, reagents that raise free intracellular calcium.^2,12 When Jurkat cells, a mature human T-cell line, or CHO cells were transfected with the pNFAT-Luc plasmid and treated with 60 ng/ml of PMA and 1 mg/ml of inomycin, luciferase activity increased by 13- and 16-fold, respectively (Figure 2). Therefore, the enhancer element in the pNFAT-Luc plasmid is responsive to calcium mobilization. Cells transfected with pNFAT-Luc and then treated with either PMA or ionomycin alone did not show a significant increase in luciferase expression (result not shown), which is consistent with previous reports.²

Fig.2

Cyclosporin is an immunosuppressive drug that was introduced in 1983 and revolutionized transplant surgery. It works primarily by inhibiting the activity of calcineurin, a protein phosphatase regulated by intracellular calcium mobilization. All the isoforms of NFAT protein contain a calcineurin-binding domain and are activated by calcineurin. Results in Figure 2 demonstrated that the luciferase expression from pNFAT-Luc in both Jurkat and CHO cells induced by PMA and ionomycin could be completely inhibited by cyclosporin. In another set of experiments, rat basophilic leukemia cells stably transfected with chemokin receptors were transfected with pNFAT-Luc and then treated with their respective ligands (data not shown). When both luciferase expression and calcium levels were monitored in these cells, luciferase expression correlated very well with calcium mobilization. Therefore, luciferase expression from pNFAT-Luc indeed reflects the activation of endogenous NFAT proteins by calcium immobilization.

Conclusions

The pNFAT-Luc plasmid is a new addition to Stratagenes PathDetect cis-reporting systems. Stratagene has developed the pNFAT-Luc cis-reporter plasmid for studying the activation of NFAT transcription factors, which play an essential role in inducible gene expression during immune responses. Additionally, this plasmid can be used for studying signal transduction pathways converging at NFATs, and for screening for activators or inhibitors of the signaling molecules along these pathways.

REFERENCES

1. Rao, A., Luo, C., and Hogan. P.G. (1997) Annu. Rev. Immunol. 15: 707-747.

2. Fiering, S., et al. (1990) Genes Dev. 4: 1823-1834.

3. Hoey, T., et al. (1995) Immunity 2: 461-472.

4. Ho, S.N., et al. (1995) J. Biol. Chem. 270: 19898-19907.

5. Ruff, V.A., et al. (1995) J. Biol. Chem. 270: 22602-22607.

6. Loh, C., et al. (1996) J. Biol. Chem. 271: 10884-10891.

7. Jain, J., et al. (1993) Nature 365: 352-355.

8. McCaffrey, P., et al. (1993) Science 262: 750-754.

9. Luo, C., et al. (1996) Mol. Cell. Biol. 16: 3955-3966.

10. Barsumian, E. L., et al. (1981) Eur. J. Immunol. 11: 317-323.

11. Hsu, M.H., et al. (1997) Immunogenetics 47: 64-72.

12. Weiss, A., et al. (1987) J. Immunol. 138: 2169-21676.