API

Relationship: 1017

Title

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Interference, nuclear localization of NFAT leads to Reduction, NFAT/AP-1 complex formation

Upstream event

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Interference, nuclear localization of NFAT

Downstream event

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Reduction, NFAT/AP-1 complex formation

Key Event Relationship Overview

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AOPs Referencing Relationship

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AOP Name Adjacency Weight of Evidence Quantitative Understanding
Inhibition of Calcineurin Activity Leading to Impaired T-Cell Dependent Antibody Response adjacent High High

Taxonomic Applicability

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Term Scientific Term Evidence Link
Homo sapiens Homo sapiens High NCBI
Mus musculus Mus musculus High NCBI

Sex Applicability

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Sex Evidence
Unspecific

Life Stage Applicability

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Term Evidence
All life stages High

Key Event Relationship Description

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Activated (dephosphorylated) nuclear factor of activated T cells (NFAT) is translocated into the nucleus through the molecular changes of exposing nuclear localization signal (NLS) and concomitant masking of nuclear export signal (NES) due to dephosphorylation of the SP motifs of NFAT. (Matsuda and Koyasu 2000, Zhu and McKeon 1999).

Nuclear localization of NFAT results in the NFAT binding with AP 1 at the IL-2 promoter region, (Schreiber and Crabtree 1992; Jain et al. 1992) and induces transcription of IL-2 (Jain et al. 1993). In addition to IL-2, NFAT localized in the nucleus of T cells also binds to the promoter region of the other classes of cytokines including IL-4 and IL-13.

Once CN phosphatase activity is inhibited, dephosphorylation of NFAT and subsequent nuclear localization of NFAT decreases, which results in a decrease of NFAT/AP-1 complex formation at the cytokine promoter sites (Rao et al. 1997).

Evidence Supporting this KER

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Biological Plausibility

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As has been mentioned, NFAT has NLS and NES among and adjacent to the N-terminal region rich in SP motifs, and once the SP region is dephosphorylated, the NLS is exposed and the NES is covered, which leads to translocation of NFAT into the nucleus (Matsuda and Koyasu 2000).

It is well known from the experiments using CN inhibitors (CNIs) that interference with the nuclear localization of NFAT in T cells leads to a reduction in the formation of NFAT/AP-1 complexes, thereby suppressing transcription of IL-2, IL-4, and a number of other cytokines (Maguire et al. 2013, Jain et al. 1992, Jain et al. 1993).

In contrast to T cells, B-cell receptor-mediated increases in intracellular concentration of calcium in B cells leads to NFAT nuclear localization, thereby producing some classes of cytokines in the same manner as T-cells (Bhattacharyya et al.2011). However, there has been no report of any evidence that CNI acts directly on B cells to effect antibody production.

Expression of IL-2 receptors in dendritic cells and NKT cells is also reported to be regulated by this CN-NFAT system (Panhans-Gross A et al. 2001; Kim et al. 2010), but there is no report showing that CNIs suppress TDAR through the changes in IL-2R expression in these cells.

Empirical Evidence

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The relationship of the interference of nuclear localization of NFAT leading to reduced NFAT/AP-1 complex formation bound at the promoter sites of cytokine genes in the presence of CNIs is well known as mentioned above.

Imaging flowcytometry revealed that concentration-dependent reduction of in vitro nuclear localization of NFAT was evident at the maximum concentration of 1 µM with minimal concentration of 0.1nM (Jurkat human T cell line) or 10nM (CD4+T cells from whole blood) after 2 hours treatment of tacrolimus (Maguire et al. 2013).

The experiment of gel mobility shift assay using Ar-5 human T cells stimulated with cross-linked anti-CD3 antibody showed that NFAT/AP-1 (cFos and Jun) complexes were found only in the nuclear extract with preexisting NFAT in the cytoplasm after T cell stimulation and that the NFAT/AP-1 complexes in the nucleus decreased after 2 hours treatment with CsA at 1µM (Jain et al. 1992). Decreased NFAT translocated to the nucleus, induced by FK506 at 100ng/mL (0.125nM) or CsA at 500ng/mL (0.416nM) for 2 hours treatment, hinders the formation of the functional NFAT/AP-1 complexes necessary to binding at the site of IL-2 promoters (Flanagan et al. 1991)NFAT/AP-1 complex formation is inhibited by CNI (Rao et al. 1997).

Quantitative understanding of NFAT/AP-1 complex formation in the nucleus is insufficient although nuclear localization of NFAT and following nuclear NFAT/AP-1 complex formation are shown to be tightly related; therefore, NFAT nuclear translocation and complex formation with AP-1 in in the nucleus deem to be suppressed by CNIs under the same conditions of dose ranges and treatment duration..

Uncertainties and Inconsistencies

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Nothing especially

Quantitative Understanding of the Linkage

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Response-response Relationship

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The relationship of the interference of nuclear localization of NFAT leading to reduced NFAT/AP-1 complex formation bound at the promoter sites of cytokine genes in the presence of CNIs is well known as mentioned above.

KE1:

Dose-dependent interference with nuclear translocation of NFAT1 was observed with increasing FK506 concentrations from 0.01nM (Jarkat T cells) up to 1 μM (1000 nM). Higher concentrations induced cellular toxicity and resulted in cell death. Dose-dependent interference of nuclear NFAT1 translocation per CN inhibition was also observed in CD4+ T cells from healthy donors, again from 10nM to maximal concentrations of 1 μM (Maguire et al. 2013). Both parameters were measured after 2 hour culture of T cells with FK506.

KE2:

Reduction in generation of NFAT/AP-1 complexes can be detected using a gel shift assay (Rao et al. 1997, Jain et al. 1992, Jain et al. 1993).

Decreased NFAT translocated to the nucleus, induced by FK506 at 100ng/mL (124nM) or CsA at 500ng/mL (416nM) after 2 hours treatment, hinders the formation of the functional NFAT/AP-1 complexes necessary to binding at the site of IL-2 promoters (Flanagan et al. 1991). As mentioned above, the gel mobility shift assay also showed that NFAT/AP-1 complexes were formed only in the nucleus after T cell activation with unchanged preexisting NFAT in the cytoplasm and that treatment of T cells with 1µM FK506 led to decease the levels of NFAT/AP-1 complex (Jain et al. 1992).

These findings suggest that nuclear translocation of NFAT after T cell stimulation is strongly related to the complex formation with AP-1 in the nucleus, and FK506 was shown to inhibit NFAT/AP-1 complex formation in the nucleus at the concentrations within the concentration range of FK506 for suppressing nuclear translocation of NFAT (Maguire et al. 2013).

Time-scale

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Nuclear translocation of NFAT was shown to be inhibited in vitro using imaging flowcytometry after 2 hours culture of T cells with FK506 (Maguire et al. 2013), and gel mobility shift assay revealed the inhibition of nuclear translocation of NFAT and following complex formation with AP-1 within the nucleus after 2 hours culture of T cells with FK506 (Jain et al. 1992, Flanagan et al. 1991).

Known modulating factors

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(To be described)

Known Feedforward/Feedback loops influencing this KER

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(To be described)

Domain of Applicability

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NFAT expresses in B cells, mast cells, neutrophils, granulocytes, dendritic cells, macrophages, and natural killer cells as well as T cells from humans, rodents, and other mammalian species (Rao et al. 1997).

CN-NFAT system functionality is common among mammalian species, including humans and rodents. It is also possible that FK506-induced interference with NFAT/AP-1 complex formation at the promoter site of the IL-2 gene is common among mammalian T cells, including those of humans and rodents (Flanagan et al. 1991).

References

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  1. Bhattacharyya, S., Deb, J., Patra, A.K., Thuy Pham, D.A., Chen, W., Vaeth, M., Berberich-Siebelt, F., Klein-Hessling, S., Lamperti, E.D., Reifenberg, K., Jellusova, J., Schweizer, A., Nitschke, L., Leich, E., Rosenwald, A., Brunner, C., Engelmann, S., Bommhardt, U., Avots, A., Müller, M.R., Kondo, E. and Serfling, E. (2011). NFATc1 affects mouse splenic B cell function by controlling the calcineurin-NFAT signaling network. The Journal of experimental medicine 208 (4): 823-39.
  2. Flanagan, W.M., Corthésy, B., Bram, R.J. and Crabtree, G.R. (1991). Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352 (6338): 803-7.
  3. Jain, J., McCaffrey, P. G., Valge-Archer, V. E. and Rao, A. (1992). Nuclear factor of activated T cells contains Fos and Jun. Nature. 356(6372): 801-4.
  4. Jain, J., Miner, Z. and Rao, A. (1993). Analysis of the preexisting and nuclear forms of nuclear factor of activated T cells. Journal of immunology. 151(2): 837-48.
  5. Kim, T., Kim, N. and Kang, H. J. (2010). FK506 causes cellular and functional defects in human natural killer cells. Journal of leukocyte biology. 88:1089-1097.
  6. Maguire O, Tornatore KM, O'Loughlin KL, Venuto RC and Minderman H. (2013) Nuclear translocation of nuclear factor of activated T cells (NFAT) as a quantitative pharmacodynamic parameter for tacrolimus. Cytometry A. 83(12):1096-104.
  7. Matsuda, S., Koyasu, S. (2000). A second target of cyclosporin A and FK506. Tanpakushitsu kakusan koso. 45(11): 1823-31.
  8. Panhans-Gross, A., Novak, N., Kraft, S., and Bieber, T. (2001). Human epidermal Langerhans’ cells are targets for the immunosuppressive macrolide tacrolimus (FK506). Journal of Allergy and Clinical Immunology 107(2): 345-52.
  9. Rao, A., Luo, C., and Hogan, PG. (1997). Transcription factors of the NFAT family: regulation and function. Annual Review of Immunology 15: 707-47.
  10. Schreiber, SL., and Crabtree, GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunology Today 13(4): 136-42.
  11. Zhu, J. and McKeon, F. (1999). NF-AT activation requires suppression of Crm1-dependent export by calcineurin. Nature. 398(6724): 256-60.