Upstream eventInhibition, Calcineurin Activity
Interference, nuclear localization of NFAT
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Inhibition of Calcineurin Activity Leading to Impaired T-Cell Dependent Antibody Response||adjacent||Moderate||Moderate|
|Homo sapiens||Homo sapiens||Moderate||NCBI|
|Mus musculoides||Mus musculoides||Moderate||NCBI|
Life Stage Applicability
|All life stages||High|
Key Event Relationship Description
The phosphatase activity of calcineurin (CN) is known to be inhibited by CN inhibitors (CNIs) such as FK506 and cyclosporin A (CsA) through the formation of complexes with immunophilins.
Immunophilins of FK506-binding protein (FKBP) and cyclophilin bind with CNIs FK506 and CsA to form complexes, which inhibit CN activity (Barik. 2006).
While FKBP12, FKBP12.6, FKBP13, and FKBP52 are all part of the FK506-binding FKBP family, FKBP12 has a significant involvement in the mechanism of action for FK506-induced immunosuppression (Siekierka et al. 1989, Kang et al. 2008).
FKBP12 is a 12-kDa protein localized in cytoplasm and has been isolated from Jurkat T-cells as a receptor that binds with the FK506 (Bram et al. 1993). FKBP12 has an FK506-binding domain (FKBD) that comprises 108 amino acids, and is expressed in T cells, B cells, Langerhans cells, and mast cells (Siekierka et al. 1990, Panhans-Gross et al. 2001, Hultsch et al. 1991).
Cyclophilin and FKBP both exhibit peptidyl propyl isomerase (PPIase) activity, but the PPlase activity and the inhibition of activity that they indicate are unrelated to CN regulation.
CN is a heterodimer that comprises a catalytic subunit (CnA) and a Ca-binding regulatory subunit (CnB). CnA handles phosphatase activity as well as calmodulin binding, and CnB regulates intracellular calcium and CnA (Klee et al. 1988, Zhang et al. 1996). CnA is a 59kDa protein with a serine-threonine phosphatase domain.
CNI-immunophilin complexes such as FK506/FKBP complexes and cyclophilin/CsA complexes bind directly to CnA in the cell, causing steric hindrance of substrate binding to CN, which in turn inhibits phosphatase activity of CN (Schreiber and Crabtree 1992, Liu et al. 1993, Bierer et al. 1993, Bram et al. 1993, Rao et al. 1997, Liu et al. 1991).
The nuclear factor of activated T cells (NFAT) is a substrate of CN (Rao et al. 1997).
When T-cell activation takes place, T-cell–receptor-mediated stimulus increases the intracellular concentration of calcium and activates CnB, which subsequently induces CnA phosphatase activation, leading to dephosphorylation of NFAT. In that process, . dephosphorylated SP motifs exposes nuclear localization signal (NLS) and covers nuclear export signal (NES), thereby promoting nuclear localization of NFAT (Matsuda and Koyasu 2000, Zhu and McKeon 1999).
When CN activity is inhibited by the binding of immunophilin complexes, dephosphorylation does not occur in NFAT, thereby interfering with nuclear localization.
Evidence Supporting this KER
The molecular structures and functions of CN and NFAT are evident based on sufficient scientific findings as mentioned above The known mechanisms for inhibition of CN phosphatase activity by FK506, CsA, or other CNIs are initiated by the formation of complexes with their respective immunophilin species. Immunophilins are general classes of proteins that exhibit PPlase activity, but modification of these functions is unrelated to inhibition of CN activity and thus thought to arise in the molecular structure of the complexes (Schreiber and Crabtree 1992, Liu et al. 1993, Bierer et al. 1993, Bram et al. 1993, Rao et al. 1997, Liu et al. 1991).
As mentioned above, inhibition of CN phosphatase activity interferes with the dephosphorylation of NFAT, which leads to the suppression of its nuclear localization.
Much experimental data is available that supports the inhibition of CN activity induced by CNI/immunophilin complexes, which subsequently suppress nuclear localization of NFAT. In addition, CN phosphatase activity is inhibited by 24 hours treatment with CNI of FK506 and CsA with IC50 values of 0.5 and 5 nM, respectively (Fruman et al.1992).
Also, concentration-dependent reduction of in vitro nuclear localization of NFAT was evident using imaging flowcytometry at the maximum concentration of 1 µM with minimal concentration of 0.1nM (Jurkat human T cell line) or 10nM (T cells from whole blood) after 2 hours treatment of tacrolimus (Maguire et al. 2013). Interference with translocation of NFAT to the nucleus is also detected using gel mobility shift assay to test nuclear extracts and cytoplasmic extracts, in which the examined concentration of FK506 was 10ng/mL (Flanagan et al. 1991).
These findings show that dose responses and temporality of MIE and KE1 seem to be the same.
Uncertainties and Inconsistencies
CN and NFAT are expressed in T cells and other immune cells including B cells, DC, and NKT cells and related to cytokine productions from these immune cells. Also, expression of IL-2 receptors (IL-2R) in DCs are lowered due to the inhibition of CN phosphatase activity by CNI treatment. Of these, reduced production of IL-2 and IL-4 from T cells plays a major role in suppression of TDAR due to lower proliferation, differentiation, and class switching of B cells. There have been no reports of CNI-induced reduction of cytokines other than IL-2 and IL-4 or reduced expression of IL-2R resulting in TDAR suppression.
FKBP12, a specific immunophilin that binds with FK506, is also an accessory molecule that binds to IP3 and Ryanodine receptors, both of which occur in Ca channels located on the membrane of the endoplasmic reticulum and participate in the regulation of intracellular Ca concentration. When binding with FK506, FKBP12 leaves these receptors to increase the influx of Ca2+ from the endoplasmic reticulum to cytoplasm, which should increase CN activity. Treatment with FK506, however, suppresses NFAT nuclear localization. In addition, FKBP12-knock out mice show no changes in immune function, including T-cell function. These facts suggest that the inhibition of CN-NFAT systems induced by FK506 treatment results from direct inhibition of CN phosphatase activity by FK506/FKBP12 complexes and not by affecting Ryanodine and IP3 receptors associated with FKBP12.
Quantitative Understanding of the Linkage
Dose-response analysis of the effects of FK506 on CN phosphatase activity in mast cell-derived KiSVMC4W cells transfected with human FKBP12 cDNA showed that increased expression of FKBP12 resulted in a greater than ten-fold increase in sensitivity to FK506-mediated inhibition, as indicated by an IC50 value of roughly 2 nM with linear inverse dose-response curve after 1 hour incuvation (Fruman et al.1995). Another phosphatase assay showed that FK506 inhibition of CN activity was concentration-dependent reverse sigmoidal and that IC50 values for CN inhibition were approximately 0.5 nM for FK 506 and 5 nM for CsA after 1 hour culture (Fruman et al.1992).
Dose-dependent interference with nuclear translocation of NFAT1 was observed with increasing CNI concentrations from 0.1 nM (Jurkat human T cells) up to 1 μM (1000 nM) using imaging flowcytometer. 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 at maximal concentrations of 1 μM with minimum concentration of 10nM (Maguire et al. 2013).
There have been no literature available to compare directly the dose response of inhibition of CN phosphatase activity with that of nuclear translocation of NFAT; however, the concentration ranges of CNIs for inhibition of CN phosphatase activity and nuclear translocation of NFAT seem to be the same range.
Inhibition of CN phosphatase activity was examined after 1 hour culture of T cells (Fruman et al.1995, Fruman et al.1992), and inhibition of nuclear translocation of NFAT was measured by imaging flowcytometry after 2 hour culture of T cells with CNI (Maguire et al. 2013).
Known modulating factors
At present, no evidence is found.
Known Feedforward/Feedback loops influencing this KER
At present, no evidence is found.
Domain of Applicability
CN is broadly distributed throughout the body, and the structure of CnA and CnB is highly conserved from yeasts to humans (Kincaid. 1993).
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).
FKBP is found in a wide variety of organisms, from prokaryotes to multicellular organisms (Siekierka et al. 1989). Multiple subfamilies of FKBP have been reported, with at least eight types having been found in mammals. FKBP12 is reported to be expressed in B-cells, Langerhans cells, and mast cells as well as in T-cells of humans, mice and other mammalian species.
Cyclophilins have been found in mammals, plants, insects, fungi and bacteria. They are structurally conserved throughout evolution and all have PPIase activity (Wang P et al. 2005).
These facts indicate that CN and immunophilins are conserved among animals and plants although they show multiple physiological functions.
In addition, CNI/immunophilin complex-induced inhibition of CN phosphatase activity resulting in suppression of immune responses is found in humans and mice.
- Barik, S. (2006). Immunophilins: for the love of proteins. Cellular and Molecular Life Sciences 63(24): 2889-900.
- Bierer, B.E., Holländer, G., Fruman, D. and Burakoff, S.J. (1993). Cyclosporin A and FK506: molecular mechanisms of immunosuppression and probes for transplantation biology. Current opinion in immunology 5 (5): 763-73.
- Bram, R.J., Hung, D.T., Martin, P.K., Schreiber, S.L. and Crabtree, G.R. (1993). Identification of the immunophilins capable of mediating inhibition of signal transduction by cyclosporin A and FK506: roles of calcimeurin binding and cellular location. Molecular and cellular biology 13 (8): 4760-9.
- 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.
- Fruman, D. A., Klee, C. B., Bierer, B. E. and Burakoff, S. J. (1992). Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A. Proceedings of the National Academy of Sciences of the United States of America. 89(9):3686-90.
- Fruman, D. A., Bierer, B. E., Benes, J. E., Burakoff, S. J., Austen, K. F. and Katz, H. R. (1995). The complex of FK506-binding protein 12 and FK506 inhibits calcineurin phosphatase activity and IgE activation-induced cytokine transcripts, but not exocytosis, in mouse mast cells. Journal of Immunology.154(4):1846-51.
- Hultsch, T., Albers, M. W., Schreiber, S.L. and Hohman, R. J. (1991). Immunophilin ligands demonstrate common features of signal transduction leading to exocytosis or transcription. Proceedings of the national academic science of the United States of America. 14: 6229-6233.
- Kang, C. B., Hong, Y., Dhe-Paganon, S. and Yoon, H. S. (2008). FKBP family proteins: immunophilins with versatile biological functions. Neurosignals. 16: 318-325.
- Kincaid, R .L. (1993). Calmodulin-dependent protein phosphatases from microorganisms to man. A study in structural conservatism and biological diversity. Adv Second Messenger Phosphoprotein Res. 27:1-23.
- Klee, C. B., Draetta, G. F. and Hubbard, M. J. (1988). Calcineurin. Advances in enzymology and related areas of molecular biology. 61:149-200.
- Liu, J., Farmer, J. D. Jr., Lane, W. S., Friedman, J., Weissman, I. and Schreiber, S. L. (1991). Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 66(4): 807-815.
- Liu, J., Albers, M. W., Wandless, T. J., Luan, S., Alberg, D. G., Belshaw, P. J., Cohen, P., MacKintosh, C., Klee, C. B. and Schreiber, S.L.. (1992). Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry. 31(16):3896-901.
- Liu, J. (1993). FK506 and cyclosporin, molecular probes for studying intracellular signal transduction. Immunology today. 14(6): 290-305.
- 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.
- Matsuda, S., Koyasu, S. (2000). A second target of cyclosporin A and FK506. Tanpakushitsu kakusan koso. 45(11): 1823-1831.
- 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.
- Rao, A., Luo, C. and Hogan, PG. (1997). Transcription factors of the NFAT family: regulation and function. Annual Review of Immunology 15: 707-47.
- Schreiber, SL. and Crabtree, GR. (1992). The mechanism of action of cyclosporin A and FK506. Immunology Today 13(4): 136-42. >
- Siekierka, JJ., Hung, SH., Poe, M., Lin, CS. and Sigal, NH. (1989). A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature 341(6244): 755-57.
- Siekierka, JJ., Wiederrecht, G., Greulich, H., Boulton, D., Hung, SH., Cryan, J., Hodges, PJ. and Sigal, NH. (1990). The cytosolic-binding protein for the immunosuppressant FK-506 is both a ubiquitous and highly conserved peptidyl-prolyl cis-trans isomerase. Journal of Biological Chemistry 265(34): 21011-5.
- Wang, P. and Heitman, J. (2005) The cyclophilins. Genome Biology 6 (7):226.
- Zhang, B.W., Zimmer, G., Chen, J., Ladd, D., Li, E., Alt, F.W., Wiederrecht, G., Cryan, J., O'Neill, E.A., Seidman, C.E., Abbas, A.K. and Seidman, J.G. (1996). T cell responses in calcineurin A alpha-deficient mice. Journal of experimental medicine 183(2): 413-20.
- Zhu, J. and McKeon, F. (1999). NF-AT activation requires suppression of Crm1-dependent export by calcineurin. Nature. 398(6724): 256-60.