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Activation of nuclear factor of activated T cells in a cyclosporin A-resistant pathway
P Ghosh1, A Sica, M Cippitelli
1Laboratory of Experimental Immunology, Division of Basic Science, SAIC, Frederick, Maryland 21702-1201, USA.
Abstract:
The mechanism of action of the immunosuppressive drug cyclosporin A (CsA) is the inactivation of the Ca2+/calmodulin-dependent serine-threonine phosphatase calcineurin by the drug-immunophilin complex. Inactive calcineurin is unable to activate the nuclear factor of activated T cells (NFAT), a transcription factor required for expression of the interleukin 2 (IL-2) gene. IL-2 production by CsA-treated cells is therefore dramatically reduced. We demonstrate here, however, that NFAT can be activated, and significant levels of IL-2 can be produced by the CsA-resistant CD28-signaling pathway. In transient transfection assays, both multicopy NFAT- and IL-2 promoter-beta-galactosidase reporter gene constructs could be activated by phorbol 12-myristate 13-acetate (PMA)/alpha-CD28 stimulation, and this activation was resistant to CsA. Electrophoretic mobility shift assay showed the induction of a CsA-resistant NFAT complex in the nuclear extracts of peripheral blood T cells stimulated with PMA plus alphaCD28. Peripheral blood T cells stimulated with PMA/alphaCD28 produced IL-2 in the presence of CsA. Collectively, these data suggest that NFAT can be activated and IL-2 can be produced in a calcineurin independent manner.
Insights
Cyclosporin A (CsA) normally blocks T-cell activation by inhibiting calcineurin. However, the CD28 pathway can activate nuclear factor of activated T cells (NFAT) and interleukin-2 (IL-2) production independently of calcineurin, even with CsA present.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- Cyclosporin A (CsA) is an immunosuppressant that inhibits T-cell activation by targeting calcineurin, a phosphatase crucial for nuclear factor of activated T cells (NFAT) activation.
- NFAT activation is essential for the transcription of the interleukin-2 (IL-2) gene, a key cytokine for T-cell proliferation and function.
- Reduced IL-2 production is a primary effect of CsA treatment in T cells.
Purpose of the Study:
- To investigate whether T-cell activation and IL-2 production can occur independently of the calcineurin pathway.
- To explore the role of the CD28-signaling pathway in overcoming CsA-mediated immunosuppression.
- To determine if NFAT can be activated through a CsA-resistant mechanism.
Main Methods:
- Transient transfection assays using reporter gene constructs (NFAT and IL-2 promoter-driven beta-galactosidase).
- Stimulation of T cells with phorbol 12-myristate 13-acetate (PMA) and anti-CD28 antibody (alpha-CD28).
- Electrophoretic mobility shift assays (EMSA) to detect NFAT complex formation in nuclear extracts.
Main Results:
- Phorbol 12-myristate 13-acetate (PMA)/alpha-CD28 stimulation activated NFAT and IL-2 promoter-reporter gene constructs in a CsA-resistant manner.
- Electrophoretic mobility shift assays confirmed the induction of a CsA-resistant NFAT complex in T cells stimulated with PMA plus alpha-CD28.
- Peripheral blood T cells stimulated via the CD28 pathway produced IL-2 even in the presence of CsA.
Conclusions:
- The CD28-signaling pathway can activate NFAT and induce IL-2 production independently of calcineurin.
- This calcineurin-independent pathway offers a potential mechanism for T-cell activation and IL-2 production that bypasses CsA inhibition.
- These findings highlight the plasticity of T-cell activation pathways and suggest alternative targets for immunomodulation.