Original Article Modulation of NFAT-5, an outlying member of the NFAT family, in human keratocytes and skin
Wael I Al-Daraji, John Afolayan, Bettina G Zelger, Adel Abdellaoui, Bernhard Zelger
Ainshams University Hospital, Dermatology and Dermatopathology Departments, Cairo, Egypt; Department of Surgery, Eastbourne District General Hospital, Kings Drive, Eastbourne, BN21 2UD; Medical University Innsbruck, Institute of Pathology, Austria; Radiology Department, Derriford Hospital, Plymouth, United Kingdom
Received November 28, 2008; accepted January, 18, 2009; available online January, 2009
Abstract: Background: Ciclosporin A (CsA) and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor Nuclear Factor of Activated T cells (NFAT). NFAT compose a family of transcription factors that are turned on during T cell activation. Aims: To study the expression of NFAT 5 mRNA and protein in normal human keratocytes and to investigate the cellular and subcellular pattern of expression of NFAT 5 in normal human skin and psoriasis, and analyse effects of different agonists and ultraviolet radiation on NFAT 5 in normal human skin. Methods: Tissue cultures, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Western analysis, immunostaing, confocal microscopy. Results: Sequencing of RT-PCR products confirmed the identity of the product that showed 100 % homology with the predicted NFAT 5 sequence. anti-NFAT 5 mainly detected a single band in cultured keratocytes and dermal fibroblasts using Western analysis. Immunohistochemistry showed that epidermal keratocytes and dermal fibroblasts in normal human and psoriatic skin express NFAT 5. NFAT 5 showed predominantly nuclear localisation in epidermal keratocytes and dermal fibroblasts within five normal adult skin biopsies. Our data also suggest that UV irradiation reduces NFAT 5 nuclear localisation within the epidermis. Unlike NFAT 1-4, NFAT 5/TonEBP was localised to both nucleus and cytoplasm of cultured keratocytes. Cyclosporin A induces nuclear membrane translocation of NFAT 5 in cultured keratocytes and raffinose (a hyertonicity inducing agent) induces more nuclear localisation of NFAT 5 compared to untreated cells. In addition, differentiation-promoting agonists that induce sustained rise in intracellular calcium did not result in changes in NFAT 5 localisation in cultured keratocytes. Conclusion: these studies provide the first observation of expression of NFAT 5/TonEBP mRNA protein in cultured keratocytes and dermal fibroblasts and possible functional regulation in cultured keratocytes. CsA and raffinose effects on NFAT 5/TonEBP in cultured keratocytes suggest diverse intracellular signalling pathways for NFAT 5/TonEBP in these cells, and that NFAT 5/TonEBP might function to translate different extracellular stimuli into appropriate functional responses.(AJTR901006).
Key Words: Ciclosporin A (CsA), human keratocytes, hyperetonicity, NFAT 5 (TonEBP), psoriasis, UVR