Localization of Calcineurin/NFAT in Human Skin and Psoriasis and Inhibition of Calcineurin/NFAT Activation in Human Keratinocytes by Cyclosporin A

Abstract

Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A 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). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis.

Nfatc2 is a primary response gene of nell-1 regulating chondrogenesis in ATDC5 cells

“…In fact, NFATs bind only weakly to DNA and require a partner protein (eg, AP1 or GATA4, but this varies among cell types) to initiate transcriptional activation. (52) NFAT transactivation involves not only its nuclear translocation but also the intrinsic function of its transactivation domain. (53) Increasing evidence supports the notion that enhancement of the N-terminal transactivating domain of Nfatc2 plays a role in its activation.…”

Ceramide-Dependent Regulation of Human Epidermal Keratinocyte CD1d Expression during Terminal Differentiation

“…KC have been demonstrated to increase their translocation of NFAT into the nucleus, in vivo during normal differentiation, in psoriatic skin lesions, as well as in vitro after exposure to agonists that induce terminal differentiation (increased extracellular CaCl 2 or 12-O-tetradecanoyl-phorbol-13-acetate plus ionomycin) (16). Cyclosporin A is an immunosuppressive agent that blocks calcineurin phosphatase in T lymphocytes as well as KC (16).…”

“…KC as well as fibroblast CD1d gene expression is inhibited by agents that block sphingolipid synthesis (fumonisin B1) (15). Cyclosporin A, which blocks KC terminal differentiation (16), also inhibited CD1d gene expression. These data suggest that both extracellular and endogenous ceramides modulate CD1d expression and provide a mechanism for the polarized expression of KC CD1d during the differentiation process in vivo within the epidermis.…”

Cyclosporine A and its non-immunosuppressive derivative NIM811 induce apoptosis of malignant melanoma cells inin vitro andin vivo studies

“…Cyclosporine A and not related immunosuppressant tacrolimus (FK506) are effective against a broad range of inflammatory skin diseases, including widespread conditions such as psoriasis and atopic dermatitis. [33][34][35][36][37] When applied topically to either mouse or human skin, CsA transport across the cutaneous barrier may be facilitated by using short oligomers of arginine CsA. 38 It raises a possibility of topical application of concentrated CsA or its derivative directly on melanoma tissue to achieve a high dose of the drug that is necessary to exert cytostatic/cytotoxic effects.…”