Expression, localisation and functional activation of NFAT-2 in normal human skin, psoriasis, and cultured keratocytes

Abstract

Ciclosporin A (CsA) is widely utilized for the treatment of inflammatory skin diseases such as psoriasis. The therapeutic effects of CsA are thought to be mediated via its immunosuppressive action on infiltrating lymphocytes in skin lesions. 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). As calcineurin and NFAT 1 have been shown to be functionally active in cultured human keratocytes, expression of other NFAT family members such as NFAT-2 and possible functional activation was investigated in human keratocytes. RT-PCR and Western Analysis were used to investigate the presence of NFAT-2 mRNA and protein in human keratocytes. Tissue culture of human keratocytes and immunostaining of cells on coverslips and confocal microscopy were used to assess the degree of nuclear localisation of NFAT-2 in cultured cells. Keratome biopsies were taken from patients with psoriasis (lesional and non-lesional skin) and normal skin and immunohistochemistry was used to assess the NFAT-2 localisation in these biopsies using a well characterized anti-NFAT-2 antibody. The NFAT-2 mRNA and protein expression was demonstrated using RT-PCR and Western blotting. Moreover, the expression of NFAT-2 in normal skin, non-lesional and lesional psoriasis showed a striking basal staining suggesting a role for NFAT-2 in keratocytes proliferation. A range of cell types in the skin express NFAT-2. The expression of NFAT-2 in human keratocytes and response to different agonists provides perhaps a unique opportunity to examine the regulation, subcellular localization and kinetics of translocation of different NFATs in primary cultured human cells. In these experiments the author assessed the expression, localization of NFAT-2 in cultured human keratocytes and measured the degree of nuclear localisaion of NFAT-2 using immunofluorescence and confocal microscopy and whether CsA and tacrolimus inhibit NFAT-2 nuclear translocation. As with NFAT 1, differentiation-promoting agents that increase intracellular calcium concentration induced nuclear translocation of NFAT-2 in cultured keratocytes but with different kinetics. These data provide the first evidence of that NFAT-2 is expressed in normal skin, psoriasis and that NFAT-2 functionally active in human keratocytes and that nuclear translocation of NFAT-2 in human skin cells has different kinetics than NFAT 1 suggesting that NFAT-2 may play an important role in regulation of keratocytes proliferation and differentiation at a different stage. Inhibition of this pathway in human epidermal keratocytes many account, in part for the therapeutic effects of CsA and tacrolimus in skin disorders such as psoriasis. Thus, supporting our previous work data that calcineurin/NFAT is functionally active not only in T cells, but in skin cells.

Keywords: Ciclosporin A (CsA); NFAT-2; Tacrolimus; calcineurin; intracellular calcium; keratocytes differentiation; psoriasis.

Figure 1

Schematic representation of T cell activation (A) and mechanism of action of ciclosporin A (CsA), tacrolimus and pimecrolimus (B). Inhibition of the phosphatase calcineurin blocks nuclear translocation of NFAT.

Figure 2

NFAT-2 mRNA expression in cultured human keratocytes, Jurkat T cells and dermal fibroblasts. Total RNA was extracted from cultured cells; reverse transcribed and PCR performed with NFAT-2-specific primers. Reaction products were separated by electro-phoresis in 1.5% agarose gels. (A), lane 1: hyperladder IV; lane 2, negative control (water); lane 3, cultured keratocytes (NFAT-2). (B) Lane 1; hyperladder I, Lane 2; Jurkat T cells (NFAT-2), lane 3; cultured dermal fibroblasts (NFAT-2), lane 4; negative control (water). Sequencing studies confirmed the expression of NFAT-2 mRNA in cultured keratocytes, dermal fibroblasts. The predicted size of the product is 224 bp for NFAT-2.

Figure 3

Western analysis confirms the expression of NFAT-2 in cultured epidermal keratocytes and dermal fibroblasts. Cell lysates were prepared from cultured keratocytes and dermal fibroblasts, separated by SDS-PAGE and immunoblotted with anti-NFAT-2 (801) antibody. This experiment confirmed that antibodies used in immunostaining detects the appropriate molecular weight of two NFAT-2 isoforms (86 kDa and 116 kDa) (A and B). (A), lane 1, medium control (keratocytes) (donor 1); lane 2, medium control (keratocytes) (donor 2); lane 3, DMSO 4 h (keratocytes) (vehicle) (donor 2); lane 4, DMSO 4 h (vehicle) (keratocytes) (donor 1); lane 5, TPA/ionomycin 4 h (donor 2); lane 6, TPA/ionomycin 4 h (donor 1). (B), lane 1, medium control (fibroblasts) (donor 1); lane 2, medium control (fibroblasts) (donor 2); lane 3, DMSO 4 h (vehicle) (fibroblasts) (donor 2); lane 4, DMSO 4 h (fibroblasts) (vehicle) (donor 1).

Figure 4

Localisation of NFAT-2 in normal human skin, lesional psoriatic skin and non-lesional psoriatic skin. Frozen sections of normal human skin (A), lesional (plaque) (B) psoriatic skin and non-lesional (uninvolved) (C) psoriatic skin were immunostained with antibody against NFAT-2 (801). NFAT-2 shows predominantly cytoplasmic and nuclear localisation in the basal layer normal and psoriatic skin (Original magnification X25). For negative control (D), equivalent dilutions of non-immune serum (normal rabbit serum) were used. (E), NFAT-2 shows predominantly nuclear and cytoplasmic localisation in the outer root sheath of hair follicles in normal skin (Original magnification X25).

Figure 5

Tacrolimus inhibits nuclear translocation of NFAT-2 induced by TPA plus ionomycin in human keratocytes. These results are representative of 3 experi-ments on keratocytes derived from 3 independent donors. Scale bar 25 μM.

Figure 6

Tacrolimus inhibits TPA induced nuclear translocation of NFAT-2 in human keratocytes. These results are representative of 3 experiments on keratocytes derived from 3 independent donors. Scale bar 25 μM.

Figure 7

Nuclear and plasma membrane translocation of NFAT-2 induced by raised extracellular calcium in human keratocytes. These results are representative of 3 experiments on keratocytes derived from 3 independent donors. Scale bar, 25μM.

Figure 8

Nuclear translocation of NFAT-2 in human keratocytes. These results are representative of 3 experiments on keratocytes derived from 3 independent donors. Scale bar, 25μM.

Figure 9

Time course of NFAT-2 subcellular localisation in response to different agonists in cultured keratocytes (A and B). Human keratocytes were cultured on coverslips and immunostained as described in Materials and Methods. The number of cells showing positive nuclear staining was counted using epifluorescence microscopy. At least 150 cells from 3 independent experiments were assessed at each time point.