Modulation of NFAT-5, an outlying member of the NFAT family, in human keratinocytes and skin

Abstract

Background: Cyclosporin 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 keratinocytes and to investigate the cellular and subcellular pattern of expression of NFAT-5 in normal human skin and psoriasis, and analyze 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, immunostaining, 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 keratinocytes and dermal fibroblasts using Western analysis. Immunohistochemistry showed that epidermal keratinocytes and dermal fibroblasts in normal human and psoriatic skin express NFAT-5. NFAT-5 showed predominantly nuclear localization in epidermal keratinocytes and dermal fibroblasts within five normal adult skin biopsies. Our data also suggest that UV irradiation reduces NFAT-5 nuclear localization within the epidermis. Unlike NFAT 1-4, NFAT-5/TonEBP was localized to both nucleus and cytoplasm of cultured keratinocytes. Cyclosporin A induces nuclear membrane translocation of NFAT-5 in cultured keratinocytes and raffinose (a hypertonicity inducing agent) induces more nuclear localization 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 localization in cultured keratinocytes.

Conclusion: These studies provide the first observation of expression of NFAT-5/TonEBP mRNA protein in cultured keratinocytes and dermal fibroblasts and possible functional regulation in cultured keratinocytes. CsA and raffinose effects on NFAT-5/TonEBP in cultured keratinocytes suggest diverse intracellular signaling pathways for NFAT-5/TonEBP in these cells, and that NFAT-5/TonEBP might function to translate different extracellular stimuli into appropriate functional responses.

Keywords: Cyclosporin A (CsA); Human keratinocytes; Hypertonicity; NFAT-5 (TonEBP); UVR; psoriasis.

Figure 1

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

Figure 2

NFAT-5 mRNA and protein expression in cultured human keratinocytes and dermal fibroblasts. Total RNA was extracted from cultured cells, reverse transcribed and PCR performed with NFAT-5-specific primers. Reaction products were separated by electrophoresis in 1.5% agarose gels. (A), lane 1 hyperladder IV; lane 2, negative control (water); lane 3, Jurkat T cells; lane 4, cultured keratinocytes. (B), lane 1, hyperladder I; lane 2, negative control (water); lane 3, cultured keratinocytes; lane 4, cultured fibroblasts. Sequencing studies confirmed the expression of NFAT-5 in cultured cells. The predicted size of NFAT-5 is 340 bp. (C), cell lysates were prepared from cultured keratinocytes and dermal fibroblasts, separated by SDS-PAGE and immunoblotted with anti-NFAT-5 antibody. This experiment confirmed that the antibody used in immunostaining detects the appropriate molecular weight of NFAT-5 (202 kDa) (arrow). Lane 1, medium control (keratinocytes) (donor 1); lane 2, medium control (keratinocytes) (donor 2); lane 3, medium control (fibroblasts) (donor 1); lane 4, medium control (fibroblasts) (donor 2).

Figure 3

Immunostaining of normal skin, lesional (plaque) and non-lesional psoriatic skin (subject 1) with an anti-NFAT-5 antibody. Frozen sections of normal human skin (A) (original magnification X25), lesional (C) (plaque) (original magnification X10) psoriatic skin and non-lesional (B) (uninvolved) (original magnification X25) psoriatic skin were stained with anti-NFAT-5 antibody. NFAT-5 shows predominantly nuclear localization in normal and psoriatic skin, but there is reduced expression in suprabasal spinous layer in psoriatic skin compared to normal skin.

Figure 4

Immunostaining of normal skin, lesional (plaque) and non-lesional psoriatic skin (Subject 2) with an anti-NFAT-5 antibody and expression of NFAT-5/TonEBP by Langerhans cells in psoriatic skin. Frozen sections of normal human skin (A), non-lesional (uninvolved) (B) psoriatic skin and lesional (plaque) psoriatic skin (C) were stained with anti-NFAT-5 antibody. NFAT-5 shows predominantly nuclear localization in normal and psoriatic skin. Langerhans cells express NFAT-5 in the suprabasal layer of lesional psoriatic skin (D). Arrowheads indicate dendritic processes of the Langerhans cell. (E) frozen sections of normal skin stained with goat IgG as negative control. (Original magnification x25).

Figure 5

Anti-NFAT-5 staining specificity in cultured human keratinocytes (A). Hypertonicity induces nuclear localization of NFAT-5/TonEBP in human keratinocytes (B). (A), human keratinocytes were cultured on coverslips in low calcium MCDB 153 medium. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, incubated sequentially with goat-polyclonal anti-NFAT-5 antibody (2 μg/ml) or goat IgG (2 μg/ml), rabbit anti-goat FITC, propidium iodide (50 μg/ml) and visualized using a Biorad confocal microscope. The images shown are mid-cell sections. Negative control coverslip (goat IgG) was scanned using the same settings (gain, black level and confocal aperture) as the positive control coverslip (anti-NFAT-5), thus ensuring that the pixel brightness values were due to antibody labelling rather than other factors such as autofluorescence or non-specific binding. Pixel brightness data were analyzed using COMOS software. (B), human keratinocytes were cultured on coverslips in low calcium MCDB 153 medium (control) and then switched to medium containing 200nM of raffinose for 24 h and 48 h as indicated. Cells were fixed and incubated sequentially with goat-polyclonal anti-NFAT-5 antibody, rabbit anti-goat FITC, propidium iodide (50 μg/ml) and visualized using a Biorad confocal microscope. The images shown are mid-cell sections. These results are representative of 3 experiments on keratinocytes derived from 3 independent donors. Scale bar 25 μM.

Figure 6

Differentiation promoting agents do not cause any change in the subcellular localization of NFAT-5/TonEBP in human keratinocytes . Human keratinocytes were cultured on coverslips in low calcium MCDB 153 medium (control) and then then treated with DMSO (vehicle control), ionomycin (1 μM), TPA (50 nM) plus ionomycin (1 μM) or TPA (50 nM) for 24 h and 48 h as indicated. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, incubated sequentially with goat-polyclonal anti-NFAT-5 antibody, rabbit anti-goat FITC, propidium iodide (50 μg/ml) and visualized using a Biorad confocal microscope. The images shown are mid-cell sections. These results are representative of 3 experiments on keratinocytes derived from 3 independent donors. Scale bar 25 μM.

Figure 7

Cyclosporin A, but not tacrolimus, induces nuclear membrane translocation of NFAT-5/TonEBP in human keratinocytes . Human keratinocytes were cultured on coverslips in low calcium MCDB 153 medium (control) and then then treated with DMSO (vehicle control), CsA (1 μM) or tacrolimus (1 μM) for 24 h and 48 h as indicated. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, incubated sequentially with goat-polyclonal anti-NFAT-5 antibody, rabbit anti-goat FITC, propidium iodide (50 μg/ml) and visualized using a Biorad confocal microscope. The images shown are mid-cell sections. These results are representative of 3 experiments on keratinocytes derived from 3 independent donors. Arrowheads indicate nuclear membrane translocation of NFAT-5/TonEBP in response to CsA. Scale bar 25 μM.

Figure 8

Effects of UVA and UVB on NFAT-5/TonEBP localization in human epidermis in vivo. 4 mm punch biopsies were taken from un-irradiated, UVA and UVB-irradiated sites of equal erythema as described in section 5.2.2. Sections were stained with goat polyclonal anti-NFAT-5 and the number of positively stained epidermal nuclei was counted as described. (Original magnification x25).