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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.

SUBMITTER: Al-Daraji WI

PROVIDER: S-EPMC2719706 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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

Al-Daraji Wael I WI Malak Tamer T TT Prescott Richard J RJ Abdellaoui Adel A Ali Mahmud M MM Dabash Tarek T Zelger Bettina G BG Zelger Bernhard B

International journal of clinical and experimental medicine 20090618 2

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 sh ...[more]

PMID: 19684889

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Project description:BackgroundPsoriasis is one of the most frequent skin diseases world-wide. The disease impacts enormously on affected patients and poses a huge financial burden on health care providers. Several lines of evidence suggest that the nuclear hormone receptor peroxisome proliferator activator (PPAR) beta/delta, known to regulate epithelial differentiation and wound healing, contributes to psoriasis pathogenesis. It is unclear, however, whether activation of PPARbeta/delta is sufficient to trigger psoriasis-like changes in vivo.Methodology/principal findingsUsing immunohistochemistry, we define the distribution of PPARbeta/delta in the skin lesions of psoriasis. By expression profiling, we confirm that PPARbeta/delta is overexpressed in the vast majority of psoriasis patients. We further establish a transgenic model allowing inducible activation of PPARbeta/delta in murine epidermis mimicking its distribution in psoriasis lesions. Upon activation of PPARbeta/delta, transgenic mice sustain an inflammatory skin disease strikingly similar to psoriasis, featuring hyperproliferation of keratinocytes, dendritic cell accumulation, and endothelial activation. Development of this phenotype requires the activation of the Th17 subset of T cells, shown previously to be central to psoriasis. Moreover, gene dysregulation in the transgenic mice is highly similar to that in psoriasis. Key transcriptional programs activated in psoriasis, including IL1-related signalling and cholesterol biosynthesis, are replicated in the mouse model, suggesting that PPARbeta/delta regulates these transcriptional changes in psoriasis. Finally, we identify phosphorylation of STAT3 as a novel pathway activated by PPARbeta/delta and show that inhibition of STAT3 phosphorylation blocks disease development.ConclusionsActivation of PPARbeta/delta in the epidermis is sufficient to trigger inflammatory changes, immune activation, and signalling, and gene dysregulation characteristic of psoriasis.

Project description:Herein we demonstrate the efficacy of an unbiased proteomics screening approach for studying protein expression changes in the KC-Tie2 psoriasis mouse model, identifying multiple protein expression changes in the mouse and validating these changes in human psoriasis. KC-Tie2 mouse skin samples (n=3) were compared with littermate controls (n=3) using gel-based fractionation followed by label-free protein expression analysis. 5482 peptides mapping to 1281 proteins were identified and quantitated: 105 proteins exhibited fold-changes ≥2.0 including: stefin A1 (average fold change of 342.4 and an average P = 0.0082; cystatin A, human orthologue); slc25a5 (average fold change of 46.2 and an average P = 0.0318); serpinb3b (average fold change of 35.6 and an average P = 0.0345; serpinB1, human orthologue); and kallikrein related peptidase 6 (average fold change of 4.7 and an average P = 0.2474; KLK6). We independently confirmed mouse gene expression-based increases of selected genes including serpinb3b (17.4-fold, P < 0.0001), KLK6 (9.0-fold, P = 0.002), stefin A1 (7.3-fold; P < 0.001) and slc25A5 (1.5-fold; P = 0.05) using qRT-PCR on a second cohort of animals (n=8). Parallel LC/MS/MS analyses on these same samples verified protein-level increases of 1.3-fold (slc25a5; P < 0.05), 29,000-fold (stefinA1; P < 0.01), 322-fold (KLK6; P < 0.0001) between KC-Tie2 and control mice. To underscore the utility and translatability of our combined approach, we analyzed gene and protein expression levels in psoriasis patient skin and primary keratinocytes vs. healthy controls. Increases in gene expression for slc25a5 (1.8-fold), cystatin A (3.0-fold), KLK6 (5.8-fold) and serpinB1 (76-fold; all P < 0.05) were observed between healthy controls and involved lesional psoriasis skin and primary psoriasis keratinocytes. Moreover slc25a5, cystatin A, KLK6 and serpinB1 protein were all increased in lesional psoriasis skin compared to normal skin. These results highlight the usefulness of preclinical disease models using readily-available mouse skin and demonstrate the utility of proteomic approaches for identifying novel peptides/proteins that are differentially regulated in psoriasis that could serve as sources of auto-antigens or provide novel therapeutic targets for the development of new anti-psoriatic treatments.

Dataset Information

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

Publications

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

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