Calcineurin/NFAT-dependent regulation of 230-kDa bullous pemphigoid antigen (BPAG1) gene expression in normal human epidermal keratinocytes.
OBJECTIVE
Cyclosporin A (CsA) is utilized widely for treatment of inflammatory skin diseases, such as psoriasis vulgaris. The therapeutic effects of CsA are thought to be mediated by its immunosuppressive action on infiltrating lymphocytes in the lesional skin. CsA also inhibits epidermal keratinocyte proliferation, suggesting a direct biological action on keratinocytes. Here we tested the hypothesis that CsA can modulate the expression of the nuclear factor of activated T-cell (NFAT) in epidermal keratinocytes. We also investigated whether the keratinocyte-specific gene expression is modified by CsA through NFAT activity in association with differentiation induction.


METHODS
RT-PCR was performed using total RNAs extracted from cultured normal human epidermal keratinocytes (NHEK), normal human dermal fibroblasts (NHDF), and normal human epidermal melanocytes (NHEM) for detecting NFAT isomolecules. Transient transfections of NHEK with a 230-kDa bullous pemphigoid antigen (BPAG1) promoter/luciferase reporter gene and the luciferase assay were conducted for examining the effect of CsA on the promoter activity of the BPAG1 gene. Electrophoretic gel mobility shift assays (EMSA) with probes containing NFAT consensus sequences for analyzing the binding activities of the nuclear proteins extracted from NHEK.


RESULTS
RT-PCR revealed expression of all of the five isoforms of NFAT in the cell lines examined. The mRNA expression levels of NFAT1, NFAT2, BPAG1, and involucrin were downregulated by CsA treatment in NHEK. The luciferase assay indicated suppression of the promoter activity by CsA. EMSA with NFAT consensus probes identified in the BPAG1 promoter region demonstrated specific binding activity in the nuclear proteins of epidermal keratinocytes.


CONCLUSIONS
As reported previously, our results indicate that epidermal keratinocytes possess calcineurin/NFAT system, which is suppressed by CsA. In addition, the data suggest that CsA can downregulate the BPAG1 gene expression perhaps via the NFAT consensus cis-elements in the BPAG1 promoter region. Such transcriptional regulatory system might be involved in the regulation of keratinocyte differentiation and proliferation.
Aizu
Takayuki
T
Department of Dermatology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
Tamai
Katsuto
K
Nakano
Hajime
H
Rokunohe
Daiki
D
Toyomaki
Yuka
Y
Uitto
Jouni
J
Sawamura
Daisuke
D
D019703:Calcineurin / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D016384:Consensus Sequence; D016572:Cyclosporine / Q000494:pharmacology*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003879:Dermatologic Agents / Q000494:pharmacology*; D000071821:Dystonin; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D008544:Melanocytes / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C486268:DST protein, human; D003879:Dermatologic Agents; D000071821:Dystonin; D050778:NFATC Transcription Factors; D009419:Nerve Tissue Proteins; D016572:Cyclosporine; D019703:Calcineurin