| Research Abstract |
1) Expression pattern of skn-1a in the normal and psoriatic skin.
Skn-1a expression pattern in the normal and psoriatic epidermis was evaluated by immunohistochemistry using skn-1a monoclonal antibody. In the normal human epidermis, skn-1a was stained mainly in the cytosol of the basal keratinocytes. In the suprabasal layers of the epidermis, skn-1a accumulated clearly in the nuclei, indicating differentiation-specific nuclear translocation of skn-1a. In the psoriatic skin, however, the epidermis of the elongated rate ridge showed sustained cytosolic staining of skn-1a, and only very upper layers revealed nuclear translocation of the skn-1a, suggesting aberrant signal transduction affecting nuclear translocation of the skn-1a in the psoriatic skin.
2) Molecular mechanism of nuclear translocation of skn-1a.
Computer search identified canonical nuclear localization signal (NLS) in the primary amino-acid sequence of skn-1a. Transient transfection studies with expression vector of the NLS-GFP
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fusion protein revealed accumulation of the fusion protein in the nuclei of cultured normal human epidermal keratinocytes (NHEK), indicating active function of the NLS in the skn-1a. Site-directed mutagenesis studies indicated that serine and threonine residues locating just beside the skn-1a NLS at C-terminal side function to regulate NLS activity. Substitution of both residues to alanine accelerates nuclear translocation, suggesting that phosphorylation/dephosphorylation regulation of the serine and/or threonine residues precisely regulates skn-1a NLS activity.
3) cDNA and genomic DNA cloning of human skn-1a.
For the characterization of exon-intron structure and regulatory region of human skn-1a gene, cDNA cloning and 5'RACE were performed. In the course of this study, novel splice variant of skn-1a, which contains an additional exon in the intervening sequence between exon 1 and exon 2 of skn-1a, was isolated, and designated as skn-1n. RT-PCR studies indicate actual expression of skn-1n in cultured normal human epidermal keratinocytes.
4) Expression analysis of skn-1a and skn-1n in vitro and in vivo.
Elevation of calcium concentration from 0.03 mM to 2.0 mM in culture media dose-and time-dependently down-regulated mRNA expression of both skn-1a and skn-1n in cultured normal human epidermal keratinocytes. At 2.0 mM calcium concentration, about 90% of the mRNA inhibition was observed by RT-PCR. In situ RT-PCR study exhibited skn-1n mRNA expression from basal to mid-spinous layers of the normal human epidermis, whereas whole epidermis showed skn-1a mRNA, suggesting differential splice regulation of skn-1a and skn-1n in vivo. Less
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