The role of POU domain transcription factors in the epidermal development and differentiation

1)正常皮肤和银屑病皮肤中skn-1a的表达模式。应用Skn-1a单克隆抗体免疫组化检测正常表皮和银屑病表皮中Skn-1a的表达谱。在正常人表皮中，skin -1a主要在基底角质形成细胞的细胞质中染色。在表皮的基上层，皮肤-1a在细胞核中明显聚集，表明皮肤-1a的分化特异性核易位。然而，在银屑病皮肤中，延长率脊的表皮显示皮肤-1a持续的细胞质染色，只有非常上层显示皮肤-1a的核易位，这表明异常的信号转导影响了银屑病皮肤中皮肤-1a的核易位。2) skn-1a核易位的分子机制。计算机检索在skn-1a的一级氨基酸序列中发现了典型核定位信号（NLS）。用NLS- gfp表达载体进行瞬时转染研究，发现在培养的正常人表皮角质形成细胞（NHEK）细胞核中有更多的融合蛋白积累，表明NLS在skn-1a中具有活性功能。定点诱变研究表明，位于skin -1a NLS c端旁边的丝氨酸和苏氨酸残基可以调节NLS的活性。将这两个残基替换为丙氨酸会加速核易位，这表明丝氨酸和/或苏氨酸残基的磷酸化/去磷酸化调控精确地调节了skn-1a NLS活性。3)人skin -1a基因cDNA及基因组DNA克隆。为了鉴定人skin -1a基因的外显子-内含子结构和调控区域，我们进行了cDNA克隆和5'RACE。在本研究过程中，分离出新的skn-1a剪接变体，该变体在skn-1a的外显子1和外显子2之间的中间序列中含有一个额外的外显子，并将其命名为skn-1n。RT-PCR研究表明，在培养的正常人表皮角质形成细胞中实际表达了skin -1n。4)体外和体内skn-1a和skn-1n的表达分析。在培养的正常人表皮角质形成细胞中，钙浓度从0.03 mM升高到2.0 mM，剂量依赖性和时间依赖性地下调了skin -1a和skin -1n的mRNA表达。在2.0 mM钙浓度下，RT-PCR观察到约90%的mRNA抑制。原位RT-PCR研究显示，正常人表皮的基底层到棘中层表达了skin -1n mRNA，而整个表皮则表达了skin -1a mRNA，这表明在体内，skin -1a和skin -1n的剪接调节存在差异。少

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 … More 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