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

POU结构域转录因子在表皮发育和分化中的作用

• 批准号: 10470185
• 负责人: TAMAI Katsuto
• 金额: $ 8.96万
• 依托单位: Hirosaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470185/
• 关键词: Keratinocytes; POU domain transcriptional factor; Skn-1n; Octanmer motif; in situ RT-PCR; POU ドメインファクター; RT-PCR; in situ; skn-1a; NLS; skn1a; 類天疱瘡抗原; sknla

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

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

项目成果

Sawamura D, Meng X, Ina S, Kon A, Tamai K, Ohe Y, Hashimoto I: "Expression vector with DNA of bovine papilloma virus 1 for keratinocyte gene therapy"J Dermatol Sci. 23. 111-116 (2000)

Sawamura D、Meng X、Ina S、Kon A、Tamai K、Ohe Y、Hashimoto I：“用于角质形成细胞基因治疗的牛乳头状瘤病毒 1 型 DNA 表达载体”J Dermatol Sci。

Tamai K, Murai T, Mayama M, Kon A, Nomura K, Sawamura D, Hanada K, Hashimoto I, Shimizu H, Masunaga T, Nishikawa T, Mitsuhashi Y, Yamamoto AI, Ikeda S, Ogawa H, McGrath JA, Pulkkine L, Uitto J: "Recurrent COL7A1 mutations in Japanese patients with dystrop

玉井 K、村井 T、真山 M、今 A、野村 K、泽村 D、花田 K、桥本 I、清水 H、增永 T、西川 T、三桥 Y、山本 AI、池田 S、小川 H、麦格拉斯 JA、普尔金 L

Hashimoto I, Kon A, Tamai K, Uitto J: "Diagnostic dilemma of "sporadie" cases of dystrophic epidermolysis bullosa : a new dominant or mitis recessive mutation?"Exp Dermatol. 18. 140-142 (1999)

Hashimoto I、Kon A、Tamai K、Uitto J：“营养不良性大疱性表皮松解症“散发”病例的诊断困境：一种新的显性突变或 mitis 隐性突变？”Exp Dermatol。

Sawamura D, Ina S, Itai K, Meng X, Kon A, Tamai K, Hanada K, Hashimoto I: "In vivo gene introduction into keratinocytes using jet injection."Gene Therapy. 6. 1785-1787 (1999)

Sawamura D、Ina S、Itai K、Meng X、Kon A、Tamai K、Hanada K、Hashimoto I：“使用喷射注射将基因体内导入角质形成细胞。”基因疗法。

Sawamura D, Meng X, Ina S, Nakano H, Tamai K, Nomura K, Hanada K, Miyazaki J, Hashimoto I: "Promoter/enhancer cassettes for keratinocyte gene therapy"J Invest Dermatol. 112. 828-830 (1999)

Sawamura D、Meng X、Ina S、Nakano H、Tamai K、Nomura K、Hanada K、Miyazaki J、Hashimoto I：“角质形成细胞基因治疗的启动子/增强子盒”J Invest Dermatol。