Molecular mechanism of the regulation of type VII collagen gene expression : Elucidation of pathogenesis and development therapy for dystophic epidermolysis bullosa

调节VII型胶原蛋白基因表达的分子机制：阐明营养不良性大疱性表皮松解症的发病机制和发育治疗

• 批准号: 11670814
• 负责人: KON Atsushi
• 金额: $ 2.3万
• 依托单位: HIROSAKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670814/
• 关键词: COL7A1; Smad; NF-κB; AP-1; basement membrane; dystrophic epidermolsis bullosa; auchoring fibril; cytokine; NF-κB

Type VII collagen is the predominat component of the anchoring fibrils, attachment structures stabilizing the association of the cutaneous basement membrane to the underlying dermis. Alterations in the type VII collagen protein structure or luck of its expression due to mutations in the corresponding gene COL7A1 are the hallmark of dystrophic epidermolysis bullosa, a mechano-bullous skin disease characterized by extreme fragility of the skin and leading to development of sub-lamina densa blisters.1) It has been previously demonstrated that various cytokines (TGF-β, TNF-α, IL-1 β) upregulate the expression of COL7A1 in dermal fibroblasts. To determine the effect of various cytokines on COL7A1 expression in keratinocytes, we performed northern blot hybridization with COL7A1 cDNA probes. The results showed the down-regulation of COL7A1 expression by TNF-α, or IL-1 β in keratinocytes, wherase TGF-β up-regulates COL7A1 expression as shown in previous paper. These results indicate that effec … More t of TNF-α or IL-1 β on COL7A1 expression is cell-type specific. 2) To gain insight into the molecular mechanism underlying the up-regulation of COL7A1 by two cytokines (TNF-α, IL-1 β) on COL7A1 transcription in dermal fibroblast, we performed transient cell transfection with a series of 5'-deletion COL7A1 promoter/luciferase reporter gene constructs, and electrophoresis mobility shift assays with an oligonucleotide spanning the response element of each cytokines. The results showed that TNF-α effect was mediated by RelA (p65) homodimer binding the TNF-α response element in the COL7A1 promoter. Furthermore, an additive effect was observed when TGF-β and INF-α were added simultaneously to the dermal fibroblasts. IL-1 β up-regulates the COL7A1 expression mediated by NF-κB and AP-1 binding the NF-κB binding site (as an enhancer) and AP-1 binding site (as a suppresor) in the COL7A1 promoter region, respectively. 3) DEB is an inherited skin disorder caused by COL7A1 mutations. Over 100 mutations within exons or exon-intron borders of COL7A1 have been publishied. However, no mutation has been detected in COL7A1 promoter region. To examine the COL7A1 mutation in promoter region, we rescreened COL7A1 of DEB patients whose mutatons have not been detected using PCR amplification, followed by heteroduplex analysis. The results of direct sequencing of all 118 exons and their flanking intronic sequences showed that no mutation was detected in COL7A1 promoter region. However, one novel pathogenic mutation 7687-6 C→T was identified in intron 102 of COL7A1. Less

VII型胶原蛋白是锚定原纤维的主要成分，锚定原纤维是稳定皮肤基底膜与下层真皮结合的附着结构。VII型胶原蛋白结构的改变或由于相应基因COL 7A 1中的突变而导致的其表达的运气是营养不良性大疱性表皮病的标志，所述营养不良性大疱性表皮病是一种机械性大疱性皮肤病，其特征在于皮肤的极端脆弱性并导致致密层下水疱的发展。（TGF-β、TNF-α、IL-1 β）上调真皮成纤维细胞COL 7A 1的表达。为了确定各种细胞因子对角质形成细胞中COL 7A 1表达的影响，我们用COL 7A 1 cDNA探针进行了北方印迹杂交。结果显示，如前所述，TNF-α或IL-1 β下调角质形成细胞中COL 7A 1的表达，而TGF-β上调COL 7A 1的表达。这些结果表明， ...更多信息 TNF-α或IL-1 β对COL 7A 1表达的影响具有细胞类型特异性。2)为了深入了解两种细胞因子（TNF-α、IL-1 β）上调真皮成纤维细胞中COL 7A 1转录的分子机制，我们用一系列5 '-缺失的COL 7A 1启动子/荧光素酶报告基因构建体进行瞬时细胞转染，并用跨越每种细胞因子反应元件的寡核苷酸进行电泳迁移率变化分析。结果表明，TNF-α的作用是由RelA（p65）同二聚体与COL 7A 1启动子中的TNF-α反应元件结合而介导的。此外，当TGF-β和INF-α同时加入真皮成纤维细胞时，观察到累加效应。IL-1 β通过NF-κB和AP-1分别结合COL 7A 1启动子区的NF-κB结合位点（作为增强子）和AP-1结合位点（作为抑制子）上调COL 7A 1的表达。3)DEB是一种由COL 7A 1突变引起的遗传性皮肤病。已经发现了COL 7A 1的外显子或外显子-内含子边界内的100多个突变。而在COL 7A 1启动子区未检测到突变。为了检测COL 7A 1基因启动子区的突变，我们对未检测到突变的DEB患者进行了COL 7A 1基因的PCR扩增，然后进行异源双链分析。对所有118个外显子及其侧翼内含子序列的直接测序结果表明，在COL 7A 1启动子区未检测到突变。然而，在COL 7A 1的内含子102中发现了一个新的致病突变7687-6 C→T。少

项目成果

Hashimoto I, Kon A, Tamai K, Uitto J: "Diagnostic dilemma of "sporadic" cases of dystrophic epidermolysis bullosa : a new dominant or mitis recessive mutation?"Exp Dermatol. 8. 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。

Tamai K: "Recurrent COL7A1 mutations in Japanese patients with dystrophic epidermolysis bullosa : Positional effects of premature termination codon mutations on clinical severity."J Invest Dermatol. 112. 991-993 (1999)

Tamai K：“日本营养不良性大疱性表皮松解症患者中复发的 COL7A1 突变：提前终止密码子突变对临床严重程度的位置影响。”J Invest Dermatol。

Kon A,Sawamura D,Itai K,Tamai K,Hashimoto I: "New direction for cellular and organ transplantation"Elsevier Science, Amsterdam. (2000)

Kon A、Sawamura D、Itai K、Tamai K、Hashimoto I：“细胞和器官移植的新方向”Elsevier Science，阿姆斯特丹。

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

玉井 K、村井 T、真山 M、Kon 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、石田山本 A、池田 S、小川 H、麦格拉斯 JA、

Kon A: "Gene therapy of dystrophic epidermolysis bullosa by introduction of type VII collagen gene to keratinocytes."New direction for cellular and organ transplantation.. 175-183 (2000)

Kon A：“通过将 VII 型胶原基因引入角质形成细胞来治疗营养不良性大疱性表皮松解症。”细胞和器官移植的新方向.. 175-183 (2000)