KLF4上调角质形成细胞中Keratin 17表达的分子机制

  目的  通过探讨转录因子KLF4在调节角蛋白17（Keratin 17, KRT17）表达中的作用，揭示银屑病患者皮损中KRT17过度表达的分子机制。  方法  以18例寻常型银屑病患者皮损组织为银屑病组，10例健康人皮肤为对照组。采用实时荧光定量PCR和Western blot检测银屑病皮损组织及正常皮肤标本中KLF4的表达水平，检测HaCat细胞中KLF4过表达叠加组蛋白乙酰转移酶EP300（E1A binding protein p300，EP300）干扰后KRT17的表达水平。染色质免疫共沉淀（chromatin immunoprecipitation, ChIP）检测银屑病皮损组织及正常皮肤样本中KRT17启动子区KLF4结合水平及组蛋白H3乙酰化水平，检测HaCat细胞中KLF4过表达叠加EP300干扰后KRT17启动子区KLF4结合水平及组蛋白H3乙酰化水平。免疫共沉淀（co-immunoprecipitation, Co-IP）检测KLF4与EP300的相互作用。  结果  银屑病组皮损组织KLF4表达水平、KRT17启动子区KLF4结合水平及组蛋白H3乙酰化水平均高于对照组皮肤标本（P<0.01）。与转染对照组相比，KLF4过表达组KRT17表达水平升高（P<0.01）；KLF4过表达叠加EP300干扰组KRT17表达水平低于KLF4过表达组（P<0.01）和转染对照组（P<0.05）。与转染对照组相比，KLF4过表达组KRT17启动子区组蛋白H3乙酰化水平升高（P<0.01）；KLF4过表达叠加EP300干扰组KRT17启动子区组蛋白H3乙酰化水平低于KLF4过表达组（P<0.01）和对照组（P<0.01）。Co-IP证实KLF4与EP300能形成蛋白复合体。  结论  过度表达的KLF4通过协同EP300上调KRT17启动子区组蛋白H3乙酰化水平，介导银屑病患者皮损中KRT17的过度表达。

Molecular Mechanism of KLF4 Up-regulating Keratin 17 Expression in Keratinocytes

  Objective  To explore the role of Kruppel-like factor 4 (KLF4) in the regulation of Keratin 17 (KRT17) expression, and to reveal the molecular mechanism of overexpression of KRT17 in psoriatic lesions.  Methods  The skin lesions of 18 patients with psoriasis vulgaris were taken as experimental group and 10 healthy persons as control group. Real time-PCR and Western blot were used to detect the expression of KLF4 in psoriasis and normal skin samples, and the changes of KRT17 expression in HaCat cells after transfection of KLF4 overexpression and EP300 interfering plasmid. ChIP-qPCR was used to detect KLF4 binding and histone H3 acetylation levels in the promoter region of KRT17 in psoriasis and normal skin samples, and the changes of KLF4 binding and histone H3 acetylation levels in the promoter region of KRT17 in HaCat cells after transfection of KLF4 overexpression and EP300 interfering plasmid. Co-IP detects the interaction between KLF4 and EP300.  Results  The expression level of KLF4, KLF4 binding level and histone H3 acetylation level in the promoter region of KRT17 in psoriasis group were significantly higher than those in normal group (P<0.01). Compared with the control group, the expression level of KRT17 was significantly higher after KLF4 overexpression (P<0.01). After KLF4 overexpression combined with EP300 interference, the expression level of KRT17 was significantly lower than that of KLF4 overexpression group (P<0.01), slightly lower than that of control group (P<0.05). Compared with the control group, the histone H3 acetylation level in KRT17 promoter region in KLF4 over-expression group was increased significantly (P<0.01). After KLF4 over-expression combined with EP300 interference, the acetylation level of histone H3 in KRT17 promoter region was significantly lower than that in KLF4 overexpression group (P<0.01) and control group (P<0.01). Co-IP confirmed that KLF4 and EP300 could form protein complexes.  Conclusion  Excessive KLF4 increases the level of histone H3 acetylation in KRT17 promoter region by synergistic EP300, and mediates the over-expression of KRT17 in psoriatic lesions.