Elucidation of pathophysiology or loricrin keratoderma

阐明兜甲角化病的病理生理学

基本信息

批准号：
18591250
负责人：
YONEDA Kozo
金额：
$ 2.82万
依托单位：
Kagawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2009
项目状态：
已结题

项目摘要

Loricrin is a major constituent of the epidermal cornified cell envelope. Recently, heterozygous loricrin gene mutations have been indentified in loricrin keratoderma. We have previously shown that the wild loricrin construct, when transiently transfected into HaCaT cells, leads to the activation of caspases and positive TUNEL staining with features of apoptosis. The apparent transfection rate is low with loricrin construct, supporting its apoptotic role but hindering further study. To bypass this problem, we generated stable HaCaT cell lines that expressed wild and mutant loricrin using an ecdysone-inducible promoter system. The cell lines expressing mutant loricrin grew more rapidly than those expressing wild loricrin. HaCaT cells with mutant loricrin express phosphorylated forms of Akt. The confocal immunofluorescence microscopic observation reveals that phospho-Akt localizes at nucleolus. The activity of Akt kinase is about 9 times higher in HaCaT cells with mutant loricrin than those in the cell lines with mock or wild loricrin. ERK1/2, epidermal growth factor receptor, vascular endothelial cell growth factor receptor (VEGFR) II and Stat 3 are all phosphorylated in mutant loricrin cells. The docking proteins, Gab1 and c-Cbl, are also tyrosine-phosphorylated in mutant loricrin cells. Neither p38 MAP kinase nor SAPK/JNK is phosphorylated in both wild and mutant loricrin cells. Next we measured the concentration of VEGF in a culture medium. VEGF in the culture medium is about 3 times more abundant in HaCaT cells with mutant loricrin compared with wild loricrin. Furthermore, chromatin immunoprecipitation assays indicate that Stat3 protein binds to the VEGF promoter in HaCaT cells with mutant loricrin. Thus, VEGF release and the subsequent activation of the VEGFR II by an autocrine/paracrine pathway link loricrin gene mutation to rapid cell proliferation in the HaCaT LK cellular model.

Loricrin 是表皮角化细胞包膜的主要成分。最近，在兜甲角化病中发现杂合兜甲基因突变。我们之前已经证明，野生兜甲蛋白构建体在瞬时转染到 HaCaT 细胞中时，会导致半胱天冬酶的激活和具有细胞凋亡特征的阳性 TUNEL 染色。 Loricrin 构建体的表观转染率较低，支持其细胞凋亡作用，但阻碍了进一步研究。为了绕过这个问题，我们使用蜕皮激素诱导启动子系统生成了稳定的 HaCaT 细胞系，该细胞系表达野生型和突变型兜甲素。表达突变型兜甲蛋白的细胞系比表达野生兜甲蛋白的细胞系生长得更快。具有突变型 loricrin 的 HaCaT 细胞表达磷酸化形式的 Akt。共聚焦免疫荧光显微镜观察表明磷酸-Akt 定位于核仁。具有突变体甲蛋白的 HaCaT 细胞中的 Akt 激酶活性比具有模拟或野生甲蛋白的细胞系中的活性高约 9 倍。 ERK1/2、表皮生长因子受体、血管内皮细胞生长因子受体 (VEGFR) II 和 Stat 3 在突变型 loricrin 细胞中均被磷酸化。对接蛋白 Gab1 和 c-Cbl 在突变型 loricrin 细胞中也被酪氨酸磷酸化。 p38 MAP 激酶和 SAPK/JNK 在野生型和突变型 loricrin 细胞中均不被磷酸化。接下来我们测量了培养基中 VEGF 的浓度。与野生兜甲蛋白相比，突变兜甲蛋白的 HaCaT 细胞中培养基中的 VEGF 含量高出约 3 倍。此外，染色质免疫沉淀分析表明，Stat3 蛋白在具有突变型 loricrin 的 HaCaT 细胞中与 VEGF 启动子结合。因此，在 HaCaT LK 细胞模型中，VEGF 释放和随后通过自分泌/旁分泌途径激活 VEGFR II 将 loricrin 基因突变与快速细胞增殖联系起来。