Elucidation of pathophysiology or loricrin keratoderma

洛丽克林是表皮角化细胞膜的主要成分。最近，在氯化角化病中发现了氯化氯化蛋白基因杂合子突变。我们先前已经证明，当野生型Clicrin结构被瞬时转染到HaCaT细胞中时，会导致caspase的激活和TUNEL染色阳性，并具有细胞凋亡的特征。Clicrin的表观转染率较低，支持其促凋亡作用，但阻碍了进一步的研究。为了绕过这个问题，我们使用蜕皮激素诱导的启动子系统建立了稳定的HaCaT细胞系，表达野生型和突变型Clicrin。表达突变型三氯氰菊酯的细胞系比表达野生型三氯氰菊酯的细胞系生长更快。突变的氯化蛋白的HaCaT细胞表达磷酸化形式的Akt。共聚焦免疫荧光显微镜观察显示，磷酸化Akt定位于核仁。在含有氯化突变蛋白的HaCaT细胞中，Akt激酶的活性约为未突变氯化蛋白或野生氯化氯化蛋白的细胞系的9倍。突变的氯蛋白细胞中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均被磷酸化。停靠蛋白GAB1和c-Cbl在突变的氯蛋白细胞中也是酪氨酸磷酸化的。野生型和突变型氯化蛋白细胞中p38MAPK和SAPK/JNK均未发生磷酸化。接下来，我们测量了培养上清液中血管内皮生长因子的浓度。在诱变后的HaCaT细胞中，培养上清液中的血管内皮生长因子的含量是野生型的3倍。此外，染色质免疫沉淀分析表明，在突变的氯化酪蛋白的HaCaT细胞中，STAT3蛋白与血管内皮生长因子启动子结合。因此，在HaCaT LK细胞模型中，血管内皮生长因子的释放和随后通过自分泌/旁分泌途径激活的VEGFR II将loricrin基因突变与细胞快速增殖联系在一起。

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.