Elucidation of pathophysiology or loricrin keratoderma

Loricrin是表皮角化细胞包膜的主要成分。近年来，在loricrin角化病中发现了loricrin基因杂合突变。我们之前已经证明，野生loricrin构建体在瞬间转染到HaCaT细胞时，会导致半胱天冬酶的激活和TUNEL阳性染色，并具有凋亡的特征。loricrin结构体转染率低，支持其凋亡作用，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启动子系统生成了稳定的HaCaT细胞系，这些细胞系表达野生和突变的loricrin。表达突变loricrin的细胞系比表达野生loricrin的细胞系生长更快。loricrin突变的HaCaT细胞表达磷酸化形式的Akt。共聚焦免疫荧光显微镜观察发现磷酸化akt定位于核仁。在含有突变loricrin的HaCaT细胞中，Akt激酶的活性比含有模拟或野生loricrin的细胞系高约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在突变的loricrin细胞中都被磷酸化。在突变的loricrin细胞中，对接蛋白Gab1和c-Cbl也被酪氨酸磷酸化。在野生和突变的loricrin细胞中，p38 MAP激酶和SAPK/JNK都没有磷酸化。接下来，我们测量了培养液中VEGF的浓度。在突变型loricrin HaCaT细胞中，培养液中VEGF的含量是野生loricrin的3倍左右。此外，染色质免疫沉淀试验表明，Stat3蛋白在loricrin突变的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基因突变与细胞快速增殖联系起来。

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.