Study of pathophysiology of keratin disease

单纯大疱性表皮松解症（EBS）是一种以蛋白质聚集为特征的皮肤起泡性疾病。在此，我们研究了EBS细胞模型中蛋白质聚集与细胞死亡的分子机制，其中HaCaT角化细胞转染表达各种角蛋白14 （K14）突变形式的质粒。在HaCaT细胞中，突变体K14形成抑制20S蛋白酶体功能的蛋白聚集体，而不是20S蛋白酶体。突变K14的角质形成细胞诱导激酶c-Jun磷酸化，以及展开蛋白Bip上调，表明内质网（ER）应激。HaCaT细胞易受caspase- 3和-8的影响而不受caspase-9和-12的影响。与野生K14相比，突变K14的角质形成细胞的培养基中肿瘤坏死因子-α(TNFα)升高，在培养基中添加中和性抗TNFα抗体可使角质形成细胞免于死亡。因此，在这种角化细胞EBS细胞模型中，TNFα释放和随后通过自分泌/旁分泌途径激活TNFα受体将蛋白聚集与细胞死亡联系起来。此外，K14突变降低了其对TNFα受体相关死亡结构域（TRADD）的亲和力，这表明由于细胞保护机制受损，突变的K14中角质形成细胞对caspase-8介导的凋亡的易感性增加

Epidermolysis bullosa simplex (EBS) is a blistering cutaneous disease featuring protein aggregates. Here we investigate the molecular mechanisms linking protein aggregates to cell death in a cellular model of EBS in which HaCaT keratinocytes are transfected with plasmids expressing various mutant forms of keratin 14 (K14). In HaCaT cells, mutant K14 was found to form protein aggregates that suppressed 20S proteasome function instead of being 20S proteasome. Keratinocytes with mutant K14 induced phosphorylation of the kinase c-Jun, as well as up-regulation of unfolding protein Bip, indicating endoplasmic reticulum (ER) stress. HaCaT cells were susceptible to apoptosis by caspases-3, and -8, but not caspase-9 or -12. Tumor necrosis factor-α(TNFα) in the culture medium was increased in keratinocytes with mutant K14 compared to wild K14, and the addition of neutralizing anti-TNFα antibody to the culture medium rescued keratinocytes from cell death. Thus, TNFα release and the subsequent activation of the TNFα receptor by an autocrine/paracrine pathway links protein aggregates to cell death in this keratinocyte EBS cellular model. Furthermore, mutation in K14 reduced its affinity to TNFα receptor-associated death domain (TRADD), suggesting that the susceptibility of keratinocytes to caspase-8-mediated apoptosis is increased in mutated K14 due to impairment of the cytoprotective mechanism