Division of Molecular Dermatology, Department of Dermatology, Paracelsus Medical University Salzburg, Müllner Hauptstrasse 48, A-5020 Salzburg, Austria.
Mech Ageing Dev. 2010 May;131(5):346-53. doi: 10.1016/j.mad.2010.04.002. Epub 2010 Apr 18.
Keratin filaments form obligatory heterodimers consisting of one type I and one type II keratin that build the intermediate filaments. In keratinocytes, type II keratin 6 (K6) interacts with type I keratin 16 (K16). We previously showed that the intermediate filament protein K16 is up-regulated in aged human skin. Here, we report that there is an obvious imbalance of K16 to K6 mRNA in in vivo and in vitro aging, which possibly leads to cellular effects. To unveil a possible biological function of K16 overexpression we investigated the migration potential of keratinocytes having up-regulated K16 expression in vitro. Two cell lines were established by transfection of human keratinocytes (HaCaT cells) with K16 or control vectors and subsequent fluorescence-activated cell sorting. By performing migration assays we were able to show a 90% reduction in the migration ability of the K16-overexpressing keratinocytes. In addition, a delay in wound closure associated with K16-overexpressing cells was shown by scratch assays. Transient overexpression of K6A in K16-overexpressing keratinocytes partially corrected the cell-migration defect. By real-time PCR we excluded co-regulation of the annotated interaction partner, K6, in the K16 cell line. Finally, we observed a decreased level of tyrosine phosphorylation in K16-overexpressing cells. Taken together, these data highlight the possibility of a physiological role for K6/K16 heterodimers in keratinocyte cell migration, in addition to the heterodimer's known functions in cell differentiation and mechanical resilience.
角蛋白丝形成必需的异二聚体，由一个 I 型和一个 II 型角蛋白组成，构成中间丝。在角质形成细胞中，II 型角蛋白 6（K6）与 I 型角蛋白 16（K16）相互作用。我们之前表明，中间丝蛋白 K16 在衰老的人类皮肤中上调。在这里，我们报告说，在体内和体外衰老中，K16 与 K6 mRNA 存在明显的不平衡，这可能导致细胞效应。为了揭示 K16 过表达的可能生物学功能，我们研究了体外表达上调的 K16 的角质形成细胞的迁移潜力。通过用 K16 或对照载体转染人角质形成细胞（HaCaT 细胞）并随后进行荧光激活细胞分选，建立了两个细胞系。通过进行迁移测定，我们能够显示 K16 过表达的角质形成细胞的迁移能力降低了 90%。此外，划痕测定显示与 K16 过表达细胞相关的伤口闭合延迟。瞬时过表达 K6A 在 K16 过表达的角质形成细胞中部分纠正了细胞迁移缺陷。通过实时 PCR，我们排除了 K16 细胞系中注释的相互作用伙伴 K6 的共调节。最后，我们观察到 K16 过表达细胞中的酪氨酸磷酸化水平降低。总之，这些数据突出了 K6/K16 异二聚体在角质形成细胞迁移中的生理作用的可能性，除了异二聚体在细胞分化和机械弹性中的已知功能。
