Kuechle M K, Presland R B, Lewis S P, Fleckman P, Dale B A
Department of Oral Biology, University of Washington, Seattle, Washington, WA 98195, USA.
Cell Death Differ. 2000 Jun;7(6):566-73. doi: 10.1038/sj.cdd.4400687.
Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. We have previously shown that filaggrin constructs, when transiently transfected into epithelial cells, lead to a collapsed keratin cytoskeletal network and dysmorphic nuclei with features of apoptosis. The apparent transfection rate is low with filaggrin constructs, supporting their disruptive role but hindering further study. To bypass this problem, we generated stable keratinocyte cell lines that express mature human filaggrin using a tetracycline-inducible promoter system. We found that cell lines expressing filaggrin, but not control cell lines, exhibited increased sensitivity to multiple apoptotic stimuli as measured by morphologic and biochemical criteria. None of the cell lines showed an increase in endogenous expression of filaggrin in response to the same stimuli. Filaggrin expression alone was insufficient to induce apoptosis in these keratinocyte cell lines. We conclude that filaggrin, due to its keratin binding ability, primes cells for apoptosis. Because filaggrin is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, we hypothesize that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.
丝聚合蛋白是一种中间丝相关蛋白，在角质形成细胞终末分化过程中辅助角蛋白丝的组装。作为无活性前体的前丝聚合蛋白定位于体内透明角质颗粒中，其表达可防止角蛋白丝过早聚集。我们之前已表明，丝聚合蛋白构建体瞬时转染至上皮细胞时，会导致角蛋白细胞骨架网络塌陷以及细胞核出现具有凋亡特征的畸形。丝聚合蛋白构建体的表观转染率较低，这虽支持了它们的破坏作用，但阻碍了进一步研究。为绕过这一问题，我们利用四环素诱导型启动子系统构建了表达成熟人丝聚合蛋白的稳定角质形成细胞系。我们发现，通过形态学和生化标准检测，表达丝聚合蛋白的细胞系对多种凋亡刺激的敏感性增加，而对照细胞系则无此现象。在相同刺激下，所有细胞系的丝聚合蛋白内源性表达均未增加。仅丝聚合蛋白表达不足以在这些角质形成细胞系中诱导凋亡。我们得出结论，丝聚合蛋白因其与角蛋白的结合能力，使细胞为凋亡做好准备。由于丝聚合蛋白在角质形成细胞处于有核颗粒层与无核角质层之间转变的表皮水平表达，我们推测丝聚合蛋白通过促进凋亡机制辅助终末分化过程。
