A role of plakophilin 3 in keratinocyte proliferation

桥粒是细胞间的黏附接触，在皮肤和心脏等易受机械应变的组织中尤其丰富。我们的数据表明亲血小板蛋白家族的成员控制桥粒的粘附强度、大小和数量。亲血小板蛋白1在体内和体外增强细胞间粘附并增加桥粒大小，而亲血小板蛋白3使桥粒更具活力。与此一致的是，我们的小鼠基因敲除研究显示，亲血小板1型KO小鼠存在严重的皮肤脆性，具有产后致命性。此外，我们已经证明嗜血小板蛋白1和3在桥粒粘附、细胞增殖、细胞迁移和锚定独立生长之间起着开关作用。细胞质嗜血小板蛋白1通过与翻译起始复合体结合以增加蛋白质的生物合成来刺激增殖。这些调节功能的激活依赖于IGF1/胰岛素信号下游的Akt2激酶对嗜血小板蛋白1的磷酸化。磷酸化的嗜血小板蛋白1通过14-3-3被困在细胞质中，这干扰了它与桥粒的结合。我们发现，敲除plakophilin 3的角质形成细胞增殖率显著降低，这与plakophilin 3 KO小鼠的生长迟缓一致，而其过表达促进了角质形成细胞的增殖。我们的比较基因表达分析显示，在plakophilin 3 KO细胞中下调的所有基因中有30%与细胞周期相关，其中许多是E2F靶点。此外，我们发现嗜血小板蛋白3易位到有丝分裂纺锤体。有丝分裂纺锤体形成和动力学所必需的几种假定相互作用蛋白的鉴定表明在细胞周期进程中具有额外的作用。基于这些数据，我们提出了嗜血小板蛋白3参与细胞周期控制的假设：(a)通过控制E2F/Rb活性调节S期进入；(b)通过微管结合蛋白调节有丝分裂纺锤体。我们的项目旨在通过验证E2F靶基因调控和分析嗜血小板蛋白3可能通过影响E2F/Rb活性促进s期进入的分子机制来验证这一假设。此外，有丝分裂过程中嗜铂蛋白3的定位和蛋白相互作用，以及以细胞周期依赖的方式控制其定位和蛋白相互作用变化的机制将被研究。我们期望我们的研究将对理解桥粒黏附和增殖之间的相互作用产生重大影响，这种相互作用必须紧密平衡以保证表皮稳态。此外，我们将深入了解嗜血小板蛋白3在调节桥粒中所起的作用，使桥粒在有丝分裂过程中能够在不干扰内聚的情况下移动。

Desmosomes are intercellular adhesive contacts that are especially abundant in tissues prone to mechanical strain such as the skin and the heart. Our data suggest that members of the plakophilin family of desmosomal proteins control desmosome adhesive strength, size and number. Whereas plakophilin 1 strengthens intercellular adhesion and increases desmosome size in vivo and in vitro, plakophilin 3 renders desmosomes more dynamic. In agreement, our mouse knockout studies reveal severe skin fragility in plakophilin 1 KO mice with postnatal lethality. Moreover, we have shown that plakophilins 1 and 3 act as switches between desmosomal adhesion, cell proliferation, cell migration and anchorage independent growth. Cytoplasmic plakophilin 1 stimulates proliferation by associating with the translation initiation complex to increase protein biosynthesis. Activation of these regulatory functions depends on plakophilin 1 phosphorylation by the Akt2 kinase downstream of IGF1/insulin signaling. Phosphorylated plakophilin 1 becomes trapped in the cytoplasm by 14-3-3 which interferes with its incorporation into desmosomes. We found that plakophilin 3 knockout keratinocytes reveal significantly reduced proliferation rates, in agreement with growth retardation of plakophilin 3 KO mice, whereas its overexpression promotes proliferation of keratinocytes. Our comparative gene expression analysis revealed that 30% of all genes downregulated in plakophilin 3 KO cells are cell cycle related, many of them being E2F targets. Moreover, we find that plakophilin 3 translocates to the mitotic spindle. The identification of several putative interacting proteins essential for mitotic spindle formation and dynamics suggests an additional role in cell cycle progression. Based on these data we propose the hypothesis that plakophilin 3 is involved in cell cycle control by (a) regulating S phase entry via control of E2F/Rb activity and (b) regulating the mitotic spindle in association with microtubule binding proteins. Our project aims to verify this hypothesis by validation of E2F target gene regulation and an analysis of the molecular mechanism by which plakophilin 3 might influence E2F/Rb activity to promote S-phase entry. Moreover, plakophilin 3 localization and protein interactions during mitosis and mechanisms controlling its changes in localization and protein interactions in a cell cycle dependent manner will be studied.We expect that our studies will have a major impact on understanding the interplay between desmosomal adhesion and proliferation which has to be tightly balanced to guarantee epidermal homeostasis. Moreover, we will gain insight into the role of plakophilin 3 in modulating desmosomes to allow for mobility without interfering with cohesion during mitosis.