Department of General, Visceral, Transplant, Vascular and Paediatric Surgery University Hospital Würzburg, Wuerzburg 97080, Germany.
Comprehensive Heart Failure Center and Department of Medicine I, University Hospital Würzburg, Würzburg, Germany.
Tissue Barriers. 2023 Oct 2;11(4):2138061. doi: 10.1080/21688370.2022.2138061. Epub 2022 Oct 24.
Previous data provided evidence for a critical role of desmosomes to stabilize intestinal epithelial barrier (IEB) function. These studies suggest that desmosomes not only contribute to intercellular adhesion but also play a role as signaling hubs. The contribution of desmosomal plaque proteins plakophilins (PKP) in the intestinal epithelium remains unexplored. The intestinal expression of PKP2 and PKP3 was verified in human gut specimens, human intestinal organoids as well as in Caco2 cells whereas PKP1 was not detected. Knock-down of PKP2 using siRNA in Caco2 cells resulted in loss of intercellular adhesion and attenuated epithelial barrier. This was paralleled by changes of the whole desmosomal complex, including loss of desmoglein2, desmocollin2, plakoglobin and desmoplakin. In addition, tight junction proteins claudin1 and claudin4 were reduced following the loss of PKP2. Interestingly, siRNA-induced loss of PKP3 did not change intercellular adhesion and barrier function in Caco2 cells, while siRNA-induced loss of both PKP2 and PKP3 augmented the changes observed for reduced PKP2 alone. Moreover, loss of PKP2 and PKP2/3, but not PKP3, resulted in reduced activity levels of protein kinase C (PKC). Restoration of PKC activity using Phorbol 12-myristate 13-acetate (PMA) rescued loss of intestinal barrier function and attenuated the reduced expression patterns of claudin1 and claudin4. Immunostaining, proximity ligation assays and co-immunoprecipitation revealed a direct interaction between PKP2 and PKC. In summary, our in vitro data suggest that PKP2 plays a critical role for intestinal barrier function by providing a signaling hub for PKC-mediated expression of tight junction proteins claudin1 and claudin4.
先前的数据提供了证据，证明桥粒对于稳定肠道上皮屏障（IEB）功能具有关键作用。这些研究表明，桥粒不仅有助于细胞间的黏附，而且还作为信号枢纽发挥作用。桥粒斑蛋白 plakophilin（PKP）在肠道上皮中的作用仍未被探索。在人类肠道标本、人类肠道类器官和 Caco2 细胞中验证了 PKP2 和 PKP3 的肠道表达，而 PKP1 未被检测到。在 Caco2 细胞中使用 siRNA 敲低 PKP2 导致细胞间黏附丧失和上皮屏障减弱。这与整个桥粒复合物的变化平行，包括桥粒蛋白 2、桥粒蛋白 2、桥粒蛋白聚糖和桥粒斑蛋白的丢失。此外，紧密连接蛋白 Claudin1 和 Claudin4 的减少紧随 PKP2 的丢失之后。有趣的是，siRNA 诱导的 PKP3 丢失不会改变 Caco2 细胞中的细胞间黏附和屏障功能，而 siRNA 诱导的 PKP2 和 PKP3 丢失会增强单独减少 PKP2 时观察到的变化。此外，只有 PKP2 和 PKP2/3 的丢失而不是 PKP3 的丢失会导致蛋白激酶 C（PKC）的活性水平降低。使用佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）恢复 PKC 活性可挽救肠道屏障功能的丧失，并减弱 Claudin1 和 Claudin4 的表达模式降低。免疫染色、邻近连接测定和共免疫沉淀显示 PKP2 和 PKC 之间存在直接相互作用。总之，我们的体外数据表明，PKP2 通过为 PKC 介导的紧密连接蛋白 Claudin1 和 Claudin4 的表达提供信号枢纽，在肠道屏障功能中发挥关键作用。
