Mattiussi Stefania, Matsumoto Kazue, Illi Barbara, Martelli Fabio, Capogrossi Maurizio C, Gaetano Carlo
Laboratorio di Patologia Vascolare, Istituto Dermopatico dell' Immacolata, Roma, Italy.
Cardiovasc Res. 2006 Jun 1;70(3):578-88. doi: 10.1016/j.cardiores.2006.02.008. Epub 2006 Feb 28.
To investigate how endothelial cells transduce intracellular signals in response to laminar shear stress (SS), we made use of the papilloma virus oncoprotein E6 which interacts with and induces degradation of numerous cellular proteins including p53 and members of the PDZ-domain family. E6 also recognizes paxillin (PXN), a fundamental component of focal adhesions, interfering with its association to focal adhesion kinase (FAK).
Human umbilical vein endothelial cells, expressing E6 or its mutated variant DeltaE6(105-110) (DeltaE6) which does not inactivate p53, were cultured under static conditions or exposed to a laminar SS of 12 dyn/cm(2) for 16h. In response to SS, cells expressing E6 or DeltaE6 failed to synthesise nitric oxide and directionally remodel their cytoskeleton, as indicated by morphology and phalloidin staining of actin microfilaments. Under these conditions, PXN association with FAK, its localization to the plasma membrane, and its phosphorylation on tyrosine-31, which partially encompasses the PXN/FAK docking site, were severely compromised. These alterations were paralleled by the impairment of important SS-dependent endothelial functions, including nitric oxide production and survival upon serum deprivation. The direct targeting of PXN expression by RNA interference partially reproduced the E6 phenotype, impairing flow-dependent cell orientation and survival but not nitric oxide production.
These results provide evidence that papilloma virus E6 protein interferes with the function of the SS-mechanosensor and suggests a potential a role for PXN in this process.
为了研究内皮细胞如何响应层流切应力（SS）转导细胞内信号，我们利用了乳头瘤病毒癌蛋白E6，它与包括p53和PDZ结构域家族成员在内的多种细胞蛋白相互作用并诱导其降解。E6还识别桩蛋白（PXN），这是粘着斑的一个基本组成部分，干扰其与粘着斑激酶（FAK）的结合。
表达E6或其不使p53失活的突变变体DeltaE6(105 - 110)（DeltaE6）的人脐静脉内皮细胞在静态条件下培养或暴露于12达因/平方厘米的层流SS下16小时。响应SS时，表达E6或DeltaE6的细胞未能合成一氧化氮，也未能如肌动蛋白微丝的形态和鬼笔环肽染色所示定向重塑其细胞骨架。在这些条件下，PXN与FAK的结合、其在质膜上的定位以及其酪氨酸-31位点的磷酸化（该位点部分包含PXN/FAK对接位点）均受到严重损害。这些改变与重要的SS依赖性内皮功能受损平行，包括一氧化氮产生和血清剥夺后的存活能力。通过RNA干扰直接靶向PXN表达部分再现了E6表型，损害了血流依赖性细胞定向和存活能力，但不影响一氧化氮产生。
这些结果提供了证据表明乳头瘤病毒E6蛋白干扰了SS机械传感器的功能，并提示PXN在此过程中可能发挥作用。
