Snyder Jessica L, McBeath Elena, Thomas Tamlyn N, Chiu Yi Jen, Clark Robert L, Fujiwara Keigi
Department of Biomedical Engineering, University of Rochester, Rochester, NY, 14611, USA.
Department of Cardiology, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA.
Biol Cell. 2017 Aug;109(8):312-321. doi: 10.1111/boc.201600079. Epub 2017 Jul 13.
Vascular endothelial cells (ECs) are a well-known cell system used in the study of mechanobiology. Using cultured ECs, we found that platelet EC adhesion molecule 1 (PECAM-1, CD31), a cell adhesion protein localised to regions of EC-EC contact, was rapidly tyrosine phosphorylated in ECs exposed to shear or cyclic stretch. Src-homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) binds phosphorylated PECAM-1 and activates the extracellular signal-regulated kinase1/2 (ERK1/2) signalling cascade, a known flow-activated signalling pathway.
Although PECAM-1 tyrosine phosphorylation is characterised in ECs exposed to fluid shear stress, it is less well demonstrated in the cells stretched cyclically. Thus, we first show that PECAM-1 is tyrosine-phosphorylated in ECs cyclically stretched. We hypothesise that when an external force is applied to a monolayer of ECs, the force is directly transmitted to PECAM-1 which is then stretched and phosphorylation sites in its cytoplasmic domain are exposed and phosphorylated. This hypothesis requires the presence of any stretchable structure within the PECAM-1 cytoplasmic domain. Force spectroscopy measurements were performed with a construct containing cytoplasmic PECAM-1 domains inserted between I27 motifs, a recombinant string of the structural elements from titin. This strategy allowed us to identify the events in which a single molecule is being pulled and to detect the unravelling of the cytoplasmic domain of PECAM-1 by force. The response by PECAM-1 to mechanical loading was heterogeneous but with magnitudes as high as or higher than the naturally force bearing I27 domains.
The PECAM-1 cytoplasmic domain has a structure that can be unfolded by externally applied force and this unfolding of PECAM-1 may be necessary for its phosphorylation, the first step of PECAM-1 mechanosignalling.
When EC monolayers are mechanically stimulated, the PECAM-1 found at EC contacts is phosphorylated. We have proposed that under these conditions, the cytoplasmic domain of PECAM-1 is unfolded, which then exposes a phosphorylation site, allowing it to be accessed. The stretch induced unfolding is essential to this model of PECAM-1 mechanosignalling. In this study, we investigate whether the cytoplasmic domain of PECAM-1 has a stretchable structure, and the results are in line with our hypothesis.
血管内皮细胞（ECs）是用于机械生物学研究的一种广为人知的细胞系统。利用培养的内皮细胞，我们发现血小板内皮细胞黏附分子1（PECAM-1，CD31），一种定位于内皮细胞-内皮细胞接触区域的细胞黏附蛋白，在受到剪切力或周期性拉伸的内皮细胞中会迅速发生酪氨酸磷酸化。含Src同源2结构域的蛋白酪氨酸磷酸酶2（SHP2）结合磷酸化的PECAM-1并激活细胞外信号调节激酶1/2（ERK1/2）信号级联反应，这是一种已知的血流激活信号通路。
尽管PECAM-1酪氨酸磷酸化在受到流体剪切应力的内皮细胞中有特征描述，但在周期性拉伸的细胞中其表现尚不充分。因此，我们首先表明PECAM-1在周期性拉伸的内皮细胞中发生酪氨酸磷酸化。我们假设，当外力施加于单层内皮细胞时，该力直接传递给PECAM-1，使其被拉伸，然后其胞质结构域中的磷酸化位点暴露并被磷酸化。这一假设要求PECAM-1胞质结构域内存在任何可拉伸结构。使用一种构建体进行力谱测量，该构建体包含插入I27基序之间的PECAM-1胞质结构域，I27基序是肌联蛋白结构元件的重组序列。这一策略使我们能够识别单个分子被牵拉的事件，并检测到通过力使PECAM-1胞质结构域解折叠。PECAM-1对机械负荷的反应是异质性的，但幅度高达或高于天然承受力的I27结构域。
PECAM-1胞质结构域具有可被外力解折叠的结构，这种PECAM-1的解折叠可能是其磷酸化所必需的，这是PECAM-1机械信号传导的第一步。
当内皮细胞单层受到机械刺激时，在内皮细胞接触处发现的PECAM-1会发生磷酸化。我们提出，在这些条件下，PECAM-1的胞质结构域会解折叠，然后暴露出一个磷酸化位点，使其能够被磷酸化。拉伸诱导的解折叠对于PECAM-1机械信号传导模型至关重要。在本研究中，我们研究了PECAM-1的胞质结构域是否具有可拉伸结构，结果与我们的假设一致。
