Vascular inflammation causes endothelial barrier disruption and tissue edema. Several inflammatory mediators act through G protein–coupled receptors (GPCRs), including protease-activated receptor-1 (PAR1), to elicit inflammatory responses. The activation of PAR1 by its ligand thrombin stimulates proinflammatory, p38 mitogen-activated protein kinase (MAPK) signaling that promotes endothelial barrier disruption. Through mass spectrometry phosphoproteomics, we identified heat shock protein 27 (HSP27), which exists as a large oligomer that binds to actin, as a promising candidate for the p38-mediated regulation of barrier integrity. Depletion of HSP27 by siRNA enhanced endothelial cell barrier permeability and slowed recovery after thrombin stimulation. We further showed that two effector kinases of p38 MAPK, MAPKAPK2 (MK2) and MAPKAPK3 (MK3), differentially phosphorylated HSP27 at Ser15, Ser78, and Ser82. Whereas inhibition of thrombin-stimulated p38 activation blocked HSP27 phosphorylation at all three sites, inhibition of MK2 reduced the phosphorylation of only Ser15 and Ser78. Inhibition of both MK2 and MK3 was necessary to attenuate Ser82 phosphorylation. Thrombin-stimulated p38-MK2-MK3 signaling induced HSP27 oligomer disassembly. However, a phosphorylation-deficient mutant of HSP27 exhibited defective oligomer disassembly and altered the dynamics of barrier recovery after thrombin stimulation. Moreover, blocking HSP27 oligomer reassembly with the small-molecule inhibitor J2 enhanced endothelial barrier permeability in vitro and vascular leakage in vivo in response to PAR1 activation. These studies reveal the distinct regulation of HSP27 phosphorylation and function induced by the GPCR-stimulated p38-MK2-MK3 signaling axis that controls the dynamics of endothelial barrier recovery in vitro and vascular leakage in vivo.

中文翻译：

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热休克蛋白 27 活性与促炎性 GPCR 诱导破坏后内皮屏障的恢复有关。


血管炎症导致内皮屏障破坏和组织水肿。多种炎症介质通过 G 蛋白偶联受体 (GPCR)（包括蛋白酶激活受体 1 (PAR1)）发挥作用，引发炎症反应。 PAR1 的配体凝血酶激活会刺激促炎性 p38 丝裂原激活蛋白激酶 (MAPK) 信号传导，从而促进内皮屏障破坏。通过质谱磷酸化蛋白质组学，我们鉴定了热休克蛋白 27 (HSP27)，它以与肌动蛋白结合的大寡聚物的形式存在，是 p38 介导的屏障完整性调节的有希望的候选者。通过 siRNA 消除 HSP27 可增强内皮细胞屏障通透性并减缓凝血酶刺激后的恢复。我们进一步表明，p38 MAPK 的两种效应激酶 MAPKAPK2 (MK2) 和 MAPKAPK3 (MK3) 在 Ser15、Ser78 和 Ser82 处差异磷酸化 HSP27。抑制凝血酶刺激的 p38 激活可阻断所有三个位点的 HSP27 磷酸化，而抑制 MK2 仅减少 Ser15 和 Ser78 的磷酸化。抑制 MK2 和 MK3 对于减弱 Ser82 磷酸化是必要的。凝血酶刺激的 p38-MK2-MK3 信号传导诱导 HSP27 寡聚体分解。然而，HSP27 的磷酸化缺陷突变体表现出有缺陷的寡聚体分解，并改变了凝血酶刺激后屏障恢复的动态。此外，用小分子抑制剂 J2 阻断 HSP27 寡聚体重新组装可增强体外内皮屏障通透性和体内响应 PAR1 激活的血管渗漏。 这些研究揭示了 GPCR 刺激的 p38-MK2-MK3 信号轴诱导的 HSP27 磷酸化和功能的独特调节，该信号轴控制体外内皮屏障恢复和体内血管渗漏的动态。