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- Title
Heat shock protein 27 activity is linked to endothelial barrier recovery after proinflammatory GPCR-induced disruption.
- Authors
Rada, Cara C.; Mejia-Pena, Hilda; Grimsey, Neil J.; Canto Cordova, Isabel; Olson, Joshua; Wozniak, Jacob M.; Gonzalez, David J.; Nizet, Victor; Trejo, JoAnn
- Abstract
Breaking down and building barriers: Ligand-dependent activation of proinflammatory GPCRs, such as the thrombin receptor PAR1, disrupts endothelial barriers, resulting in tissue edema. GPCR-mediated activation of the kinase p38 MAPK mediates endothelial barrier permeability. Rada et al. used mass spectrometry to show that the chaperone protein HSP27 was differentially phosphorylated by kinases downstream of p38 MAPK in thrombin-stimulated endothelial cells. Phosphorylation shifted HSP27 from an oligomeric to a monomeric state, resulting in enhanced endothelial barrier disruption. However, reassembly of HSP27 oligomers was required for endothelial barrier recovery. In mice, pharmacological inhibition of HSP27 function increased PAR1-induced vascular leakage. Together, these data suggest that the regulation of HSP27 phosphorylation and function affects the dynamics of endothelial barrier function. 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.
- Subjects
MOLECULAR chaperones; HEAT shock proteins; INFLAMMATORY mediators; MITOGEN-activated protein kinases; G protein coupled receptors; THROMBIN receptors
- Publication
Science Signaling, 2021, Vol 14, Issue 698, p1
- ISSN
1945-0877
- Publication type
Article
- DOI
10.1126/scisignal.abc1044