Thrombin selectively engages LIM kinase 1 and slingshot-1L phosphatase to regulate NF-κB activation and endothelial cell inflammation

Abstract

Endothelial cell (EC) inflammation is a central event in the pathogenesis of many pulmonary diseases such as acute lung injury and its more severe form acute respiratory distress syndrome. Alterations in actin cytoskeleton are shown to be crucial for NF-κB regulation and EC inflammation. Previously, we have described a role of actin binding protein cofilin in mediating cytoskeletal alterations essential for NF-κB activation and EC inflammation. The present study describes a dynamic mechanism in which LIM kinase 1 (LIMK1), a cofilin kinase, and slingshot-1Long (SSH-1L), a cofilin phosphatase, are engaged by procoagulant and proinflammatory mediator thrombin to regulate these responses. Our data show that knockdown of LIMK1 destabilizes whereas knockdown of SSH-1L stabilizes the actin filaments through modulation of cofilin phosphorylation; however, in either case thrombin-induced NF-κB activity and expression of its target genes (ICAM-1 and VCAM-1) is inhibited. Further mechanistic analyses reveal that knockdown of LIMK1 or SSH-1L each attenuates nuclear translocation and thereby DNA binding of RelA/p65. In addition, LIMK1 or SSH-1L depletion inhibited RelA/p65 phosphorylation at Ser(536), a critical event conferring transcriptional competency to the bound NF-κB. However, unlike SSH-1L, LIMK1 knockdown also impairs the release of RelA/p65 by blocking IKKβ-dependent phosphorylation/degradation of IκBα. Interestingly, LIMK1 or SSH-1L depletion failed to inhibit TNF-α-induced RelA/p65 nuclear translocation and proinflammatory gene expression. Thus this study provides evidence for a novel role of LIMK1 and SSH-1L in selectively regulating EC inflammation associated with intravascular coagulation.

G Protein–Coupled Receptor and RhoA-Stimulated Transcriptional Responses: Links to Inflammation, Differentiation, and Cell Proliferation

“…This leads to phosphorylation and inactivation of the cofilin phosphatase slingshot. Cofilin phosphorylation was recently implicated in thrombin-induced NFkB activation in endothelial cells (Leonard et al, 2013). Since slingshot is regulated by PKD phosphorylation (Fu and Rubin, 2011;Xiang et al, 2013), PKD effects on cofilin-mediated responses could play a role in the activation of NFkB.…”

“…Agonists shown to activate NFkB through RhoA signaling include neurotensin (Zhao et al, 2003), bradykinin (Pan et al, 1998), gastrin-releasing peptide , angiotensin (Cui et al, 2006), fMLP (Huang et al, 2001), thrombin (Kang et al, 2005;Kawanami et al, 2011;Dusaban et al, 2013;Leonard et al, 2013), S1P (Siehler et al, 2001), and LPA (Hwang et al, 2006). S1P activates NFkB in HEK293 cells through collaborative effects of RhoA and protein kinase C (PKC) activation (Siehler et al, 2001).…”

Novel regulators of endothelial barrier function

“…Conversely, endothelial activation and expression of adhesion molecules are essential prerequisites for neutrophil invasion and emigration. For example, endothelial knock-down or inhibition of calpains, a family of Ca 2ϩ -dependent, nonlysosomal cysteine proteases, reduces endothelial NO synthase (NOS3)-mediated NO production, subsequent phosphorylation of ICAM-1, and thereby, neutrophil recruitment as well as lung edema and protein extravasation (80). Similarly, deficiency in the polymodal cation channel TRPV4, which is highly expressed in pulmonary endothelial (95) and smooth muscle cells (29, 157) but seemingly absent in neutrophils (8), prevented both edema formation and neutrophil invasion in murine models of chlorine gas-and acid aspiration-induced lung injury (8).…”

“…An abundant literature has documented that triggers such as thrombin, H 2 O 2 , or LPS can increase endothelial permeability in vitro in the absence of immune cells. Recent data by Leonard and colleagues (80) demonstrate that the underlying dynamic regulation of the actin cytoskeleton not only mediates the endothelial permeability response to thrombin but also modulates in turn proinflammatory signaling pathways including NF-B. Modulation of actin filament stability through phosphorylation or dephosphorylation of the actin binding protein cofilin regulates not only endothelial barrier properties but also NF-B activity and expression of its target genes such as ICAM-1.…”

Activation of PAR-1/NADPH Oxidase/ROS Signaling Pathways is Crucial for the Thrombin-Induced sFlt-1 Production in Extravillous Trophoblasts: Possible Involvement in the Pathogenesis of Preeclampsia

“…Thrombin is a multifunctional serine protease generated at the site of vascular injury that transforms fibrinogen into fibrin, activates blood platelets and elicits multiple effects on a variety of cell types including extravillous trophoblast cells [11], endothelial cells [12], vascular smooth muscle cells [13], and monocytes [14]. It is widely known that uteroplacental hemorrhage [15], fibrin deposition [16], and infarction [17] are commonly observed in established preeclampsia.…”