Abstract
Microtubules (MT) play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC) barrier by hepatocyte growth factor (HGF) which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics.
Highlights
Enhancement of the endothelial cell (EC) peripheral actin cytoskeleton and increased assembly of cell adhesive complexes by barrier protective agonists provide a structural basis for the maintenance of vascular barrier integrity and prevent catastrophic consequences of uncontrolled vascular leakiness in the lung or other organs caused by bacterial pathogens, cytokine storm accompanying sepsis, trauma, or excessive mechanical forces [1,2,3,4,5,6].Hepatocyte growth factor (HGF) is a multifunctional mesenchyme-derived pleiotropic factor secreted by several cell types
Antibodies to phospho-myosinassociated phosphatase (MYPT), GEF-H1, PAK1, phosphoMLC, phospho-Y421 cortactin were from Cell Signaling (Beverly, MA); stathmin, and End-Binding protein-1 (EB1) were from BD Transduction Laboratories (San Diego, CA); Rac1, RhoA, His-tag were from Santa Cruz Biotechnology (Santa Cruz, CA)
To assess HGF effects on peripheral MT density and growth, control and HGF-stimulated EC were fixed with methanol and subjected to immunofluorescence staining with antibody to btubulin or End-Binding protein-1 (EB1) which tracks the growing plus end of microtubules
Summary
Enhancement of the endothelial cell (EC) peripheral actin cytoskeleton and increased assembly of cell adhesive complexes by barrier protective agonists provide a structural basis for the maintenance of vascular barrier integrity and prevent catastrophic consequences of uncontrolled vascular leakiness in the lung or other organs caused by bacterial pathogens, cytokine storm accompanying sepsis, trauma, or excessive mechanical forces [1,2,3,4,5,6].Hepatocyte growth factor (HGF) is a multifunctional mesenchyme-derived pleiotropic factor secreted by several cell types. Enhancement of the endothelial cell (EC) peripheral actin cytoskeleton and increased assembly of cell adhesive complexes by barrier protective agonists provide a structural basis for the maintenance of vascular barrier integrity and prevent catastrophic consequences of uncontrolled vascular leakiness in the lung or other organs caused by bacterial pathogens, cytokine storm accompanying sepsis, trauma, or excessive mechanical forces [1,2,3,4,5,6]. HGF enhances basal EC monolayer barrier properties and exhibits potent protective effects against vascular endothelial barrier compromise induced by agonists and pathologically relevant mechanical forces [10,11,12]. HGF-induced EC barrier protection is mediated by PI3-kinase-dependent activation of RacGTPase signaling [13], which causes downregulation of the barrier disruptive RhoA pathway [11]. The precise mechanism of HGF-induced negative Rac-Rho crosstalk remains poorly understood
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