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      RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response

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          Abstract

          Background

          The role of advanced glycation end products (AGEs) in the development of diabetes, especially diabetic complications, has been emphasized in many reports. Accumulation of AGEs in the vasculature triggers a series of morphological and functional changes in endothelial cells (ECs) and induces an increase of endothelial permeability. This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by AGEs.

          Methods

          Using human dermal microvascular endothelial cells (HMVECs), the effects of human serum albumin modified-AGEs (AGE-HSA) on the endothelium were assessed by measuring monolayer permeability and staining of F-actin in HMVECs. Activations of RhoA and ROCK were determined by a luminescence-based assay and immunoblotting. Transfection of recombinant adenovirus that was dominant negative for RhoA (RhoA N19) was done to down-regulate RhoA expression, while adenovirus with constitutively activated RhoA (RhoA L63) was transfected to cause overexpression of RhoA in HMVECs. H-1152 was employed to specifically block activation of ROCK. Co-immunoprecipitation was used to further confirm the interaction of ROCK and its downstream target moesin. To identify AGE/ROCK-induced phosphorylation site in moesin, two mutants pcDNA3/HA-moesinT 558A and pcDNA3/HA-moesinT 558D were applied in endothelial cells.

          Results

          The results showed that AGE-HSA increased the permeability of HMVEC monolayer and triggered the formation of F-actin-positive stress fibers. AGE-HSA enhanced RhoA activity as well as phosphorylation of ROCK in a time- and dose-dependent manner. Down-regulation of RhoA expression with RhoA N19 transfection abolished these AGE-induced changes, while transfection of RhoA L63 reproduced the AGE-evoked changes. H-1152 attenuated the AGE-induced alteration in monolayer permeability and cytoskeleton. The results also confirmed the AGE-induced direct interaction of ROCK and moesin. Thr558 was further identified as the phosphorylating site of moesin in AGE-evoked endothelial responses.

          Conclusion

          These results confirm the involvement of RhoA/ROCK pathway and subsequent moesin Thr558 phosphorylation in AGE-mediated endothelial dysfunction.

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          Regulation of endothelial permeability via paracellular and transcellular transport pathways.

          Yulia Komarova, Asrar B. Malik (2010)
          The endothelium functions as a semipermeable barrier regulating tissue fluid homeostasis and transmigration of leukocytes and providing essential nutrients across the vessel wall. Transport of plasma proteins and solutes across the endothelium involves two different routes: one transcellular, via caveolae-mediated vesicular transport, and the other paracellular, through interendothelial junctions. The permeability of the endothelial barrier is an exquisitely regulated process in the resting state and in response to extracellular stimuli and mediators. The focus of this review is to provide a comprehensive overview of molecular and signaling mechanisms regulating endothelial barrier permeability with emphasis on the cross-talk between paracellular and transcellular transport pathways.
          Article 0 comments Cited 191 times – based on 0 reviews     Review now
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            Rho-Kinase Phosphorylates COOH-terminal Threonines of Ezrin/Radixin/Moesin (ERM) Proteins and Regulates Their Head-to-Tail Association

            Takeshi Matsui, Masato Maeda, Yoshinori Doi … (1998)
            The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho- associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and ∼30 and ∼100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase–dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase–dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.
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              The actin cytoskeleton in endothelial cell phenotypes.

              Nutan Prasain, Troy Stevens (2009)
              Endothelium forms a semi-permeable barrier that separates blood from the underlying tissue. Barrier function is largely determined by cell-cell and cell-matrix adhesions that define the limits of cell borders. Yet, such cell-cell and cell-matrix tethering is critically reliant upon the nature of adherence within the cell itself. Indeed, the actin cytoskeleton fulfills this essential function, to provide a strong, dynamic intracellular scaffold that organizes integral membrane proteins with the cell's interior, and responds to environmental cues to orchestrate appropriate cell shape. The actin cytoskeleton is comprised of three distinct, but inter-related structures, including actin cross-linking of spectrin within the membrane skeleton, the cortical actin rim, and actomyosin-based stress fibers. This review addresses each of these actin-based structures, and discusses cellular signals that control the disposition of actin in different endothelial cell phenotypes.
              Article 0 comments Cited 80 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): Cardiovasc Diabetol
                Title: Cardiovascular Diabetology
                Publisher: BioMed Central
                ISSN (Electronic): 1475-2840
                Publication date Collection: 2012
                Publication date (Electronic): 17 January 2012
                Volume: 11
                Page: 7
                Affiliations
                [1 ]Department of Pathophysiology, Key Lab for Shock and Microcirculation Research, Southern Medical University, Guangzhou, 510515, P. R. China
                Article
                Publisher ID: 1475-2840-11-7
                DOI: 10.1186/1475-2840-11-7
                PMC ID: 3280169
                PubMed ID: 22251897
                SO-VID: 3933cb01-d09f-4335-86cc-481cfffafcdf
                Copyright © Copyright ©2012 Wang et al; licensee BioMed Central Ltd.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 3 December 2011
                Date accepted : 17 January 2012
                Categories
                Subject: Original Investigation

                ScienceOpen disciplines: Endocrinology & Diabetes
                Keywords: rhoa/rock pathway,moesin,advanced glycation end products (ages),vascular permeability
                Data availability:
                ScienceOpen disciplines: Endocrinology & Diabetes
                Keywords: rhoa/rock pathway, moesin, advanced glycation end products (ages), vascular permeability

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