Elsevier

Brain Research

Volume 1373, 10 February 2011, Pages 1-10
Brain Research

Research Report
Advanced glycation end products induce moesin phosphorylation in murine brain endothelium

https://doi.org/10.1016/j.brainres.2010.12.032Get rights and content

Abstract

Advanced glycation end products (AGEs) have been found to play an important role in the development of diabetes, and AGE levels are correlated with the severity of diabetic complications. We have demonstrated that moesin, a protein linker between actin filaments and the plasma membrane, undergoes phosphorylation of its threonine 558 residue by AGE stimulation in human dermal microvascular endothelial cells through activation of p38 and Rho kinase (ROCK) pathways. In this study, we observed in situ whether AGEs caused phosphorylation of vascular endothelial cells in the brains of AGE-stimulated mice. The animals were injected with AGE-modified mouse serum albumin (AGE-MSA) for 7 consecutive days. Immunohistochemistry was conducted to assess the phosphorylation of moesin in brain vessels. The level of moesin protein phosphorylation was also assessed in cerebral microvessels by western blotting. The effects of p38 and ROCK activation were determined by application of a p38 inhibitor (SB203580) and a ROCK inhibitor (Y27632) at 30 min before each AGE administration. The results showed specific expression of moesin in murine brain vascular endothelial cells. AGE treatment induced a significant increase of threonine 558 phosphorylation in moesin, while inhibition of p38 and ROCK remarkably attenuated the phosphorylation of moesin. The level of moesin protein phosphorylation was also increased in cerebral microvessels, along with an increased permeability of the blood–brain barrier, while inhibition of the p38 and ROCK attenuated these responses. These results demonstrate that AGEs cause the phosphorylation of moesin in murine brain microvascular endothelial cells, with p38 and ROCK being involved in this process.

Research Highlights

► Moesin (one of ERM family proteins) is expressed in murine brain endothelial cells. ► Injection of AGE to mice induced the phosphorylation of moesin in brain endothelial cells. ► Activation of ROCK and p38 pathways are involved in inducing the phosphorylation of moesin.

Introduction

Diabetes mellitus is a major risk factor for cerebrovascular disease and the most common diabetic cerebrovascular complications are ischemic stroke and cerebral atrophy. Ischemic stroke is due to accelerated macrovascular atherosclerosis (Sonnen et al., 2009), while cerebral atrophy is known to result from microvascular lesions (Messier et al., 2004). The resulting alterations of cognitive function are a diabetes-related disability. Endothelial dysfunction and damage are believed to play an important role in the pathogenesis of these diabetic macrovascular and microvascular complications (Bakker et al., 2009, Huber, 2008). Disruption of the blood–brain barrier and an increase of brain vascular permeability are early changes during the development of diabetes in both animal models (Huber et al., 2006) and clinical observation (Hovsepyan et al., 2004). However, the pathological features and molecular pathogenesis of cerebrovascular complications related to diabetes are not fully understood.

Hyperglycemia and its related pathological alterations lead to the onset of vascular complications (Geraldes et al., 2009). There is growing evidence that advanced glycation end products (AGEs) produced by non-enzymatic reactions between reducing sugars and amino reactive groups of proteins and lipids are especially relevant to the processes of macrovascular and microvascular damages (Takeuchi and Yamagishi, 2009). It has been noticed that microvascular endothelial cells in the brain are more susceptible to AGE-induced inflammatory damage than aortic endothelial cells (Niiya et al., 2006). By binding to a specific receptor known as RAGE, AGEs trigger oxidative stress and the activation of the mitogen-activated protein kinases (MAPKs) and RhoA kinase (ROCK) signaling pathways in endothelial cells, leading to up-regulation of pro-inflammatory molecules and disruption of endothelial barrier function (Forbes et al., 2003, Guo et al., 2005). The activation of MAPK and ROCK signaling strengthens the construction of F-actin and opens inter-endothelial junctions (Mehta and Malik, 2006). It has been noted in numerous studies that the ezrin/radixin/moesin (ERM) family of plasma membrane–actin linking proteins plays an important role in mediating the response to kinase signals and F-actin (Niggli and Rossy, 2008). Moesin is regarded as the most important player in this endothelial response since it is the ERM dominantly expressed by endothelial cells (Berryman et al., 1993), as well as cerebrovascular endothelial cells (Hayashi et al., 1999, Johnson et al., 2002). Our previous study demonstrated that moesin is phosphorylated at threonine residue 558 by AGE-induced signaling, and this change plays an important role in modulating the endothelial cytoskeleton and barrier function in human microvascular endothelial cells (HMVECs) (Guo et al., 2009). We have already detected an increase of murine retinal microvascular permeability in AGE-stimulated mice (unpublished data). The purpose of the present study was to assess the changes of blood–brain barrier function after AGE stimulation and to explore the role of threonine phosphorylation in moesin in this response. The involvement of MAPK and ROCK in AGE-induced phosphorylation of moesin in brain microvascular endothelial cells was also assessed in this study.

Section snippets

Effect of AGE-MSA on immunohistochemical staining of moesin and threonine 558-phosphorylated moesin in the murine brain

Cellular expression of moesin was immunohistochemically identified with an antibody against moesin or an antibody against 558 threonine-phosphorylated moesin. Immunohistochemistry of brain tissue showed that moesin was strongly and predominately expressed in murine brain microvascular endothelial cells (Fig. 1A, arrow). There was almost no phosphorylated moesin in control brain endothelial cells. Administration of AGE-MSA did not alter total moesin expression, but it significantly strengthened

Discussion

The present study demonstrated that AGEs induced the phosphorylation of moesin at threonine residue 558 in murine brain microvascular endothelial cells, and that phosphorylation of moesin was accompanied by an increase of cerebral microvascular permeability. Inhibition of p38 MAPK and ROCK not only attenuated this AGE-evoked moesin phosphorylation, but also preserved blood–brain barrier function, indicating involvement of the p38 MAPK and ROCK pathways in this AGE-induced cerebral microvascular

Materials

Mouse serum albumin (MSA, fraction V), D-glucose, and Evans blue were purchased from Sigma (St. Louis, MO, USA). The antibody specifically recognizing phospho-moesin (T558) was obtained from Santa Cruz Biotechnology (CA, USA). Antibodies recognizing total moesin, total MAPK, and phospho-p38 MAPK were purchased from CST (USA). Antibody against ROCK I was obtained from Chemicon (USA) and antibody against phospho-ROCK I was from Upstate (USA). The p38 inhibitor SB203580 and the Rho kinase

Acknowledgments

This work was supported by General Program from Natural Science Foundation of China (30771028, 30971201); Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT) (No. IRT0731); and National Key Foundation for Basic Science Research of China (G2005CB522601).

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