Research ReportAdvanced glycation end products induce moesin phosphorylation in murine brain endothelium
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).
References (44)
- et al.
Beta(2)-Microglobulin modified with advanced glycation end products delays monocyte apoptosis
Kidney Int.
(2001) - et al.
Astrocytic and neuronal biochemical markers in the sera of subjects with diabetes mellitus
Neurosci. Lett.
(2004) - et al.
NGF-induced moesin phosphorylation is mediated by the PI3K, Rac1 and Akt and required for neurite formation in PC12 cells
J. Neurochem Int.
(2010) - et al.
Ezrin/radixin/moesin, versatile controllers of signaling molecules and of the cortical skeleton
Int. J. Biochem. Cell Biol.
(2008) - et al.
Susceptibility of brain microvascular endothelial cells to advanced glycation end products-induced tissue factor upregulation is associated with intracellular reactive oxygen species
Brain Res.
(2006) - et al.
Phosphorylation of moesin by rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures
J. Biol. Chem.
(1998) - et al.
Upregulation of RAGE and its ligands in proliferative retinal disease
Exp. Eye Res.
(2006) - et al.
Expression of ezrin radixin moesin proteins in the adult subventricular zone and the rostral migratory stream
Neuroscience
(2010) - et al.
Extra-nuclear signaling of ERalpha to the actin cytoskeleton in the central nervous system
Steroids
(2010) - et al.
The molecular basis of vascular lumen formation in the developing mouse aorta
Dev. Cell
(2009)
A simple method for isolation and characterization of mouse brain microvascular endothelial cells
J. Neurosci. Methods
Cell endothelial dysfunction and diabetes: roles of hyperglycemia, impaired insulin signaling and obesity
Tissue Res.
Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells
J. Cell Sci.
Involvement of microtubules, p38, and Rho kinases pathway in 2-methoxyestra-diol-induced lung vascular barrier dysfunction
Am. J. Physiol. Lung Cell. Mol. Physiol.
Neurally mediated increase in vascular permeability in the rat trachea: onset, duration, and tachyphylaxis
Exp. Lung Res.
Activation of the ROCK1 branch of the transforming growth factor-beta pathway contributes to RAGE-dependent acceleration of atherosclerosis in diabetic ApoE-null mice
Circ. Res.
TIMAP is a positive regulator of pulmonary endothelial barrier function
Am. J. Physiol.: Lung Cell Mol. Physiol.
Role of advanced glycation end products in diabetic nephropathy
J. Am. Soc. Nephrol.
Activation of PKC-delta and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy
Nat. Med.
Advanced glycation end products, sparking the development of diabetic vascular injury
Circulation
ERM protein moesin is phosphorylated by advanced glycation end products and modulates endothelial permeability
Am. J. Physiol.: Heart Circ. Physiol.
Mechanism of advanced glycation end products-induced hyperpermeability in endothelial cells
Sheng Li Xue Bao
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