The Mechanism Of Moesin Phosphorylation In Advanced Glycation End Products Induced Responses In Endothelial Cells

Advanced glycation end products(AGEs) are formed irreversibly in serum and tissue as the result of a chain of chemical non-enzymatic reactions.Accumulation of AGEs in vivo has been found to increase with age and to occur at an accelerated rate in subjects with particular pathological conditions.Especially,AGEs are found to play an important role in the development of diabetes,where levels of AGEs are correlated with the severity of complications elicited.Deposited AGEs may have the capacity to alter cellular properties by a number of mechanisms.Direct effects of AGEs in the extracellular space include formation of crosslinks that may also trap neighboring unrelated macromolecules.Furthermore,AGEs and receptor for AGEs (RAGE) signaling exert complex effects on cellular functions via complicated transduction pathways,such as p21ras,MAPKs and nuclear factor-κB in endothelial cells(ECs),monoeytes,and vascular smooth muscle cells.Our previous study has demonstrated that AGEs significantly altered endothelial F-actin cytoskeleton morphology and increased EC monolayer permeability through the activation of p38 MAPK signaling pathway.But there is still a missing link between kinases and cytoskeleton since those signals are not directly act on F-actin and other cytoskeletal molecules.ERM(ezrin/radixin/moesin) proteins are emerging as the potential candidates that likely to mediate this process.Serving as cross-linkers between actin filaments and plasma membrane,ERM molecules are engaged in cell adhesion, microvilli formation,cell motility,etc.The encoding genes constitute a gene family, which is now called the ERM family,and homologues are found in various species. The sequence of their amino-terminal halves is highly conserved,and this sequence is also found in the amino-terminal ends of various submembranous proteins,including the erythrocyte membrane protein band 4.1 protein,indicating that the ERM family is included in the band 4.1 superfamily.Since moesin is the main ERM molecule expressed by endothelia,this present study focuses on the effects of moesin in mediating the AGE-induced interaction of signaling molecules and cytoskeleton in ECs.ERM proteins are highly homologous in their primary structures and functions,while those structures allow variable protein-protein interactions.The COOH-terminal domain may form an intramolecular band to the NH2-terminal FERM(four-point-one,ezrin,radixin,moesin homology) domain or may bind to F-actin depending on the phosphorylation state of a conserved threonine residue(Thr567 in ezrin,Thr564 in radixin,Thr558 in moesin).On the basis of in vitro studies,it is suggested that phosphorylation of the Thr residue suppresses the intramolecular binding.Rho kinase and p38 MAPK are postulated to phosphorylate this residue,although the identity of kinases that directly phosphorylate moesin remains to be clearly defined.This study tested the hypothesis that moesin is phosphorylated on this critical threonine residue by AGE-induced signaling events and plays an important role in modulating the endothelial cytoskeleton arrangement and barrier function.Objective:This study is to explore the role of moesin phosphorylation in AGE-induced endothelial F-actin rearrangement and barrier dysfunction.Methods:A series of experiments were accomplished in cell and in tissue levels by using cell culture,western blot,siRNA,RT-PCR,fluorescent staining,endothelial monolayer permeability assay,and isolated venual permeability assay.AGE-modified human serum albumin(AGE-HSA) was prepared by incubation of 150 mM human serum albumin with 250 mM of D-glucose for 8 weeks.Human dermal microvaseular endothelial cell line(HMVEC) were grown to 90%confluent and starved for 2 hours before being stimulated with AGE-HSA in indicated doses and times.In the case of receptor antibody or inhibitor(Y-27632, PD203580,SB203580) treatment,HMVECs were pretreated with 100μg/ml RAGE antibody for 1 h or 25μmol/L inhibitor for 30 min,then cultured in fresh complete medium with 50 or 100μg/ml AGE-HSA for 1 h.ECs were also pre-infected with recombinant adenovirus of constitutive dominant negative forms of MKK6b,p38αorβand then subjected to AGE-HSA stimulation.Ceils were then harvested and proteins were extracted.The expression of total or phosphorylation moesin was monitored by western blot.The morphological changes of F-actin were visualized by fluorescent staining.Endothelial permeability was assessed by measuring the flux TRITC-albumin across ECs monolayer,siRNA was used to down-regulate moesin expression.Venules were isolated from rat skin and cannulated with micropipettes.The venular permeability was measured with a fluorescence ratio technique and expressed as the permeability coefficient to albumin(Pa).Results:1.Stimulating the isolated venules with AGE-HSA induced a significant elevation of Pa for FITC-albumin in dose-and time-dependent patterns.In isolated vessels exposed to 12.5,25,50 or 100μg/ml of AGE within 2 h,Pa increased gradually as the multiplication of exposure dose and the prolongation of exposure time.The isolated vessels treated with HSA alone showed change in Pa.2.Knockdown of moesin using siRNA suppressed AGE-induced endothelial response(1) Treatment with siRNA against moesin inhibited moesin mRNA expression in HMVECs,but had no effect on the expression of GAPDH(Fig.1A).Treatment with moesin siRNA also efficiently inhibited protein expressions of moesin and ERM in HMVECs,but had no effect onβ-aetin expression.(2) The role of moesin in modulating ECs responses induced by AGEs was then examined by comparing the distribution of F-actin in ECs,as well as the fluxes of albumin across ECs monolayer between control siRNA and moesin siRNA treatments. Without the stimulation of AGE-HSA,the organizations of F-actin in moesin siRNA treated-ECs were similar to control ECs.Exposure of control siRNA-treated ECs to AGE-HSA caused a shift in F-actin distribution from a web-like structure to polymerized stress fibers.However,this change was prevented in ECs treated with siRNA against moesin.The results indicated that moesin protein is required in this F-actin rearrangement process.To further address the role of moesin in endothelial permeability increases induced by AGE-HSA,a transwell system was used to examine the fluxes of albumin across HMVECs monolayer.In each group,ECs were treated with either control buffer,100μg/ml HSA or 100μg/ml AGE-HSA for 1 h before TRITC-labeled albumin was added to the top wells.EC monolayer treated with control buffer or moesin siRNA alone had similar permeability to albumin.In response to AGE-HSA stimulation,ECs treated with control siRNA showed a significant increase in Pa while this increase was notably attenuated by moesin siRNA treatment.This data indicated that moesin was essential for AGE-induced increase in EC monolayer permeability.3.AGEs induced threonine phosphorylation of moesin and ERM proteins(1) Treatment of HMVEC with 50μg/ml AGE-HSA resulted in a significant increase in moesin phosphorylation in a time-dependent manner.This phosphorylation of moesin occurred without detectable changes in the protein expression of total moesin.A dose-response study indicated that the notable increase of moesin phosphorylation appeared in applications of AGE-HSA at 50μg/ml for 1 h. Incubation HMVECs with HSA alone had no effect on moesin threonine phosphorylation.(2) Furthermore,threonine phosphorylation of ERM proteins was also estimated by immunoblotting with p-Ezrin(Thr567)/Radixin(Thr564)/Moesin(Thr558) antibody.Increases in ERM threonine phosphorylations induced by AGE-HSA were also detected in time-and concentration-dependent patterns.(3) AGE-induced moesin phosphorylation was completely diminished by down-regulation of moesin expression with moesin siRNA. 4.Involvement of RAGE in AGE-induced moesin phosphorylation(1) HMVECs were pretreated with 100μg/ml RAGE Ab,anti-RAGE IgG,1h before exposure to 50μg/ml AGE-HSA for another 1h.RAGE Ab blocked AGE-induced moesin phosphorylation and rearrangement in cytoplasma.Furthermore, pretreatment of HMVECs with 100μg/ml RAGE Ab for 1 h also attenuated 100μg/ml AGE-induced elevation in Pa,from 152.6±8.7%to 118±5.5%(P＜0.05).This data suggests that binding of AGEs to RAGE plays an important role in the mediation of AGE-induced moesin phosphorylation and increase of vascular permeability.(2) Pretreatment of isolated venules with soluble anti-RAGE IgG for 1 h attenuated AGE-HSA-induced elevation in Pa in a concentration-dependent manner. Anti-RAGE IgG(50μg/ml and 100μg/ml) reduced Pa from 268.1±35.4%to 181.6±a:33.2%and 147.1±25.2%of control,respectively,in the presence of 50μg/ml AGE-HSA(P＜0.05),there was no change on Pa in isolated vessels treated with anti-RAGE IgG alone.5.The roles of ROCK and p38 MAPK were then further confirmed by the results that AGE-HSA treatment did induce significantly phosphorylations of ROCK and p38 proteins in HMVECs.These evidences approve the specific effects of those kinases on AGEs signaling pathways.6.Involvement of Rho kinase in AGE-induced moesin phosphorylation(1) Pretreated HMVECs with 25μmol/L ROCK inhibitor Y-27632 blocked AGE-induced moesin phosphorylation.Pretreatment with Y-27632 attenuated AGE-induced elevations in Pa,from 152.6±8.7%to 124.9±7.2%(P＜0.05).(2) Y-27632 blocked AGE-induced venular hyperpermeability in a concentration-dependent manner.In the presences of Y-27632(10,25μmol/L) AGE treatment yielded Pa of 188.2±26.2%and165.7±30.2%compared with268.1±35.4%without Y-27632(P＜0.05).Incubation with Y-27632 alone had no effect on venular permeability.7.Involvement of p38 MAPK in AGE-induced moesin phosphorylation (1) HMVECs were pretreated with MAPK inhibitors SB203580,PD98059 or SP600125,respectively,before exposing to 50μg/ml AGE-HSA.p38 inhibitor SB203580 blocked AGE-induced moesin phosphorylation.Pretreatment of SB203580 attenuated 100μg/ml AGE-induced elevations in Pa,from 152.6±8.7%to 122.9±6.2%(P＜0.05).Constructs of dominant negative mutation of p38 upstream kinase MKK6b, MKK6b(A),were used to further specify the role of p38 MAPK pathway on AGE-induced endothelial barrier dysfunction.The results showed that the blockage of p38 pathway by MKK6b(A) abolished AGE-induced threonine phosphorylation and the hyperpermeability response in endothelial monolayer,while the activation of p38 pathway by a construct of constitutive active mutation of MKK6b,MKK6b(E), sufficiently mimicked EC alterations induced by AGE-HSA application.The p38 isoforms participating in AGE-induced moesin phosphorylation were then identified by pre-infecting ECs with adenoviruses of constructs of dominant negative form of p38αor p38β,respectively,24 h before the addition of AGE-HSA.The results demonstrated that the suppression of p38αactivation remarkably eliminated AGE-mediated moesin threonine phosphorylation and barrier dysfunction in ECs, while the inhibition of p38βactivation showed no effects on AGE-induced endothelial malfunction.The activation of MKK6b by MKK6b(E) transfection mimicked the effects of AGEs on ECs and this mimicry was blocked by p38α,but not p38βsuppression.(2)Pretreatment of isolated venules with SB203580(10,25μmol/L) abolished AGE-HSA induced hyperpermeability,as Pa were reduced from 268.1±35.4%to 195.7±42.4%and 163.5±30.7%,respectively(P＜0.05).The effect of upstream kinase of p38(MKK6b) was detected by infecting rats with recombinant virus of dominant negative forms of MKK6b(MKK6b(A)) 24 h prior to AGE-HSA treatment. Consistently,the results showed potent inhibitory effect upon the stimulation of AGE. This AGE-induced hyperpermeability response was also attenuated by infecting the cells with recombinant virus of dominant negative form ofp38α(p38α(A)). 8.The distribution of threonine-phosphorylated moesin in ECs was then examined by immunological fluorescent staining.Phosphorylated moesin was localized primarily at the cell periphery without AGE-HSA treatment.Treated the cells with AGE-HSA induced an increase in density of fluorescent staining of phosphorylated moesin,consistent with the western blotting studies.The phosphorylated moesin was presented in EC cytoplasm and formed polymerized sarciniform fibers after 50μg/ml AGE-HSA administration for 1 h.Distribution of total moesin within these ECs was not altered by AGE-HSA.Pretreatment of HMVECs with RAGE Ab,Y-27632 and SB203580 attenuated 100μg/ml AGE-induced increase and redistribution of phosphorylated moesin.MKK6b(E) sufficiently mimicked EC alterations induced by AGE-HSA application.While MKK6b(A) or p38α(A) abolished AGE-induced threonine phosphorylation and relocalization of moesin in ECs.Conclusion:1.AGE-HSA could increase the permeability in endothelial monolayer and isolated venules in time-and dose-dependent manners.2.Moesin and ERM protein were involved in AGE-induced endothelial responses.Knockdown moesin expression by siRNA could abolish endothelial cell dysfunction induced by AGEs,3.AGE-HSA could increase moesin and ERM phosphorylation in time-and dose-dependent manners,suggesting that moesin phosphorylation plays a central role in regulating F-actin rearrangement and EC permeability.4.RAEG Ab could attenuate AGE-induced moesin phosphorylation and hyperpermeability in ECs,suggesting that AGE-RAGE interaction plays a critical role in mediating the signal into cells.5.ROCK inhibitor could abolish AGE-induced moesin phosphorylation and hyperpermeability in ECs,supporting the hypothesis that Rho/Rho kinase signaling transduetion pathway takes part in this pathological process.6.p38 MAPK is involved in the signaling mechanisms in AGE-induced moesin phosphorylation and dysfunction of endothelial cells.Additionally,p38αis the major p38 subtype involved in this response.