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ERM protein Moesin is phosphorylated by advanced glycation end products and modulate vascular permeability PDF

ERM protein Moesin is phosphorylated by advanced glycation end products and modulate vascular permeability PDF
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   doi:10.1152/ajpheart.00196.2009 297:238-246, 2009. First published Apr 24, 2009;  Am J Physiol Heart Circ Physiol Wu, Ping Zhu, Xuliang Huang and Qiaobing Huang Xiaohua Guo, Lingjun Wang, Bo Chen, Qiang Li, Jiping Wang, Ming Zhao, Wei   You might find this additional information useful... 41 articles, 19 of which you can access free at: This article cites http://ajpheart.physiology.org/cgi/content/full/297/1/H238#BIBLincluding high-resolution figures, can be found at: Updated information and services http://ajpheart.physiology.org/cgi/content/full/297/1/H238 can be found at:  AJP - Heart and Circulatory Physiology about Additional material and information http://www.the-aps.org/publications/ajpheartThis information is current as of June 30, 2009 . http://www.the-aps.org/.ISSN: 0363-6135, ESSN: 1522-1539. Visit our website at Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. intact animal to the cellular, subcellular, and molecular levels. It is published 12 times a year (monthly) by the Americanlymphatics, including experimental and theoretical studies of cardiovascular function at all levels of organization ranging from the publishes srcinal investigations on the physiology of the heart, blood vessels, and  AJP - Heart and Circulatory Physiology   on J  un e 3  0  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om   ERM protein moesin is phosphorylated by advanced glycation end productsand modulates endothelial permeability Xiaohua Guo, 1,2 Lingjun Wang, 1,2 Bo Chen, 1,2 Qiang Li, 1,2 Jiping Wang, 1,2 Ming Zhao, 1,2 Wei Wu, 1,2 Ping Zhu, 3 Xuliang Huang, 1,2 and Qiaobing Huang 1,2 1  Department of Pathophysiology,  2 Key Lab for Shock and Microcirculation Research, and   3  Department of Immunology,Southern Medical University, Guangzhou, People’s Republic of China Submitted 2 March 2009; accepted in final form 23 April 2009 Guo X, Wang L, Chen B, Li Q, Wang J, Zhao M, Wu W, ZhuP, Huang X, Huang Q.  ERM protein moesin is phosphorylated byadvanced glycation end products and modulates endothelial perme-ability.  Am J Physiol Heart Circ Physiol  297: H238–H246, 2009.First published April 24, 2009; doi:10.1152/ajpheart.00196.2009.—Advanced glycation end products (AGEs) accumulated in differentpathological conditions have the potent capacity to alter cellularproperties that include endothelial structural and functional regula-tions. The disruption of endothelial barrier integrity may contribute toAGE-induced microangiopathy and macrovasculopathy. Previousstudies have shown that AGEs induced the rearrangement of actin andsubsequent hyperpermeability in endothelial cells (ECs). However,the mechanisms involved in this AGE-evoked EC malfunction are notwell understood. This study directly evaluated the involvement of moesin phosphorylation in AGE-induced alterations and the effects of the RhoA and p38 MAPK pathways on this process. Using immor-talized human dermal microvascular ECs (HMVECs), we first con-firmed that the ezrin/radixin/moesin (ERM) protein moesin is requiredin AGE-induced F-actin rearrangement and hyperpermeability re-sponses in ECs by knockdown of moesin protein expression withsmall interfering RNA. We then detected AGE-induced moesin phos-phorylation by Western blot analysis. The mechanisms involved inmoesin phosphorylation were analyzed by blocking AGE receptorbinding and inhibiting Rho and MAPK pathways. AGE-treatedHMVECs exhibited time- and dose-dependent increases in the Thr 558 phosphorylation of moesin. The increased moesin phosphorylationwas attenuated by preadministrations of AGE receptor antibody, Rhokinase (ROCK), or p38 inhibitor. Suppression of p38 activation viathe expression of dominant negative mutants with Ad.MKK6b orAd.p38   also decreased moesin phosphorylation. The activation of the p38 pathway by transfection of HMVECs with an adenoviralconstruct of dominant active MKK6b resulted in moesin phosphory-lation. These results suggest a critical role of moesin phosphorylationin AGE-induced EC functional and morphological regulations. Acti-vation of the ROCK and p38 pathways is required in moesin phos-phorylation.vascular permeability; receptor for advanced glycation end products;Rho kinase; mitogen-activated protein kinase; ezrin/radixin/moesin ADVANCED GLYCATION END PRODUCTS  (AGEs) are formed irre-versibly in serum and tissue as the result of a chain of chemicalnonenzymatic reactions (35). The accumulation of AGEs invivo has been found to increase with age and to occur at anaccelerated rate in subjects with particular pathological condi-tions (10, 33). Especially, AGEs have been found to play animportant role in the development of diabetes, where levels of AGEs are correlated with the severity of complications elicited(11, 27, 28, 39). Deposited AGEs may have the capacity toalter cellular properties by a number of mechanisms. Directeffects of AGEs in the extracellular space include the forma-tion of cross links that may also trap neighboring unrelatedmacromolecules. Furthermore, AGE and receptor for AGEs(RAGE) signaling exert complex effects on cellular functionsvia complicated transduction pathways, such as p21 Ras,MAPKs, and NF-  B in endothelial cells (ECs), monocytes,and vascular smooth muscle cells (3, 32, 40). Our previousstudy (13) has demonstrated that AGEs significantly alteredendothelial F-actin cytoskeleton morphology and increased ECmonolayer permeability through the activation of the p38MAPK signaling pathway. However, there is still a missinglink between kinases and cytoskeleton since those signals donot directly act on F-actin and other cytoskeletal molecules.Ezrin/radixin/moesin (ERM) proteins are emerging as the po-tential candidates that are likely to mediate this process. Serv-ing as cross linkers between actin filaments and the plasmamembrane, ERM molecules are engaged in cell adhesion,microvilli formation, cell motility, etc. (21, 25).Since moesin is the main ERM molecule expressed by theendothelium (4, 18), the present study focused on the effects of moesin in mediating the AGE-induced interaction of signalingmolecules and the cytoskeleton in ECs. ERM proteins arehighly homologous in their primary structures and functions,but those structures allow variable protein-protein interactions(7). The COOH-terminal domain may form an intramolecularband to the NH 2 -terminal four-point-one ERM homology do-main or may bind to F-actin depending on the phosphorylationstate of a conserved threonine residue (Thr 567 in ezrin, Thr 564 in radixin, and Thr 558 in moesin). On the basis of an in vitrostudy (23), it was suggested that the phosphorylation of thethreonine residue suppresses the intramolecular binding. Rhokinase (ROCK) and p38 MAPK have been postulated tophosphorylate this residue, although the identity of the ki-nase(s) that directly phosphorylates moesin remains to beclearly defined (19, 20, 29, 41). This study tested the hypoth-esis that moesin is phosphorylated on this critical threonineresidue by AGE-induced signaling events and plays an impor-tant role in modulating the endothelial cytoskeleton arrange-ment and barrier function. MATERIALS AND METHODS  Materials.  Chemicals were purchased from Sigma (St. Louis, MO)unless otherwise stated. Antibodies recognizing total moesin andphosphorylated (p-)moesin (Thr 558 ), human moesin small interfering(si)RNA, and control nonsense siRNA were from Santa Cruz Biotech-nology (Santa Cruz, CA). Antibodies recognizing total ERM, p-ERM Address for reprint requests and other correspondence: Q. Huang, Dept. of Pathophysiology, Key Lab for Shock and Microcirculation Research, SouthernMedical Univ., Guangzhou 510515, People’s Republic of China (e-mail:Huangqb2000@yahoo.com).  Am J Physiol Heart Circ Physiol  297: H238–H246, 2009.First published April 24, 2009; doi:10.1152/ajpheart.00196.2009.0363-6135/09 $8.00 Copyright  ©  2009 the American Physiological Society http://www.ajpheart.orgH238   on J  un e 3  0  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om   [ezrin (Thr 567 )/radixin (Thr 564 )/moesin (Thr 558 )], and   -actin werefrom Cell Signaling (Beverly, MA). The ROCK inhibitor Y-27632,MEK1 inhibitor PD-98059, and p38 inhibitor SB-203580 were fromCalbiochem (San Diego, CA). TNF-   antibody was obtained fromAbcam (Cambridge, MA). Rhodamine-phalloidin was from Molecu-lar Probes (Carlsbad, CA). MCDB 191 medium, FCS, trypsin, glu-tamine, penicillin, and streptomycin were from GIBCO-BRL (GrandIsland, NY). Monoclonal antibodies against the different epitopes of RAGE (anti-RAGE IgG) were prepared and characterized accordingto Zhu et al. (42). Adenoviruses were kindly provided by Dr. HanJiahuai (Scripps Research Institute, La Jolla, CA). Preparation of AGE-modified human serum albumin.  AGE-modi-fied human serum albumin (AGE-HSA) was prepared according to theprotocol of Hou et al. (15, 16). Briefly, HSA (150 mmol/l, pH 7.4) wasincubated in PBS with  D -glucose (250 mmol/l) at 37°C for 8 wk.Control albumin was incubated without glucose. At the end of theincubation period, both solutions were extensively dialyzed againstPBS and purified. The endotoxin content was detected by a  Limulusamebocyte  lysate assay (Sigma) and was found to be  500 U/l in bothsolutions. AGE-specific fluorescence was determined using ratio spec-trofluorometry. AGE-HSA contained 70.2 U/mg protein of AGEs,whereas native albumin contained 0.9 U/mg protein of AGEs. Cells and culture conditions.  The human dermal microvascularECs (HMVECs) from an immortalized HMVEC cell line were pur-chased from Cell Applications (San Diego, CA) (30). Cells weregrown on 100-mm dishes or six-well plates and maintained in MCDB131 medium containing with EC growth supplements, 10% FCS, and2 mmol/l  L -glutamine at 37°C in a humidified atmosphere containing5% CO 2 . siRNA-mediated knockdown of moesin in HMVECs.  Transfectionwas performed according to the protocols provided by the manufac-turer with slight modifications. Briefly, HMVECs were transfectedwith optimized concentrations of either human moesin siRNA, controlnonsense siRNA, or with mock conditions using siRNA transfectionreagent alone. Forty-eight hours after transfection, cells were lysed,and mRNA was extracted and subjected to RT-PCR to evaluatemoesin mRNA expression. Whole cell lysates were subjected toWestern blot analysis with anti-moesin antibody to confirm siRNA-mediated knockdown in moesin expression.  RNA extraction and RT-PCR.  Total RNA was isolated fromHMVECs using TRIzol reagent (Invitrogen, Life Technologies) ac-cording to the manufacturer’s protocol. RT-PCR amplification wasperformed with moesin-specific primers [moesin (h)-PR, Santa CruzBiotechnology]. Two microliters of RNA at a concentration of 0.5 g/lwere used as templates for cDNA synthesis in the reverse transcrip-tase reaction. After an initial 4-min denaturation at 95°C, cDNA wasamplified for 30 cycles and then annealed at 58°C for moesin andGAPDH. At the end of the annealing process, 2 min of the elongationphase was followed by a single extension phase of 3 min at 72°C. PCRproducts were separated on 1.5% agarose gels. Stimulation.  In all experiments, HMVECs were grown to 90%confluence and starved for 2 h before being stimulated with AGE-HSA at the indicated doses and times. The preferences of effectiveconcentrations of AGE-HSA were based on our previous studies (13,38) in freshly cultured primary human umbilical vein ECs and wereconsistent with those from Talmor et al. (31). In the case of receptorantibody or inhibitor (Y-27632, PD-203580, or SB-203580) treat-ment, HMVECs were pretreated with 100 mg/l RAGE antibody for1 h or 25   mol/l inhibitor for 30 min and then cultured in freshcomplete medium with 50 or 100 mg/l AGE-HSA for 1 h. Cells werethen harvested, and proteins were extracted.  Endothelial monolayer permeability assay.  Endothelial monolayerpermeability was measured as described by Tinsley et al. (34). ECswere grown to confluence on 1% gelatin-coated transwell-clear poly-ester membranes (Corning Costar, Acton, MA). Cells were exposed todifferent reagents as indicated before being subjected to 100 mg/lAGE-HSA. The tracer protein TRITC-albumin (1 g/l) was then addedto the upper chambers for 45 min. Samples were collected from boththe upper (luminal) and lower (abluminal) chambers for fluorometryanalysis. Albumin concentrations were detected using a HTS 7000microplate reader (Perkin-Elmer, Yokohama, Japan) with a standardcurve. The permeability coefficient for albumin ( P a ) was calculated asfollows:  P a    [A]/  t     1/   A    V/[L], where [A] is the abluminalalbumin concentration,  t   is time (in s),  A  is the area of the membrane(in cm 2 ), V is the volume of the abluminal chamber, and [L] is theluminal albumin concentration. Western blot analysis.  Total cellular extracts from AGE-treatedcells were prepared by lysing and sonicating the cells in lysis buffer[20 mmol/l Tris (pH 7.4), 2.5 mmol/l EDTA, 1% Triton X-100, 1%deoxycholic acid, 0.1% SDS, 100 mmol/l NaCl, 10 mmol/l NaF, 1mmol/l Na 3 VO 4 , and an anti-protease cocktail pill]. Samples weresubjected to SDS-PAGE, and proteins were transferred to polyvinyli-dene difluoride membranes. Blots were blocked with 5% BSA in PBScontaining 0.5% Tween 20 (PBS-T) for 1 h and then incubated witha 1:1,000 dilution of the primary antibody of interest overnight at 4°Con a rocker. After being washed three times for 5 min each withPBS-T, blots were incubated with a 1:1,000 dilution of horseradishperoxidase-conjugated species-specific respective secondary antibody(Dako, Ely, UK ) for 1 h at room temperature. After being washedthree times for 5 min each with PBS-T, protein bands were visualizedby chemiluminescence. Fluorescent staining.  ECs were plated in gelatin-coated glass-bottom microwells (MatTek) and cultured to confluence. After theappropriate treatments, cells were fixed and permeated for 15 min atroom temperature in PBS with 3.7% formaldehyde and 0.5% TritonX-100. Cells were blocked in 5% BSA in PBS for 1 h and subse-quently incubated with moesin or p-moesin antibody at room temper-ature for 2 h. After a thorough wash in PBS, cells were stained withFITC-conjugated secondary antibody (Zymed, San Francisco, CA).For F-actin staining, cells were incubated with rhodamine-phalloidin(2,000 U/l) for 40 min at room temperature. Finally, cells werewashed three times with PBS, mounted to allow observation, andimaged with a Leica TCS SP2 laser confocal scanning microscope(Wetzlar, Germany).  Infections with adenovirus constructs.  Recombinant adenovirusconstructs were generated as previously described (24, 37). Briefly,we used Ad.MKK6b(E), the constitutively active form of MKK6encoded by a recombinant adenovirus, and Ad.MKK6(A), the consti-tutively negative form of MKK6, and inactive forms of the differentp38 isoforms to activate or block the p38 MAPK pathway. Cells wereseeded in 100-mm dishes to confluency before the infection withadenoviruses. Cells were then incubated for 24 h before being exposedto AGE-HSA.  Data analysis and statistical methods.  Data were normalized tocontrol and are reported as percentages of the basal value in means  SD of at least three independent experiments. Data were analyzedusing one-way ANOVA followed by post hoc comparisons. The levelof statistical significance was taken as  P  0.05. RESULTS Knockdown of moesin using siRNA suppressed the AGE-induced endothelial response.  Our previous study (13) showedthat AGE-HSA significantly evoked an endothelial responsewith F-actin stress fiber formation and a decrease in EC barrierfunction in human umbilical vein ECs. To evaluate the role of moesin in these responses, siRNA targeting moesin and controlsiRNA were used in HMVECs. Treatment with siRNA againstmoesin inhibited moesin mRNA expression in HMVECs buthad no effect on the expression of GAPDH (Fig. 1  A ). Treat-ment with moesin siRNA also efficiently inhibited the proteinexpression of moesin and ERM in HMVECs but had no effecton  -actin expression (Fig. 1  B ). This result is consistent withH239 MOESIN PHOSPHORYLATION IN THE AGE-INDUCED ENDOTHELIAL RESPONSE  AJP-Heart Circ Physiol  •  VOL 297  •  JULY 2009  •  www.ajpheart.org   on J  un e 3  0  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om   the report that moesin is the major ERM protein expressed byECs, because treatment with moesin siRNA in total ERMexpression caused the same decrease as in moesin.The role of moesin in modulating EC responses induced byAGEs was then examined by comparing the distribution of F-actin in ECs as well as the fluxes of albumin across the ECmonolayer between control siRNA and moesin siRNA treat-ments. Without the stimulation of AGE-HSA, the organizationof F-actin in moesin siRNA-treated ECs was similar to controlECs (Fig. 2,  A  and  B ). Exposure of control siRNA-treated ECsto 50 mg/l AGE-HSA for 1 h caused a shift in the F-actindistribution from a web-like structure to polymerized stressfibers (Fig. 2 C  ). However, this change was prevented in ECstreated with siRNA against moesin (Fig. 2  D ). These resultsindicate that moesin protein is required in this F-actin rear-rangement process. To further address the role of moesin inincreasing endothelial permeability by AGE-HSA, a transwellsystem was used to examine the fluxes of albumin across theHMVEC monolayer. In each group, ECs were treated witheither control buffer, 100 mg/l HSA, or 100 mg/l AGE-HSAfor 1 h before TRITC-labeled albumin was added to the top Fig. 2. Downregulation of the expression of moesin by siRNA preventedAGE-induced cytoskeletal changes and permeability increases.  A–D : the effectof moesin siRNA on the distribution of F-actin in the cytoskeleton before orafter AGE-HSA treatment. ECs treated with control or moesin siRNA weretreated with either buffer or 50 mg/l AGE-HSA for 1 h, and F-actin in thecytoskeleton was examined using confocal microscopy.  A : control siRNA;  B : moesin siRNA;  C  : control siRNA    AGE-HSA;  D : moesin siRNA   AGE-HSA. Scale bar    30   m.  E  : the effect of moesin siRNA on AGE-induced increases in the fluxes of albumin across ECs. TRITC-labeled albuminwas added to the top chamber, and the amount of albumin appearing in the topand bottom chambers was measured. ECs seeded on filters were treated witheither control or moesin siRNA. ECs were then stimulated with either bufferor 100 mg/l AGE-HSA for 1 h before the concentration of albumin wasmeasured. Transfection with moesin siRNA decreased the hyperpermeabilityinduced by AGE-HSA. Permeability of albumin ( P a ) values are shown aspercentages of control (means    SD;  n    4 for each sample). * P    0.05compared with no AGE-treated control; # P    0.05 compared with thecorresponding samples treated with AGE-HSA only.Fig. 1. Knockdown of moesin expression in human dermal microvascular endo-thelial cells (HMVECs) with small interfering (si)RNA. ECs were either untreated(U) or treated with control siRNA (C) or siRNA targeting moesin.  A : cells wereharvested 48 h after transfection and then lysed, and mRNA was extracted andsubjected to RT-PCR to evaluate moesin mRNA expression.  B : the effect of siRNA on the protein expression of moesin, ezrin/radixin/moesin (ERM), or  -actin was examined using Western blot analysis.  C  : ECs were either untreatedor treated with control siRNA or siRNA targeting moesin. ECs were thenstimulated with 50 mg/l advanced glycation end product (AGE)-modified humanserumablumin(AGE-HSA)for1h.TheeffectofsiRNAontheproteinexpressionof phosphorylated (p-)moesin or  -actin was examined using Western blot anal-ysis. Data are presented as fold changes over the buffer or AGE-treated controlafter normalization by  -actin level and are presented as means  SD;  n  3 foreach sample. * P  0.05 compared with buffer-treated controls. H240  MOESIN PHOSPHORYLATION IN THE AGE-INDUCED ENDOTHELIAL RESPONSE  AJP-Heart Circ Physiol  •  VOL 297  •  JULY 2009  •  www.ajpheart.org   on J  un e 3  0  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om   wells. The EC monolayer treated with control buffer or moesinsiRNA alone had a similar permeability to albumin (Fig. 2  E  ).In response to AGE-HSA stimulation, ECs treated with controlsiRNA showed a significant increase in the permeability toalbumin, whereas this increase was notably attenuated bymoesin siRNA treatment. These data indicate that moesin isessential for the AGE-induced increase in EC monolayer per-meability.  AGEs induced threonine phosphorylation of moesin and  ERM proteins in HMVECs.  After the essential role of moesinin AGE-induced EC responses was confirmed by suppressingmoesin expression, the effect of AGEs on the threonine phos-phorylation of moesin in confluent ECs was then examined.Treatment of HMVECs with 50 mg/l AGE-HSA resulted in asignificant increase in moesin phosphorylation in a time-de-pendent manner (Fig. 3  A ). A dose-response experiment indi-cated that a notable increase of moesin phosphorylation ap-peared in applications of AGE-HSA at 50 mg/l for 1 h (Fig.3  B ). Incubation of HMVECs with HSA alone had no effect onmoesin threonine phosphorylation. AGE-induced moesin phos-phorylation also significantly diminished in ECs pretreatedwith moesin siRNA (Fig. 1 C  ). Furthermore, threonine phos-phorylation of ERM proteins was also estimated by immuno-blot analysis with p-ERM [ezrin (Thr 567 )/radixin (Thr 564 )/ moesin (Thr 558 )] antibody. Increases in ERM threonine phos-phorylation induced by AGE-HSA were also detected in time-and concentration-dependent patterns (Fig. 4).The distribution of p-moesin in ECs was then examined byimmunological fluorescent staining. p-Moesin was localizedprimarily at the cell periphery without AGE-HSA treatment(Fig. 5  A ). Treatment of the cells with AGE-HSA induced anincrease in the density of fluorescent staining of p-moesin,consistent with the Western blot experiments. p-Moesin waspresented in the cytoplasm of ECs and formed polymerizedsarciniform fibers after the administration of 50 mg/l AGE-HSA for 1 h (Fig. 5  B ). The distribution of total moesin withinthese ECs was not altered by AGE-HSA (data not shown).It has been shown that moesin phosphorylation is involvedin TNF-  -evoked responses in pulmonary microvascular ECs(18). To exclude the possibility of LPS-induced TNF-   con-tamination in this AGE-HSA application, a specific antibodyfor neutralizing TNF-   was added at doses of 25 mg/l toAGE-HSA solution 4 h before being applied to ECs. Theresults showed no differences in moesin phosphorylation andEC monolayer permeability between groups with or withoutTNF-  antibody (Fig. 6,  A  and  B ).  Involvement of RAGE in AGE-induced moesin phosphory-lation.  To test the role of RAGE in AGE-induced moesinphosphorylation, HMVECs were pretreated with 100 mg/lRAGE antiboy (anti-RAGE IgG) 1 h before exposure to 50mg/l AGE-HSA for another 1 h. RAGE antibody blockedAGE-induced moesin phosphorylation and rearrangement inthe cytoplasm (Figs. 7 and 5 C  ). Furthermore, pretreatment of HMVECs with 100 mg/l RAGE antibody for 1 h also attenu-ated the 100 mg/l AGE-induced elevation in the permeabilityof albumin (Fig. 8  B ). These data suggest that binding of AGEsto RAGE plays an important role in the mediation of AGE-induced moesin phosphorylation and the increase of vascularpermeability.  Involvement of the ROCK and p38 MAPK pathways in AGE-induced moesin phosphorylation.  To elucidate the signalpathways that might be involved in AGE-induced moesinphosphorylation, HMVECs were pretreated with 25   mol/lROCK inhibitor Y-27632 and MAPK inhibitors SB-203580,PD-98059, or SP-600125, respectively, before being exposedto 50 mg/l AGE-HSA. Moesin threonine phosphorylation andthe morphological rearrangement in ECs were remarkablyprevented by Y-27632 or SB-203580, whereas ERK or JNKinhibition with PD-98059 or SP-600125, respectively, did notattenuate this AGE-evoked alteration (Figs. 8  A  and 5,  D  and Fig. 3. AGEs induced threonine phosphorylation of moesin in HMVECs.HMVECs were treated with either buffer, AGE-HSA, or HSA at the indicateddoses for the indicated times, and threonine-phosphorylated moesin or totalmoesin was examined using Western blot analysis.  A : time-dependent changesin the threonine phosphorylation of moesin induced by 50 mg/l AGE-HSA(solid bars;  n  4 for each sample) or 50 mg/l HSA (open bars;  n  3 for eachsample).  B : dose-dependent changes in the threonine phosphorylation of moesin after AGE-HSA (solid bars;  n  5 for each sample) or HSA (open bars; n  3 for each sample) treatment for 1 h. Data are presented as fold changesover the buffer-treated control after normalization by total moesin level andexpressed as means  SD. * P  0.05 compared with buffer-treated controls. H241 MOESIN PHOSPHORYLATION IN THE AGE-INDUCED ENDOTHELIAL RESPONSE  AJP-Heart Circ Physiol  •  VOL 297  •  JULY 2009  •  www.ajpheart.org   on J  un e 3  0  ,2  0  0  9  a j   ph  e ar  t  . ph  y  s i   ol   o g y . or  gD  ownl   o a d  e d f  r  om 
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