Advanced glycation end product (AGE) receptor 1suppresses cell oxidant stress and activation signalingvia EGF receptorWeijing Cai*, John C. He†, Li Zhu*, Changyong Lu*, and Helen Vlassara*‡

*Division of Experimental Diabetes and Aging, Brookdale Department of Geriatrics, and †Division of Nephrology, Department of Medicine,Mount Sinai School of Medicine, New York, NY 10029

Edited by Anthony Cerami, The Kenneth S. Warren Institute, Ossining, NY, and approved July 24, 2006 (received for review January 13, 2006)

Advanced glycation end product receptors (AGERs) play distinctfunctional roles in both the toxicity and disposal of advancedglycation end products (AGEs), substances that are linked todiabetes and aging. Overexpression of AGER1 in murine mesangialcells (MCs) (MC-R1) inhibited AGE-induced MAPK1,2 phosphoryla-tion and NF-�B activity and also increased AGE degradation. Themechanism of the inhibitory effects of AGER1, upstream of MAPK,was explored in MCs and HEK293 AGER1-expressing cells. AGE-induced Ras activation was found to be linked to Shc�Grb2 complexformation and Shc phosphorylation in MCs, responses that weremarkedly reduced in MC-R1 cells. AGE responses also included EGFreceptor (EGFR) phosphorylation in MCs or HEK293 cells, but thislink was blocked in both MC-R1 and HEK293-R1 cells. Coexpressionof AGER1 and EGFR in HEK293 cells decreased AGE-mediated EGFRand p44�p42 phosphorylation but not EGF-induced p44�p42 acti-vation. AGE, S100�calgranulin, or H2O2 promoted MAPK phosphor-ylation in EGFR� cells in a manner that was inhibitable by an EGFRinhibitor, AG1478. Also, in AGER1 cells, AGE-induced H2O2 forma-tion and AGE- or S100-induced p44�p42 phosphorylation weresuppressed, and these effects were restored by R1 siRNA. Thesedata confirm that R1 negatively regulates AGE-mediated oxidantstress-dependent signaling via the EGFR and Shc�Grb2�Ras path-way. AGER1 could serve as a model for developing therapeutictargets against vascular and kidney disorders related to diabetesand aging.

phosphorylation � reactive oxygen species � mesangial cells � MAPK �diabetes

Increased levels of advanced glycation end products (AGEs),generated by the nonenzymatic reaction of amino groups and

reducing sugars, have been implicated in several chronic diseasescharacterized by sustained oxidant stress (OS) and multiorganlow-level inflammatory injury, including diabetes-related car-diovascular and renal disease, as well as aging (1–3). At thecellular level, the damaging effects of AGE have been attributedto several AGE-binding proteins, most notably to RAGE (re-ceptor for AGE), as well as others, including, but not limited to,AGE receptor (AGER) 1, R2, and R3 and the scavengerreceptors class A type II (MSR-AII) and class B type I (MSR-BI,CD36) (4–7). These receptors have been shown to play distinctfunctional roles in AGE toxicity or AGE detoxification. Al-though RAGE is known to mediate cellular OS and inflamma-tory effects through the activation of the Ras-MAPK pathway,some receptors may have other functions [e.g., in AGE disposal(8–10)].

AGER1, an �50-kDa type A integral membrane protein witha short internal domain, a single transmembrane segment, anda long intracellular tail, was initially isolated with other membersof the oligosaccharyltransferase complex and was later found tobe active in AGE-specific ligand binding and degradation (11).Low expression of AGER1 in the kidneys of nonobese diabeticmice was associated with high tissue AGE levels and with kidneydisease (12). Human circulating mononuclear cells from diabetic

subjects also show an association between low expression ofAGER1 and high serum AGE along with severe diabetic com-plications (13). Recently, we found that overexpression ofAGER1 in mouse mesangial cells (MCs) led to inhibition ofAGE- and RAGE-induced MAPK phosphorylation and NF-�Bactivity (9), suggesting that AGER1 may mitigate AGE-inducedcellular toxicity. The purpose of this study was to elucidate themechanism by which this property of AGER1 is mediated.

AGE, by means of reactive oxygen species (ROS) generation,could stimulate multiple receptors, including the EGF receptor(EGFR) (14, 15). EGFR belongs to a large family of tyrosinekinase receptors and is involved in the regulation of multiplecellular processes, such as cell growth, motility, differentiation,survival, and death (16). The EGFR binds many growth factors,including EGF, heparin-binding EGF, transforming growth fac-tor-�, amphiregulin, neuroregulin, �-cellulin, and epiregulin(17). Ligand-triggered activation of its intrinsic tyrosine kinaseand autophosphorylation of its tyrosine residues induces Ras-MAPK1,2 phosphorylation through activation of the Grb2�Shc�Sos complex (18–20). ROS may also block EGFR dephosphor-ylation, possibly by inhibition of protein-tyrosine phosphataseactivity (21, 22). AGE can generate ROS, in part through RAGE(15), but there has been no direct evidence that AGE can interactwith EGFR or with the Grb2�Shc�Sos complex. Therefore, weinvestigated whether AGE induces EGFR phosphorylation and,if so, what is the mechanism of induction. We then examined theeffect of AGER1 overexpression on the AGE-induced EGFRpathway of activation.

We found that Shc�Grb2 and EGFR phosphorylation are in-duced by AGE in WT MCs and kidney epithelium-like cells(HEK293) but are markedly attenuated by overexpressing AGER1.The observations provide vital mechanistic information about thispreviously uncharacterized AGE�OS negative regulatory pathway,which may be important in AGE�OS homeostasis.

ResultsOverexpression of AGER1 Leads to Down-Regulation of AGE-InducedRas Activation and of Shc�Grb2 Complex Formation. MCs andHEK293 cells were stably transfected with a V5-tagged vectorencoding AGER1 at its C terminus (V5-AGER1), and, afterenrichment by antibiotic selection, they were assessed by West-ern blot analysis using anti-V5 antibody. The V5-AGER1 proteinwas overexpressed in both transfected cell types but not in mocktransfectants or WT cells (Fig. 1). Because Ras activation occurs

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: AGE, advanced glycation end product; AGER, AGE receptor; EGFR, EGFreceptor; ROS, reactive oxygen species; IB, immunoblotting; MC, mesangial cell; RAGE,receptor for AGE; OS, oxidant stress; PAO, phenylarsine oxide.

‡To whom correspondence should be addressed at: Division of Experimental Diabetes andAging, Brookdale Department of Geriatrics, Mount Sinai School of Medicine, Box 1640,One Gustave Levy Place, New York, NY 10029. E-mail: helen.vlassara@mssm.edu.

© 2006 by The National Academy of Sciences of the USA

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upstream of MAPK1,2 phosphorylation, we investigatedwhether Ras activity was altered in AGER1-overexpressing andmock MCs after stimulation with AGE-BSA (50 and 200 �g�ml,respectively) for 15 min. Using a Ras activation assay, Rasactivation was found to be blocked by AGER1 overexpression inMC-R1 cells when compared with MC-mock at both concen-trations of AGE-BSA (P � 0.05) (Fig. 2). As a control, nativeBSA did not affect Ras activation in either MC-R1 or MC-mockcells (Fig. 2). Activation of Ras occurs as a result of Shc�Grb2complex formation. Therefore, the Shc�Grb2 complex was as-sessed by immunoprecipitation with anti-Grb2 antibody fol-lowed by immunoblotting (IB) with anti-Shc antibody in MC-R1and MC-mock cells after AGE stimulation. We found that, inmock cells, AGE-BSA increased Shc�Grb2 complex formation,

as shown by coimmunoprecipitation, whereas Shc�Grb2 wascompletely blocked in MC-R1 cells (P � 0.05) (Fig. 3). Again,EGF activation of Shc was preserved, suggesting that there wasnot a competitive interaction between the AGE and EGFligands.

AGE-Induced Shc Phosphorylation Is Reduced in AGER1-ExpressingCells. Because Shc�Grb2 complex formation was significantlylower in AGER1-overexpressing cells, we examined Shc phos-phorylation. Tyrosine phosphorylation of Shc protein was de-termined by using anti-phospho-Shc (Y317) antibody, becauseY317 tyrosine phosphorylation has been shown to cause MAPKactivation through Grb2 and Sos (23). AGE stimulation ofMC-mock cells markedly enhanced Shc52 tyrosine phosphory-lation between 5 min (P � 0.01) and 15 min (P � 0.05) ofstimulation (Fig. 4). However, in 293-R1 cells, there was signif-icantly less phosphorylated Shc52 after stimulation with the sameamount of AGE (P � 0.05). Total Shc protein levels did notchange between 293-mock and 293-R1 cells.

EGFR Tyr Phosphorylation Is Blocked After AGE Stimulation, but NotAfter EGF Stimulation, in HEK293-R1-Expressing Cells. Because Shc isrecruited to phospho-residues of growth factor receptors [e.g.,EGFR (24)], we tested the effect of AGE on phosphorylation ofEGFR. We found that AGE caused EGFR phosphorylation inboth HEK293 and HEK293-R1 cells (Fig. 5A). In addition, weobserved a striking difference in the levels of EGFR Tyrphosphorylation between HEK293-mock and HEK293-R1 cellsin response to AGE-BSA. EGFR was maximally Tyr phosphor-ylated at 5 min in HEK293-mock cells, whereas it was attenuatedin HEK293-R1 cells (Fig. 5A). Of note, this suppressed responseto AGE was somewhat recovered or counterbalanced after

Fig. 2. AGE-induced Ras activation is blocked in MC-R1 cells. (A and B) Rasactivation in serum-starved MC-R1 and MC-mock cells was examined by Raspull-down assays after exposure to AGE-BSA (50–200 �g�ml) or BSA (200�g�ml) for 15 min at 37°C. EGF (30 ng�ml) was used as a positive control. (C)Densitometry of data shown in A and B. **, P � 0.01 vs. unstimulated cells (CL);#, P � 0.05 vs. MC-mock cells. The results were consistent among threeindependent experiments.

Fig. 3. AGE-induced Shc�Grb2 complex formation is blocked in MC-R1 cells.(A and B) Serum-starved MC-R1 and MC-mock cells were exposed to AGE-BSA(50–200 �g�ml) for 15 min at 37°C. Cell lysates were subjected to immuno-precipitation with anti-Grb2 antibody and IB with anti-Shc antibody. Cellstreated with EGF (30 ng�ml) for 15 min were used as a positive control. (C) Datafrom densitometric analysis. *, P � 0.05; **, P � 0.01 vs. unstimulated condi-tions; #, P � 0.05 vs. MC-mock cells. The results were consistent among threeindependent experiments.

Fig. 1. V5-AGER1 expression in mouse MCs and HEK293 cells. (A and B) MouseMCs and HEK293 cells (293) were transfected with V5-tagged full-lengthhuman cDNA encoding AGER1. Cell lysates from these stable cell lines weresubjected to Western blotting by using anti-V5 antibody as described.

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cotransfection of 293-R1 cells with EGFR (Fig. 5 A and B, rightblots), suggesting a dependence on receptor availability. How-ever, when, instead of AGE, EGF was used as a stimulant,neither EGFR protein expression nor phosphorylation levelsdiffered between HEK293-R1 and mock cells (Fig. 5 C and D).

AGER1 Interacts with EGFR. Experiments were performed to exam-ine the relationship between AGER1 and EGFR. Cell lysates wereprepared from 293-R1 and MC-R1 cells before or after exposureto AGE-BSA (200 �g�ml) and were immunoprecipitated withanti-V5-R1 antibody before IB with anti-EGFR antibody. AGER1was associated with EGFR in AGER1-expressing cells under both

resting and AGE-stimulated conditions (Fig. 6 A and C). This resultwas confirmed by reverse immunoprecipitation (with anti-V5-R1antibody and with anti-EGFR antibody) (Fig. 6 B and D). Coim-munoprecipitation between cells expressing control vector with V5alone and with EGFR showed that there was no interactionbetween V5 and EGFR (data not shown). The association betweenAGER1 and EGFR was further investigated by transiently trans-fecting 293-AGER1 cells with an expression vector containing thehuman EGFR (WT) under the control of the CMV promoter.After 48 h of transient transfection, cell lysates were analyzed byimmunoprecipitation with anti-EGFR antibody and IB with thesame antibody or with anti-V5-R1antibody. A greater amount ofAGER1 was found in 293 cells overexpressing EGFR and AGER1compared with mock cells (Fig. 6E), providing further confirmationof a direct association between EGFR and AGER1.

AGE-Induced p44�p42 Phosphorylation Is Constant After Overexpress-ing EGFR but Not After Coexpressing AGER1 and EGFR. HEK293-mock or EGFR� cells were stimulated by AGE (200 �g�ml for15 min); S100, a RAGE ligand agonist (5 �g�ml for 15 min); orH2O2 (0.1 mM for 15 min) in the presence or absence of theEGFR-specific tyrosine phosphorylation inhibitor AG1478 (10�M). All three stimulants caused rapid MAPK phosphorylation(Fig. 7A), which was not further increased after EGFR overex-pression. These data were consistent with EGFR activationbecause this phosphorylation was largely blocked by AG1478(Fig. 7B). However, when AGER1 was coexpressed in 293-EGFR� cells, AGE and S100-mediated MAPK phosphorylationwas abolished (Fig. 7C) but not when a more potent ROS source,H2O2, was used. Furthermore, the suppression of AGE and S100was restored by silencing R1 by using siRNA (Fig. 7C), con-firming the fact that the inhibitory effects depended on thepresence of AGER1.

AGE-Induced ROS Generation Is Suppressed in R1-Expressing Cells.After incubation of 293-mock cells to AGE (200 �g�ml for 4 h),intracellular ROS (measured as H2O2) increased significantlybut not after exposure to BSA (Fig. 8). In contrast, in R1-expressing cells, AGE-induced ROS generation was inhibited,suggesting that AGER1 negatively regulates ROS generation,the latter being a potent stimulus for EGFR phosphorylation. Toassess whether EGFR activation indirectly influences AGE-

Fig. 4. AGE-induced Shc phosphorylation is decreased in MC-R1 cells. (A)Serum-starved MC-R1 and MC-mock cells were treated with AGE-BSA (200�g�ml) for 5, 15, and 30 min at 37°C. Cell lysates were subjected to IB with anantibody specific for phospho-Shc (Y317), and the same membrane wasreprobed with anti-Shc antibody for normalizing loading amounts. (B) Den-sitometry of data shown in A. *, P � 0.05; **, P � 0.01 vs. unstimulatedconditions; #, P � 0.05 vs. MC-mock cells. Data represent three independentexperiments.

Fig. 5. Overexpression of R1 in HEK293 cells suppresses AGE-induced ty-rosine phosphorylation of the EGFR by AGE, but it does not decrease EGFRtyrosine phosphorylation by EGF. Serum-starved 293-R1 cells were transientlytransfected by human EGFR for 48 h. These cells and corresponding mock cellswere treated with 200 �g�ml AGE-BSA or BSA for 5, 15, and 30 min at 37°C orwith EGF (100 ng�ml) for 5 min. (A and C) Cell extracts were prepared andimmunoprecipitated with an anti-EGFR antibody and immunoblotted withPY20 phosphotyrosine antibody. (B and D) The membranes were then re-probed with anti-EGFR antibody. Representative data from three indepen-dent experiments are shown.

Fig. 6. EGFR interacts with R1. Serum-starved 293-R1 and MC-R1 cells wereincubated with or without 200 �g�ml AGE-BSA for 5, 15, and 30 min at 37°C.(A–D) Cell extracts were used for coimmunoprecipitation with anti-V5 anti-body (A and C) or anti-EGFR antibody (B and D). The immunoprecipitates wereimmunoblotted with anti-EGFR antibody (A and C) or anti-V5 antibody (B andD). (E) After transient transfection with WT human EGFR, 293-R1 cell lysateswere analyzed by immunoprecipitation with anti-EGFR antibody and IB withanti-EGFR antibody or anti-V5 antibody. The results obtained were consistentin three independent experiments.

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induced ROS generation, 293-mock and R1-expressing cellswere pretreated with AG1478 for 2 h before exposure to AGE(200 �g�ml for 4 h), and ROS were measured as above. AG1478did not alter AGE-induced ROS generation in either mock orR1-expressing cells, indicating that EGFR activation does notcontribute to AGE-mediated OS (Fig. 8).

To further assess whether enhanced AGE clearance is respon-sible for reduced ROS in AGER1 cells, ROS production by AGEwas assessed after inhibiting endocytosis by using phenylarsineoxide (PAO). PAO partially reversed the inhibitory effect ofAGER1 on ROS generation in R1-expressing cells, possibly dueto a blockade of AGE endocytosis and degradation in these cells(data not shown).

DiscussionAGER1 may play a protective role in the AGE-induced cellulartoxicity that is seen in diabetes and aging by enhancing AGE

disposal and by countering RAGE-promoted OS and subsequentMAPK�NF-�B-dependent cell activation (9, 12, 13). In the presentreport, we found that EGFR and the Shc�Grb2 complex have apreviously unidentified role in the AGE signaling activation path-way. The negative regulatory effects of AGER1 on AGE signalingare mediated by means of the inhibition of EGFR phosphorylationand Shc�Grb2 complex formation. These effects may be related toa direct molecular interaction between AGER1 and EGFR.AGER1 also exhibits significant suppressive effects on intracellularROS generation, which may in part be due to increased AGEdegradation by AGER1.

We found that AGE causes Ras�MAPK1,2 activation by meansof tyrosine phosphorylation of EGFR in two different types ofkidney cells, MCs and epithelial (HEK293) cells. This interaction,as with the S100�RAGE interaction, depends on the generation ofROS. The mechanism by which AGE induces ROS generation,resulting in Ras�MAPK1,2 phosphorylation, remains unclear, be-cause no AGERs, including RAGE, have known autophosphory-lation sites or specific kinase activity (15, 25).

ROS and H2O2 are known triggers of Tyr phosphorylation ofEGFR, which leads to downstream MAPK or Akt activation (26,27). Importantly, this can happen in the absence of EGFstimulation. As with H2O2, here, AGE and S100 also induced Tyrphosphorylation of EGFR. Because the EGFR activation inhib-itor AG1478 inhibited the p44�42 Tyr phosphorylation inducedby all three ROS-triggering ligands, these inhibitions appeared toact via an EGFR-specific signal pathway. However, when thecells overexpressing EGFR were also transfected with AGER1,only AGE- and S100-induced p44�42 Tyr phosphorylation wasinhibited, and not that due to H2O2. Thus, AGER1 can coun-teract some of the downstream effects of AGE and S100stimulation, both of which are ROS generators. Although thedata do not suggest a direct interaction of either R1 or EGFRwith RAGE, such a possibility requires further study. However,the findings remain consistent with a dependence of RAGEsignaling on ROS and with a lack of specificity for AGE. Inaddition, AGER1 did not counteract exogenous H2O2-inducedMAPK phosphorylation, indicating that this phosphorylationinvolves a different mechanism.

Activation of Ras occurs through GDP�GTP exchange, cat-alyzed by the guanine nucleotide-exchange factor Sos proteins,which are translocated to the plasma membrane as a result of

Fig. 7. AGE-induced MAPK (p44�p42) phosphorylation was enhanced in EGFR-expressing cells but was suppressed in R1-EGFR-coexpressing cells. (A)Serum-starved 293-mock and 293-R1 cells transiently transfected with or without vector containing WT human EGFR. Cells were exposed to AGE-BSA (200 �g�ml),S100 (5 �g�ml), or H2O2 (0.1 mM) for 15 min at 37°C. Lysates were subjected to Western blot analysis using anti-phospho-p44�p42 antibody. Stripped membraneswere reprobed by using anti-p44�p42 antibody. (B) 293-mock cells were preincubated with a specific inhibitor of EGFR tyrosine kinase AG1478 at 10 �M for 1 hand then treated as indicated. (C) EGFR��R1� cells were transiently transfected with pooled R1 siRNA at 100 nM for 48 h before adding the agonists. The resultsobtained were consistent in three independent experiments. Data from densitometric analyses are shown above the blots. *, P � 0.05; **, P � 0.01 vs.unstimulated conditions.

Fig. 8. Effects of AGE-BSA on intracellular ROS in AGER1-expressing 293cells. Levels of dichlorofluorescein (DCF) were measured with 2�,7�-dichlorofluorescein diacetate after adding AGE or BSA (200 �g�ml for 4 h) byusing a fluorescence spectrophotometer at 485-nm excitation and 530-nmemission wavelengths as described. Nonstimulated R1-expressing and mock-293 cells served as controls. Intracellular ROS production was also measured incells pretreated with AG1478 before stimulation with AGE or BSA. Data areexpressed as means � SD of three independent experiments. *, P � 0.05; **,P � 0.01 vs. control; #, P � 0.05 vs. 293-mock cells treated with AGE-BSA.

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Shc�Grb2 complex formation (28). Shc, an important group ofadaptor proteins, plays a critical role in the Ras�ERK signalingcascade (29). After undergoing phosphorylation, Shc provideshigher affinity docking sites for Grb2 complex formation (24).Shc phosphorylation usually occurs after Grb2 complex recruit-ment to phosphotyrosine residues of growth factors such asEGFR (24). We found that AGE-mediated Ras and MAPK1,2activation (30) progressed in a stepwise fashion through theEGFR-triggered sequence of events described above. Althoughwe have not explored all of the relationships in this proximalphase of AGE signal induction, we noted that brief exposure toAGE preferentially stimulates Tyr phosphorylation of the 52-kDa Shc and, to a lesser extent, the 46-kDa Shc, but not the66-kDa Shc, in kidney epithelial and MC lines. These studiesmade use of an anti-phosphotyrosine antibody that is specific forTyr-317. In several cell types, EGFR activation involves all threeShc isoforms (31), including tyrosine and serine�threoninephosphorylation. Thus, further studies are needed to determinewhether these discrepancies are due to differences in the celltypes or in the immunoreagents used. Based on previous reportsthat have linked phosphorylation of Shc66 to EGFR inactivation(31), its possible role in AGER1–AGE signaling inactivation isworthy of further investigation (31).

Our findings suggest that EGFR is an important contributorto the AGE cell activation pathway and also reveal a potentialmechanism by which AGE signal suppression can limit AGEtoxicity after AGER1 overexpression.

Interestingly, AGER1 overexpression did not affect EGF-induced signaling. Instead, EGF stimulation led to enhancedEGFR protein expression and signaling, remaining independentof the inhibitory action of AGER1. This observation suggeststhat AGER1 does not interfere with EGFR gene regulation andevents induced by EGF [i.e., dimerization, adaptor recruitment,or signaling (16)]. Rather, the AGER1 effects on EGFR activityappear to be AGE�ROS-specific. In fact, AGER1-mediatedinhibition of AGE-induced EGFR activation (32, 33) was re-stored by R1 siRNA. Furthermore, AGE-induced intracellularROS generation was inhibited in AGER1-overexpressing cells,supporting the pivotal role of AGER1 in this interaction.

Although AGE has been shown to stimulate heparin-bindingEGF production (34), the relationship that we found betweenAGERs and EGFR has not been suggested previously. Thepresent studies clearly demonstrate a direct interaction betweenAGER1 and EGFR, although the molecular domains and thepossibility that additional cellular components are involved inthis interaction remain to be further explored. A possibledependence of EGFR responsiveness on the level of EGFR vs.R1 cell surface availability was suggested during EGFR andAGER1 single expression and coexpression studies. These datamay point to a potential explanation for the low efficacy ofAGER1 (12) under conditions of sustained ROS generation[e.g., in diabetes, when EGFR expression and activity might beexpected to be elevated (32, 33)].

Our previous report showed that AGER1 played a role in AGEclearance by mediating enhanced AGE uptake and degradation (9).In the current study, we found that AGE-induced ROS levels in293-R1 cells were no different from mock cells after partialblockade of endocytosis with the endocytosis inhibitor PAO. Thisobservation indicated that, when AGER1 is overexpressed, it maymore effectively clear AGE, causing attenuation of AGE�ROS-mediated signaling, and that this finding could serve in part as anadditional mechanism for reduced EGFR phosphorylation in R1cells (35, 36). However, a nonspecific PAO action could not beruled out. Also, these data do not account for AGE ROS that aregenerated in cell types lacking AGE-specific endocytosis, andfurther studies are needed to address this issue in greater detail,including mapping of the AGER1-binding site and�or of sites ofinteraction with EGFR.

Based on these findings, we speculate that, in a normalenvironment, AGER1 may sequester EGFR, the end resultbeing to minimize undue EGFR activation by uncoupling EGFRfrom Shc�Grb2�Sos and Ras, thereby preventing inappropriatecell activation. Under conditions of sustained excess of AGE andROS, however, the AGE-saturated AGER1 may be renderedinactive and no longer accessible to EGFR. Thus, excess ROScould lead to sustained EGFR and MAPK�NF-�B stimulation,which may account in part for an ineffective AGER1 function inchronic unregulated oxidant or carbonyl stress (e.g., in diabetesand age-related vascular�renal diseases, also known as inflam-matory states).

In conclusion, our findings help elucidate aspects of both thepositive and negative regulatory steps of AGE- and AGER-mediated activation pathways involved in the inflammatoryresponse. Although we studied murine or human kidney cells,the data may apply to other cells and tissues that are shown toexpress AGER1. These data may improve the understanding bywhich AGE-caused cell toxicity can be regulated and could serveas a guide for developing therapeutic targets for vascular andkidney disorders related to diabetes and aging.

Materials and MethodsMaterials. Anti-EGFR and Ras activation assay kits were obtainedfrom Upstate Biotechnology (Lake Placid, NY). Anti-phosphoty-rosine antibody PY20 was obtained from Santa Cruz Biotechnology(Santa Cruz, CA). Antibodies to Shc and Grb2 were obtained fromBD Biosciences Transduction Laboratories (San Jose, CA). Theexpression vector containing WT human EGFR under the controlof a CMV promoter was obtained from Upstate Biotechnology.Antibody to MAPK1,2 was purchased from New England BioLabs(Beverly, MA). Anti-V5 antibody was from Invitrogen (Carlsbad,CA). S100 was from Calbiochem (San Diego, CA). There was nocross-reaction between anti-V5 and EGFR, or anti-EGFR and R1,or between other reagents used. EGFR inhibitor AG1478 was fromCalbiochem. N-acetylcysteine and hydrogen peroxide (H2O2) wereobtained from Sigma (St. Louis, MO), as was the endocytosisinhibitor PAO. Endotoxin-free AGE-BSA was prepared fromendotoxin-free, lyophilized BSA (fraction IV; Sigma) and glucoseas described in ref. 37 after extensive dialysis and passing througha filter (0.22-�M pore size; Millipore, Billerica, MA). An endotox-in-binding affinity column (Pierce, Rockford, IL) was used toremove endotoxin from AGE-BSA and native BSA (tested by aLimulus amebocyte lysate assay; Bio Whittaker, Walkerville, MD).

Cell Culture. Human embryonic kidney epithelium-like cells(HEK293 or 293) were chosen because EGFR has been extensivelystudied in these cells (38–40). Mouse MCs were identical to thoseused in our recent AGER1 studies (9). MCs (CRL-1927; AmericanType Culture Collection) were maintained in DMEM�F12 medium(3:1) containing 100 units�ml penicillin, 100 �g�ml streptomycin,14 mmol�liter Hepes, and 5% FBS. HEK293 cells (CRL-1573;American Type Culture Collection) were maintained in MEMsupplemented with 2 mM L-glutamine�100 units/ml penicillin�100�g/ml streptomycin�10% FBS. Both cell lines were cultured at 37°Cunder a humidified atmosphere (5% CO2 and 95% air). Beforetreatment, cells were washed with PBS and incubated with serum-free media for 12 h (quiescent cells). The addition of native BSA(300 �g�ml) under serum starvation conditions produced nodiscernible changes in cell reactivity.

Stable Expression of AGER1 in MCs and HEK293 Cells. Stable MC and293 cell lines expressing human AGER1 protein were obtainedafter transfection of cells with a pcDNA3.1 (Invitrogen) vectorcontaining a V5-tagged AGER1 construct (V5-AGER1) andsubsequently selected by culturing cells in the presence of theneomycin analogue G418 (1 mg�ml) (Invitrogen) for at least 3weeks before use (9). G418-resistant cells containing

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pcDNA3.1-R1 were used for the study. Mock-transfected MCsand HEK293 cells expressing the neomycin resistance gene wereestablished as described in ref. 9. Expression of V5-AGER1-tagged protein in transfected cells was confirmed by Westernblot by using monoclonal anti-V5 antibody (Invitrogen).

Immunoprecipitation. Quiescent cells were treated with agonists at37°C for the indicated times. Cells were rinsed twice in ice-coldPBS and scraped in 500 �l of RIPA lysis buffer. The cells werethen disrupted by brief sonication and centrifuged at 10,000 �g for 5 min. The protein concentration was determined, and 1 mgof cell lysate was immunoprecipitated by incubating with 10 �gof anti-EGFR antibody (Upstate Biotechnology) or 4 �g ofanti-V5 antibody (Invitrogen) at room temperature for 1 h,followed by further incubation at 4°C with 60 �l of protein A�Gplus agarose beads (Santa Cruz Biotechnology), and gentlyrocked overnight. The immunoprecipitates were collected inloading buffer after washing three times in RIPA lysis buffer.

Western Blot Analysis. Equal amounts of samples were separatedon 8% SDS-polyacrylamide gels and transferred onto nitrocel-lulose membranes. The membranes were blocked in TTBS(Tris-buffered saline with 0.1% Tween 20) containing 5% drymilk for 1 h. Incubation with primary antibodies was performedin TTBS with 5% dry milk overnight at 4°C. After washing, themembranes were incubated with the appropriate secondaryperoxidase-conjugated antibody for 1 h in TTBS. Immunoreac-tive proteins were visualized by using the enhanced chemilumi-nescence system (Roche, Indianapolis, IN). After stripping witha buffer containing 50 mM Tris�HCl (pH 6.8), 2% SDS, and 0.1M 2-mercaptoethanol, membranes were reprobed with an ap-propriate secondary antibody.

Ras Activity Assay. Ras activity assays were carried out as de-scribed in ref. 41. Briefly, the soluble cell lysates were incubatedin Mg2� lysis buffer with Raf-1–RBD agarose, a GST fusionprotein corresponding to the human Ras binding domain (RBD)of Raf-1, on glutathione-Sepharose beads at 4°C for 30 min. TheSepharose beads were recovered by gentle centrifugation andrapidly washed three times with 1 ml of ice-cold lysis buffer.Protein recovered with the Raf-1–RBD fusion protein wasseparated by SDS�PAGE and subjected to Western blot analysiswith a monoclonal antibody against Ras.

RNAi. Quiescent cells were transfected with pooled AGER1siRNA by using Lipofectin reagent (GibcoBRL, Carlsbad, CA).The sequences were (i) GACCAUCAGUGCCUUUAUU, (ii)CGACGUGUAUGGUGUAUUC, (iii) GGAAUUCCUC-UAUGACAAU, and (iv) GACAGGCAACUAUGAACUA.After 48-h transfection, cells were exposed to the appropriateagonists for the time period appropriate for each experiment.The inhibitory efficiency of R1 siRNA was determined byWestern blotting for MAPK1,2 phosphorylation.

Intracellular ROS. ROS were measured by using the probe 2�,7�-dichlorofluorescein diacetate (DCFDA) (Molecular Probes, Eu-gene, OR) as described in ref. 28. In brief, cells were treated withor without 200 �g�ml AGE-BSA at 37°C for 4 h, washed withPBS, and incubated with 5 �M DCFDA for 45 min. Thedichlorofluorescein was measured by using a fluorescence spec-trophotometer with 485-nm excitation and 530-nm emissionwavelengths.

We thank Ina Katz for invaluable editorial assistance. This work wassupported by National Institutes of Health Grants NIA AG09453 (toH.V.), NHL73417 (to H.V.), and DK-65495 (to J.C.H.).

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