Free Radical Biology and Medicine 74 (2014) 294–306

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Original Contribution

Nε-carboxymethyllysine-mediated endoplasmic reticulum stress promotes endothelial cell injury through Nox4/MKP-3 interaction Wen-Jane Lee a,b,1, Wayne Huey-Herng Sheu c,d,e,2, Shing-Hwa Liu f,1, Yu-Chiao Yi g, Wei-Chih Chen g, Shih-Yi Lin h, Kae-Woei Liang i, Chin-Chang Shen j, Hsiang-Yu Yeh k, Li-Yun Lin l, Yi-Ching Tsai c,d, Hsing-Ru Tien d, Maw-Rong Lee m, Tzung-Jie Yang m, Meei-Ling Sheu a,d,e,n a

Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan Department of Social Work, Tunghai University, Taichung, Taiwan Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan d Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan e Rong Hsing Research Center for Translational Medicine, and National Chung Hsing University, Taichung 402, Taiwan f Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan g Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan h Division of Endocrinology and Metabolism, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan i Cardiovascular Center, Taichung Veterans General Hospital, Taichung, Taiwan j Institute of Nuclear Energy Research, Atomic Energy Council, Longtan, Taoyuan, Taiwan k Department of Nutrition and Institute of Biomedical Nutrition and Hung-Kuang University, Taichung, Taiwan l Department of Food and Nutrition, Hung-Kuang University, Taichung, Taiwan m Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan b c

art ic l e i nf o

a b s t r a c t

Article history: Received 15 February 2014 Received in revised form 29 May 2014 Accepted 17 June 2014 Available online 8 July 2014

Nε-carboxymethyllysine (CML) is an important driver of diabetic vascular complications and endothelial cell dysfunction. However, how CML dictates specific cellular responses and the roles of protein tyrosine phosphatases and ERK phosphorylation remain unclear. We examined whether endoplasmic reticulum (ER) localization of MAPK phosphatase-3 (MKP-3) is critical in regulating ERK inactivation and promoting NADPH oxidase-4 (Nox4) activation in CML-induced endothelial cell injury. We demonstrated that serum CML levels were significantly increased in type 2 diabetes patients and diabetic animals. CML induced ER stress and apoptosis, reduced ERK activation, and increased MKP-3 protein activity in HUVECs and SVECs. MKP-3 siRNA transfection, but not that of MKP-1 or MKP-2, abolished the effects of CML on HUVECs. Nox4-mediated activation of MKP-3 regulated the switch to ERK dephosphorylation. CML also increased the integration of MKP-3 with ERK, which was blocked by silencing MKP-3. Exposure of antioxidants abolished CML-increased MKP-3 activity and protein expression. Furthermore, immunohistochemical staining of both MKP-3 and CML was increased, but phospho-ERK staining was decreased in the aortic endothelium of streptozotocin-induced and high-fat diet-induced diabetic mice. Our results indicate that an MKP-3 pathway might regulate ERK dephosphorylation through Nox4 during CML-triggered endothelial cell dysfunction/injury, suggesting that therapeutic strategies targeting the Nox4/MKP-3 interaction or MKP-3 activation may have clinical implications for diabetic vascular complications. & 2014 Elsevier Inc. All rights reserved.

Keywords: AGEs CML Nox4 MKP-3 ERK Human endothelial cells Free radicals

Nε-carboxymethyllysine (CML), one of the better characterized advanced glycation end-products, is implicated in several steps

n Corresponding author at: Institute of Biomedical Sciences, National Chung Hsing University, Taichung 402, Taiwan. Fax: þ 886 4 22853469. E-mail addresses: [email protected], [email protected] (M.-L. Sheu). 1 These authors contributed equally to this work. 2 W.H.-H. Sheu equally to corresponding author.

http://dx.doi.org/10.1016/j.freeradbiomed.2014.06.015 0891-5849/& 2014 Elsevier Inc. All rights reserved.

leading to the development of diabetic vascular complications and is frequently used as an advanced glycation end-product (AGE) marker [1]. It is deposited in human intramyocardial blood vessels and abundant in Bruch's membrane, the extracellular matrix separating the retinal pigment epithelium from the blood-bearing choriocapillaries [2]. Evidence indicates that higher serum CML levels are involved in the development of chronic diabetic microvascular complications and reflect the degree of glycemic control

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for a long duration in type 1 diabetic children and adolescents, suggesting it as a predicting factor for the development of microvascular complications [3]. Previous reports show that CML is a general marker of oxidative stress and tissue damage through protein alteration, which is associated with acute and chronic inflammatory diseases, including rheumatoid arthritis [4]. Reports also link AGEs to the vascular matrix. These AGEs can chemically interfere with the bioavailability of nitric oxide (NO) by quenching NO and mediating defective endothelium-dependent vasodilatation in experimental diabetes [5]. Recently, CML has been shown to induce NADPH oxidase/ROS-activated SHP-1 signaling, which in turn initiates the dephosphorylation of VEGFR-2 to cause human endothelial cell dysfunction [6]. However, intricate cellular and molecular events underlying the pathophysiologic changes in vascular endothelial cells induced by CML remain unclear. Impaired endothelial nitric oxide synthase (eNOS) activity may be involved in the pathogenesis of diabetic macrovascular and microvascular complications [7]. Decreases in vascular bioavailability of NO have long been proposed to be the common pathogenetic mechanism of endothelial dysfunction, resulting from diverse cardiovascular risk factors [8]. eNOS expression is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Several signaling pathway systems are involved in regulating eNOS activity, such as PI3K/AKT, mitogen-activated protein kinase (MAPK), and rho kinase [8]. Specially, the MAPK extracellular signal-regulated kinase-1/2 (ERK1/2) exerts a prosurvival function. Recently, it was shown that the ERK1/2–eNOS Ser-635 phosphorylation pathway modulated by endoplasmic reticulum (ER) Ca2 þ may have a broad role in the regulation of endothelial function [9]. The endothelial mitogen netrin-1 potently stimulates NO production via an ERK1/2–eNOS Ser-1177-dependent mechanism [10]. However, the mechanisms by which AGEs/CML influence the ERK1/2 phosphorylation process are not clearly elucidated in endothelial cell dysfunction or damage. Recent evidence indicates that the activation of the ER stress pathway known as the unfolded protein response (UPR) can lead to adverse affects on ER function and cell pathology and, later, to tissue dysfunction such as hyperglycemia, hypoxia, and oxidative stress. Emerging evidence show that the UPR is chronically activated in atherosclerotic lesion cells, particularly in endothelial cells with advanced injury [11]. In type 2 diabetes, ER stress and reactive oxygen species (ROS) production are important mechanisms that underlie many of the serious consequences. Thus, these may be potential targets for a novel therapeutic approach to preventing impaired vascular pathology. MAPK phosphatase-3 (MKP-3) is a dual-specificity phosphatase that plays an important role in attenuating MAPK signaling. MKP-3 can antagonize the insulin effect of repressing gluconeogenesis in cultured Fao hepatoma cells [12]. An early study also demonstrated that MKP-3 has a role in regulating glucose metabolism in vivo and elucidated that FOXO1 is the downstream mediator of MKP-3 in promoting gluconeogenesis, suggesting that inhibition of MKP-3 activity may provide new therapies for type 2 diabetes [13]. Interestingly, the controversial role of the loss of MKP-3 by an intracellular ROS is a crucial factor in the aberrant activation of ERK1/2 in the progression and chemoresistance of human ovarian cancer [14]. Diabetes-evoked NADPH oxidase activity-derived ROS have been suggested to regulate phosphatase, which affects the cell function of phosphorylation and leads to signaling-related gene induction. However, the role of MKP-3 in CML-induced vascular injury is still unclear. This study hypothesizes that the pathway of ROS-triggered MKP-3-regulated ERK dephosphorylation may be involved in CMLtriggered ER stress and endothelial injury. Human umbilical vein endothelial cells (HUVECs), which played a major role as a model

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system in the development of the field of vascular biology; mouse microvascular endothelial cells (SVECs); primary rat aorta endothelial cells (RAECs); and diabetic animal models were used to test this hypothesis.

Material and methods Cell culture Primary HUVECs were obtained by collagenase treatment of umbilical cord veins as described previously [6,15,16]. SV-40 immortalized SVECs were cultured in Dulbecco’s modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Diabetic mouse models The protocol of the animal study was approved by the Institutional Animal Care and Use Committee, and the care and use of laboratory animals were conducted in accordance with the guidelines of the Animal Research Committee of the Taichung Veterans General Hospital. Six-week-old db/db mice displayed significant obesity and hyperglycemia. Male db/db mice (C57BLKS/J-leprdb/ leprdb) and male nondiabetic db/m mice (C57BLKS/J-leprdb/ þ) as controls were donated by S.H. Liu (n¼ 6–8 per group). The streptozotocin (STZ)-induced type 1 diabetic (n ¼20–22 per group) and high-fat diet (HFD)-induced obesity diabetic (n ¼26–28 per group) models were performed as described previously [6,15,16]. Measurement of CML or AGEs in patients with type 2 diabetes Serum CML and AGE levels were measured in 48 newly diagnosed type 2 diabetic patients (16 women, 32 men) with a mean age of 64.4 79.9 years, body mass index (BMI) of 26.0 73.4 kg/m2, and hemoglobin A1c (HbA1c) 4 8.6% at diagnosis. In addition, 48 apparently healthy subjects (17 women, 31 men) with a mean age of 62.5 76.4 years, BMI of 25.3 72.5 kg/m2, fasting glucose o92 mg/dl, and HbA1c o5.8% were enrolled to serve as the control group. The study was approved by the Institutional Review Board of Taichung Veterans General Hospital, and written informed consent was obtained from all participants. Analysis of CML and AGE production The CML and AGE concentrations were measured using an Nεcarboxymethyllysine ELISA kit (OxiSelect ELISA Kit, Cell Biolabs), an advanced glycation end product ELISA kit (OxiSelect ELISA Kit, Cell Biolabs), and a Uscn ELISA kit (Uscn Life Science). Synthesis of CML–collagen and AGE–bovine serum albumin (BSA) identified by LC/MS/MS and biochemical study CML–collagen was prepared by chemical modification of acidsoluble bovine skin collagen (Sigma), which was dissolved fresh in 1 mM HCl and incubated at 37 1C with occasional mixing overnight. Then sodium cyanoborohydride and glyoxylic acid in sterile phosphate-buffered saline (PBS) were mixed in. Simultaneously, control collagen was produced except that glyoxylic acid was omitted. The mixtures were incubated at 37 1C for 24 h and then extensively dialyzed (Spectrum Medical Industries, Inc.; Laguna Hills, CA). CML–collagen was converted to CML lysine residues, detected by the trinitrobenzenesulfonic acid assay (Pierce). For AGE–BSA, BSA was incubated under sterile conditions with D-glucose and 5 mM diethylenetriaminepentaacetic acid in 0.2 M phosphate buffer (pH 7.4) at 37 1C for 8 weeks. Low-molecular-weight reactants and

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D-glucose

were separated out using a PD-10 desalting column and dialysis against PBS (pH 7.4). Analysis of the synthesis of CML– collagen and AGE–BSA was performed in the selected-reaction monitoring mode using two transitions: m/z 205-m/z 130 (CE: 25) and m/z 205-m/z 84 (CE: 25). The amount of endotoxin contamination was measured using the Pyrochrome Limulus amoebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA, USA) and found to be low (0.05570.010 ng/mg lipopolysaccharide in control collagen and 0.02270.011 ng/mg lipopolysaccharide in CML–collagen). First, in the analytical chemistry, capillary electrophoresis was used to separate CML–collagen into ionic species by their charge and frictional force and hydrodynamic radius (data not shown). Then, we further characterized the CML–collagen by liquid chromatography tandem mass spectrometry. The commercial standard CML was purchased from Symo-Chem (Eindhoven, The Netherlands). With regard to the analytical biochemistry, CML–collagen was highly reactive with both anti-CML monoclonal antibodies 6D12 (KH001) and NF-1 G (KH024) (Trans Genic, Tokyo, Japan). Monoclonal antibodies specific for CML or AGEs were used for Western blots or immunohistochemical staining, and the control collagen did not react with them. Transmission electron microscopy (TEM) TEM was performed as described previously [17]. Immunohistochemistry Immunohistochemistry was performed as previously described [6,18]. The expression of antibodies in the aortas and kidneys of diabetic mice was determined. Annexin V–FITC and propidium iodide (PI) double staining Annexin V/PI assay (Clontech, Mountain View, CA, USA) was used to quantify the numbers of apoptotic cells, as described previously [15,16]. Assay of MKP-3 phosphatase activity The MKP-3 activity was measured using immunoprecipitated MKP-3. Phosphatase activities of immunoprecipitated MKP-3 were analyzed using the pNPP protein phosphatase assay kit (AnaSpec) according to the manufacturer's instructions. Absorbance was measured at 405 nm. Immunofluorescence staining Cells were prepared and immunofluorescence was determined by laser scanning confocal microscopy as previously described [6,15,16]. The slides were incubated for anti-mouse immunoglobulin–RPE, anti-rabbit immunoglobulin–FITC, or ER-Tracker (Invitrogen). Images were background-subtracted and merged using the Confocal Assistant MetaMorph software program and processed with Adobe Photoshop software.

Western blot analysis and immunoprecipitation Protein levels were analyzed by Western blot as previously described [6,15,16]. For immunoprecipitation, proteins (500 μg) were incubated with specific antibodies and immobilized onto protein A–Sepharose beads.

Transient transfection The delivery of MKP-3 and NADPH oxidase subfamily siRNA pools into HUVECs and SVECs was performed using Lipofectin. Cell transfection was conducted for 24 h at a final siRNA concentration of 100 pM. Normal growth medium control cells were mock transfected without siRNA. All experimental results were confirmed using scrambled siRNA. The commercial siRNA products for human MKP-1 (sc-35937), MKP-2 (sc-38998), MKP-3 (sc-39000), Nox4 (sc-41586), p91phox (Nox2; sc-35503) and p22phox subunits (sc-36149), cytosolic p47phox (sc-29422), p67phox (sc36163), p40phox (sc-36155), and Rac (sc-36351-SH) and scramble control (control siRNA; sc-37007) were used and purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Statistical analyses The values are presented as means 7SEM. Analysis of variance, followed by Fisher’s least significant difference test, was performed for all data. Statistical significance was set at p o0.05.

Results Expression of CML in type 2 diabetes mellitus (T2DM) patients, STZinduced T1DM mice, and HFD-induced diabetic animals and db/db mice The CML expression was evaluated using ELISA kits on the sera of T2DM patients, STZ-induced T1DM mice, and HFD-induced diabetic mice (Figs. 1A–C). Compared to the control group, these three groups had higher CML expression by more than 50%. In the evaluation of CML expression and AGE expression (data not shown) on aortic ring tissues in diabetic animal models, immunohistochemical staining was increased and specific staining was localized in the lining layer of aortic endothelial cells isolated from diabetic db/db, STZ-induced, and HFD-induced mice (Figs. 1D–F). The CML and AGEs were identified by capillary electrophoresis as separate ionic species by their charge and frictional forces and hydrodynamic radius (data not shown). Biochemical study (Fig. 1G) and LC/MS/MS analysis (Fig. 1H) were used for in vitro study. HUVECs incubated with CML supernatant were concentrated and evaluated using anti-CML monoclonal antibody. Purified CML acted as the positive control. However, control HUVECs did not show any effect per se (Fig. 1G). There was also increased endothelial cell sloughing in the aortic rings of diabetic mice. AGE expression was also detected and paralleled the CML production. The result simultaneously verified the persistent higher CML levels in clinical studies of T2DM patients, in mice with STZ-induced T1DM or HFD-induced diabetes, and in diabetic db/db mice. This implied that the elevated CML levels might be correlated with endothelial cell dysfunction.

Small interfering MKP-3, but not MKP-1 or MKP-2, RNA abolished CML-induced apoptosis Flow cytometry was used to investigate if endothelial cell dysfunction was generated in CML-induced apoptosis. CML significantly increased cell apoptosis in HUVECs (Fig. 2A), which was reduced by siRNA-MKP-3 but not siRNA-MKP-1 or siRNA-MKP-2, based on annexin V staining. Apoptosis was also abolished by antioxidant (N-acetyl-L-cysteine (NAC) and apocynin) treatment after CML induction in HUVECs. The inhibitory effect of

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Fig. 1. The expression of Nε-carboxymethyllysine (CML) in type 2 diabetic (T2DM) patients, streptozotocin (STZ)-induced type 1 DM mice, and high-fat diet (HFD)-induced diabetic mice. CML expression was determined by ELISA in samples from (A) T2DM patients, (B) C57BL/6 J mice with STZ-induced T1DM, and (C) C57BL/6 J mice with HFDinduced diabetes. Immunohistochemical staining for CML or advanced glycation end-products (AGEs) in aortas isolated from (D) diabetic db/db mice, (E) STZ-induced diabetic mice, and (F) HFD-induced diabetic mice is shown. Increased endothelial cell sloughing in mouse aortic rings and increased expression of CML is seen. (G) Western blot analysis for CML. The samples in lane 1, lane 2, and lane 3 are purified CML, 0.1 mg (positive control), untreated control HUVECs, and HUVECs treated with CML (50 μg/ml) for 24 h, respectively. (H) LC/MS/MS analysis for CML and AGE–BSA. Samples of CML standard (a), CML–collagen (b), and AGE–BSA (c) were detected. Spectra were averaged over the early eluting and late eluting peaks. Elution time for CML was 16.8 min. CML–collagen and AGE–BSA monitored at m/z 205 (CE: 25) to m/z 130 (CE: 25) and m/z 84 were detected in the condition buffer mixture by selected-ion monitoring as described under Material and methods.

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Fig. 2. Annexin V staining revealed that small interference MKP-3, but not MKP-1 or MKP-2, RNA abolished CML-induced apoptosis. (A) Treatment with CML (50 μg/ml) in HUVECs for 24 h induced apoptosis, as determined by annexin V/PI staining. HUVECs were transfected with MKP-1, MKP-2, or MKP-3 siRNA (50 nmol/L) or nontargeting siRNA scramble (50 nmol/L). After 12 h, the cells were treated with CML (50 μg/ml) and then incubated for 24 h. Phosphatidylserine expression level was detected by flow cytometry and quantitative analysis was determined by annexin V/PI staining. Bars represent the mean 7 SEM fragment condensed staining/field for nucleus (p o 0.05). (B) CML-induced apoptosis was determined by the Hoechst 33285 fluorescent dye method. Images a, control; b, CML 50 μg/ml; c, scrambleþCML; d, scramble; e, siMKP1þ CML; f, siMKP-2þ CML; g, siMKP-3 þ CML; h, siMKP-3 þ CML; i, APO þCML; j, APO 0.5 mM; k, NAC þ CML; l, NAC 0.5 mM. (C) HUVECs were transfected with pcDNA or pcDNA-MKP-3 for 24 h and evaluated by annexin V/PI staining. Results are representative of three independent experiments and relative quantification.

pharmacologic antioxidant and gene silencing on CML-induced apoptosis was also demonstrated in morphologic characteristic staining by Hoechst 33258.

SiRNA-MKP-3 and antioxidant treatment directly suppressed the increase in condensed pyknotic nuclei in CML-induced apoptotic cells. Condensed nuclei were quantified by manual counting

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Fig. 3. CML, AGEs, or high glucose (HG) enhanced ER stress biomarkers in activated human endothelial cells (HUVECs). (A) HUVECs were cultured in the presence or absence of CML, AGEs, or HG. Cell lysates were blotted and immunostained with signature ER stress marker antibodies as indicated. Results are representative of at least four independent experiments. (B) HUVECs were collected and visualized by electron microscopy. (a) Control-, (b) CML-, (c) AGE-, and (d) HG-treated cells displayed ER dilation and serious ER dilation with increased distention and a fragmented organelle. (e) Thapsigargin (1 μM) was a positive control for ER stress induction. (f) CMLþ siMKP-3 dampened profound ER swelling in the ultrastructural morphology. Arrows indicate dilated ER; original magnification,  800 K. Results are typical representatives of independent experiments.

(Fig. 2B; Supplementary Fig. 1A). To determine whether MKP-3 was involved in cellular apoptosis in the CML exposure condition, transfection of pcDNA-MKP-3 in HUVECs was examined. The results revealed that pcDNA-MKP-3 evoked a statistically and proportionally significant increase in cell death (Fig. 2C; Supplementary Fig. 1B), implying that CML-induced MKP-3 elevation might trigger endothelial cell dysfunction. CML, AGEs, or high-glucose-enhanced ER stress biomarkers in activated HUVECs Signature ER stress markers, phospho-PERK, GRP78, GRP94, calpain-II, and phospho-eIf2-α, were used to study the effects of CML, AGEs, or high glucose (HG) on the unfolded protein response. Western blotting of cells treated with CML, AGEs, or HG at various

concentrations or dosages showed that all of the ER stress markers were significantly induced by CML in a dose-dependent manner (Fig. 3A; Supplementary Fig. 2). The effects of CML on two ER stress-associated proapoptotic markers, cleaved or intermediate forms of caspase-7 and CHOP/GADD153, revealed that the cleavage of caspase-7 rose steadily with the duration of CML, AGE, or HG treatment. To more directly investigate the enhancement of CML, AGEs, or HG on ER stress induction in dysfunctional endothelial cells, the ER morphology was examined by transmission electron microscopy. There was ER dilation in CML-, AGE-, and HG-treated cells (Fig. 3B). The combination of siMKP-3 and CML reduced serious ER dilation with decreased distention and fragmentation of organelles, which was in contrast to the observation in endothelial cells under thapsigargin treatment (as a positive control for ER stress

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blocked phosphorylation of ERK per se, but did not affect the expression/constitutive form of ERK (Fig. 6A; Supplementary Fig. 4A). Furthermore, transfected siRNA targeting MKP-3 effectively attenuated CML inhibition, but not MKP-1 or MKP-2. The pharmacologic inhibitors NAC and apocynin (data not shown) reversed the CML-induced phosphorylation of ERK in HUVECs (Fig. 6B; Supplementary Fig. 4B). Recombinant protein MKP-3 significantly decreased the phosphorylation of ERK as a positive control. Protein interaction by immunoprecipitation assay or confocal microscopy detection revealed the intimate colocalization of endogenous MKP-3 with phosphorylation of ERK in CMLor AGE-treated HUVECs, which was abolished by silencing MKP-3 (Figs. 6C and D). Knockdown of MKP-3 also reversed AGE-treated ERK dephosphorylation (Fig. 6E). The quantification of intensity is shown in Supplementary Fig. 5. These results demonstrated that CML enhanced the interaction between MKP-3 and ERK phosphorylation and that both endogenous MKP-3 activation and the subsequent gene regulation affected endothelial cell dysfunction. Antioxidants abolished CML- or AGE-activated MKP-3 activity and protein expression in HUVECs

Fig. 4. CML increased MKP-3 mRNA expression in HUVECs, SVECs, and RAECs, but not that of MKP-1 or MKP-2. (A) HUVECs were treated with CML (25 μg/ml) for 12 h. The MKP-3 mRNA expression was detected by qPCR. (B) MKP-3 mRNA was detected in HUVECs, SVECs, and RAECs with or without CML (25 μg/ml) treatment for 3–12 h. All data are presented as means 7 SEM of three independent experiments. np o0.05 compared to control.

induction). These demonstrated that CML, AGEs, or HG enhanced the function of MKP-3, indicating that MKP-3 activation and the following gene regulation affected endothelial cell dysfunction. CML activated MKP-3 activity and protein expression in HUVECs Exposure of HUVECs to CML or AGEs significantly increased the expression of MKP-3 mRNA, but not MKP-1 and MKP-2 mRNA, by 4.2- to 5.1-fold, in a dose-dependent manner (Fig. 4A). SVECs and RAECs also had similar results (Fig. 4B). CML strengthened MKP-3 activity in HUVECs or SVECs starting at 8 h, peaking at 16–20 h, and then dropping to a lower level at 26 h, with statistical significance (Fig. 5A). CML treatment also evoked MKP-3 activity in a dose-dependent manner at 20 h. Moreover, exposure of HUVECs to recombinant protein MKP-3 (0.1 U/ml) induced a statistically significant increase in MKP-3 activity at 30 min compared to the controls (4.8 70.12-fold of controls) (data not shown). The result simultaneously confirmed the CML-specific targeting of MKP-3 activity and protein expression in a time- and dose-dependent manner (Figs. 5B–D; Supplementary Figs. 3A and 3B), implying that CML-induced elevation in MKP-3 activity might trigger endothelial cell dysfunction. Primary RAECs showed similar results (data not shown). CML inhibited ERK phosphorylation and enhanced MKP-3/ERK interaction in HUVECs To investigate if CML-induced MKP-3 activation affected the expression or phosphorylation of ERK in HUVECs, siMKP-3 knockdown function was first checked (Fig. 5E). CML significantly

Further examination of whether antioxidants were capable of decreasing CML- or AGE-induced MKP-3 activity in HUVECs showed that CML or AGEs caused a significant 2.7- to 3.2-fold increase in MKP-3 activity in 20 h, which was completely abolished by the antioxidants NAC and apocynin (Fig. 7A). CML also triggered MKP-3 production in HUVECs, which was attenuated by NAC and apocynin (data not shown). The AGE-increased MKP-3 expression in HUVECs was also effectively attenuated by apocynin (Fig. 7B; Supplementary Fig. 6) or PDTC (data not shown). Moreover, regulated NADPH oxidase protein subunits were measured by silencing RNA. The specific knockdown of protein expression of membrane-bound Nox4 completely inhibited MKP-3 activity (Fig. 7C; Supplementary Fig. 7), compared to gp91phox (Nox2) and gp22phox subunits, cytosolic gp47phox, gp67phox, and small GTPase Rac, in CML- or AGE-treated HUVECs. These results showed that Nox4 plays a crucial role in CML- or AGE-induced MKP-3 activity or protein expression. CML, AGEs, or HG enhanced the interaction between Nox4 and MKP-3 in the ER in HUVECs Depending on the duration or severity of ER stress and the fact that Nox4 is located in the ER, this study tested whether a fraction of MKP-3 was associated with the ER subcellular organelle structure, using established biochemical techniques to isolate the ER lumen. The presence of MKP-3 in the ER lumen and the membrane fractions was determined by Western blot analysis. As expected, MKP-3 was enriched in the ER lumen by CML or AGE treatment (Fig. 8A) compared to the membrane fractions (data not shown). MKP-3 localization at the ER was also verified by laser confocal microscopy image staining. Consistent with earlier studies, Nox4 was reported as an ER-residing protein [19]. Furthermore, a fraction of Nox4 was copurified with the ER membrane, whereas the majority of MKP-3 and Nox4 was fractionated into the lumen. To gain insight into the MKP-3 and Nox4 subcellular localization in the ER, whether MKP-3 or Nox4 was associated with the ER was determined for protein–protein interaction by laser confocal microscopy detection and immunoprecipitation assay. CML, AGEs, or HG enhanced the interaction of MKP-3 with Nox4 in HUVECs (Figs. 8B and C and Supplementary Fig. 8). Consistent with this prediction and the immunoprecipitation assay, confocal microscopy revealed a perinuclear and cytoplasmic distribution of activated MKP-3, with colocalization with Nox4 at the ER

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Fig. 5. CML activated MKP-3, but not MKP-1 or MKP-2, activity and protein expression in HUVECs. (A) Cells were treated with CML for 4–24 h for the time-course study or (B) with CML for 20 h for the dose-dependency study. MKP-3 activity was then detected with pNPP as substrate. (C) Protein expression of MKP-1, MKP-2, and MKP-3 in HUVECs or SVECs treated with or without CML and AGEs for 24 h was analyzed. (D) MKP-3 protein expression in HUVECs or SVECs treated with or without CML and AGEs was analyzed up to 36 h. Results are representative of four independent experiments. The numbers below the images represent the fold increase in MKP-3 protein expression relative to the untreated group after normalization to the loading control. (E) Detection of siMKP-3 function. HUVECs were transfected with siRNA-MKP-3 (50 nmol/L) or scramble (50 nmol/L). The activity is representative of at least three independent experiments.

(Fig. 8D). The quantification of the immunoprecipitation assay intensity is shown in Fig. 8E. These data were confirmed using transfection with siRNANox4 and siRNA-MKP-3 in SVECs. There was a direct reverse phosphorylation of ERK by CML exposure (Fig. 8F). Collectively, these data established that enhanced MKP-3/Nox4 interaction was localized to the ER and that CML induction regulated ERK activation in endothelial cell dysfunction.

was detected (data not shown). The increased endothelial cell staining in aortic rings and the smooth muscle layer of the media of diabetic mice revealed condensed CML, AGEs, and MKP-3 and reduced phospho-ERK activation (Figs 9A, a and b).

Immunohistochemical staining of MKP-3 and phospho-ERK in aortas isolated from STZ-induced or HFD-induced diabetic animals

This study demonstrates that CML and AGEs, at concentrations found in patients with diabetes, are capable of activating MKP-3 activity through a NADPH oxidase Nox4-related ROS pathway, which triggers ERK dephosphorylation and consequently leads to vascular endothelial cell injury and dysfunction. In particular, this study reveals a new relationship between MKP-3, an abundant ER stress-inducible long-term hyperglycemia or complicated formation of AGEs associated with the ER, and Nox4, a major source of oxidative stress in cardiovascular disease. The signaling cascade for CML-induced endothelial cell dysfunction is shown in Fig. 9B. This study expands the pharmacologic potential of targeting MKP-3 to

In further testing of the expression of MKP-3, phospho-ERK, CML, and AGE in aortas isolated from STZ-induced or HFD-induced diabetic mice (Fig. 9A), the immunohistochemical staining for CML, AGEs, and MKP-3 increased, but phospho-ERK staining decreased in the aortic endothelium of both types of diabetic mice (Fig. 9A, a and b). The CML staining in the endothelium of the tunica intima of renal vascular tissue isolated from STZ-induced diabetic mice, HFD-induced diabetic mice, or diabetic (db/db) mice

Discussion

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Fig. 6. MKP-3 was required for CML-mediated dephosphorylation of ERK. (A) HUVECs were transfected with siRNA-MKP-1, siRNA-MKP-2, or siRNA-MKP-3 or scramble. The levels of ERK protein phosphorylation and the constitutive form of ERK were detected by immunoblot analysis. (B) HUVECs pretreated with or without antioxidants NAC (0.5 mM) or apocynin (data not shown) were stimulated with CML for 60 min, and ERK dephosphorylation was assayed by Western blotting. (C) HUVECs were treated with or without CML or AGEs for 12 h. MKP-3 was immunoprecipitated by anti-MKP-3 antibody from the cell lysates. The immunoblot was probed with the antibodies for p-ERK and MKP-3. Results are representative of four independent experiments. (D) Laser confocal microscopy for p-ERK and MKP-3 interaction in HUVECs. The images are representative of at least five independent experiments. (E) HUVECs were transfected with siRNA-MKP-3 or scramble. The levels of ERK protein phosphorylation, the constitutive form of ERK, and MKP-3 were detected by immunoblot analysis.

combat metabolic disease, particularly complications of diabetic vascular dysfunction. To date, the role of Nox4/MKP-3 in mediating oxidative stress and pathological vascular dysfunction has not been clearly demonstrated. This study demonstrates for the first time that CML and AGEs correlate with MKP-3 activity and result in dephosphorylation of ERK signaling, leading to an increase in endothelial cell apoptosis and induction of endothelial cell injury. These findings suggest that MKP-3 may play a crucial role in CML- or AGEtriggered endothelial cell dysfunction and may be a promising therapeutic target. Furthermore, small-molecule inhibitors of MKP-3 may be useful in attenuating vascular dysfunction, apoptosis in endothelial cells, and eventual macrovascular complications in response to diabetes. MKP-3 reportedly activates PEPCK gene transcription and increases gluconeogenesis in rat hepatoma cells [12]. In insulin resistance and type 2 diabetes, there is an elevation of hepatic

gluconeogenesis that contributes to hyperglycemia. Evidence shows that in an insulin-resistant obese mouse model, MKP-3 is expressed in insulin-responsive tissues and that its expression is markedly elevated in the liver [13]. The results demonstrate that dysregulation of MKP-3 expression function in the liver may contribute to the pathogenesis of insulin resistance and type 2 diabetes. Moreover, MKP-3-mediated dephosphorylation of FOXO1 at Ser-256 promotes its nuclear translocation and evokes PGC-1α-coactivated FOXO1 in Pepck and G6pase transcription, which in turn increases glucose output. Although the studies show that MKP-3 promotes gluconeogenic gene transcription in hepatoma cells, little is known about the vascular dysfunction of MKP-3 in vivo. Consistent with this, MKP-3 expression is highly elevated in STZ-induced and HFDinduced diabetic animals and diabetic (db/db) mice. In a previous report, AGEs and CML induced endothelial cell injury. In the present study, the role of MKP-3 in vascular complications in

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Fig. 7. Antioxidants abolished CML-activated MKP-3 activity and protein expression in HUVECs. HUVECs were pretreated with or without antioxidants NAC (0.1– 0.5 mM) or apocynin (0.1–1 mM) for 60 min and then stimulated with CML (25–50 μg/ml) or AGEs (100 μg/ml) for 20 h. The cell lysates were then used to analyze (A) MKP-3 activity or (B) protein expression. Results shown are representative of three independent experiments. (C) HUVECs were transfected with siRNA for Nox4, gp91phox (Nox2) and gp22phox subunits, cytosolic gp47phox, gp67phox, gp40phox, small GTPase Rac, and scramble. After 12 h, the cells were treated with CML (25 μg/ml) or AGEs (100 μg/ml) and then incubated for 24 h. The MKP-3 activity was detected by enzymatic analysis.

diabetic animals and the underlying molecular mechanism were studied. Vascular dysfunction is particularly important to consider in diabetic complications because ER stress can adversely affect both systemic atherosclerotic risk factors and cell biological processes occurring at the level of the arterial wall. There is increasing in vitro and in vivo evidence that ER stress-induced apoptosis of intimal cells, notably endothelial cells and macrophages, plays an important role in atherosclerotic plaque progression. In addition, chronic ER stress can adversely exaggerate endothelial cell function. Gargalovic and colleagues have reported that blocking the gene silencing of ATF4 and/or XBP1 (XhoI site-binding protein 1) can abolish cultured human aortic endothelial cells with the UPR activator tunicamycin. There is also a reduction in interleukin-8, interleukin-6, MCP-1 (monocyte chemoattractant protein 1), and CXCL3 (chemokine CXC motif ligand 3) [20]. Moreover, inducers of pathological ER stress in endothelial cells include hyperglycemia, advanced glycation end products, modified forms of low-density lipoprotein, and homocysteine, through a mechanism involving disturbed ER metabolism [21,22]. In this work, high glucose, CML, a major advanced glycation end-product, and complicated AGEs enhance ER stress, MKP-3

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activity and MKP-3 gene regulation, cleavage of caspase-7, and apoptosis in activated endothelial cells. Furthermore, subcellular organelles clearly demonstrate the exchange in anatomic structure. Actually, subjects with type 2 diabetes mellitus and the diabetic mouse model have high CML, AGEs (Fig. 1), IL-8, IL-6, MCP-1, and CXCL3 production (data not shown). Immunolabeling of CML in tissues indicated elevated posttranslation modification of several proteins with implications for diabetes-mediated elevation in AGEs. SiRNA-MKP-3 effectively reverses apoptosis in CMLinduced dysfunction of endothelial cells. SiRNA-MKP-3, but not siRNA-MKP-1 or siRNA-MKP-2, effectively reverses the apoptosis enhanced by CML in vascular function. These provide evidence that CML-triggered apoptosis is induced in activated endothelial cells under ER stress induction, which can be regulated by MKP-3. The results from these studies reveal, for the first time, a critical role for CML-induced MKP-3 in regulating endothelial cell injury in vitro and in vivo. Endothelial cell exposure to CML increases MKP-3 activity and gene expression. Gene knockdown of MKP-3 effectively reduces endothelial cell apoptosis by CML activation. MKP-3 is associated with the ER subcellular organelle and is located on the ER. These are consistent with the findings that ER stress is an important episode in the composition. Moreover, endothelial cells of both the intima and the smooth muscle layer of the media have elevated MKP-3 expression in both STZ-induced mice and HFD-induced obese mice. Examining MKP-3 protein levels in the diabetic mouse model and by direct immunohistochemistry staining, we found that MKP-3 is significantly increased in the vascular lumen of diabetic mice but is only mildly increased in control mice fed a chow diet with normal calories. The db/db mouse is a model of obesity that shows similar patterns. One of the substrates for MKP-3 is ERK, which does play a significant role in mediating endothelial cell proliferation. Exogenous NO interferes with ERK1/2 dephosphorylation in response to TNF-α-induced MKP-3 upregulation [23]. A large accumulation of data documents the important role of adipose TNF-α in the development of insulin resistance in obesity and diabetes. Subjects with obesity-related insulin resistance have a higher local expression of TNF-α in adipose tissue [24]. These imply that MKP-3 may have potential reactivity in response to endothelial cell activation in diabetes. Most importantly, reduction of MKP-3 expression in both NO donor and HO-1 activator alleviates CML, AGEs, or HGinduced endothelial cell dysfunction (data not shown). Elevation of vascular MKP-3 expression in the hyperglycemic or insulinresistant state may contribute to the inappropriate activation of endothelial cells in type 1 and type 2 diabetes. The results demonstrate that MKP-3 is an important operator of vascular cell dysfunction in vitro and in vivo. Oxidative stress-induced abnormal activation of signaling pathways can lead to disorder. Previous reports have shown that increasing ROS production causes the activation of five major pathways involved in the pathogenesis of complications: polyol pathway flux, increased formation of AGEs, increased expression of the receptor for AGEs and its activating ligands, activation of protein kinase C isoforms, and overactivity of the hexosamine pathway. However, collectively, for the most part, clinical trials have failed to demonstrate a beneficial effect of antioxidant supplements on cardiovascular disease morbidity and mortality. Hence, understanding closely intracellular signal transduction cascades can provide a stratagem to develop pharmacological agents to block the above pathways. Our previous study has shown that tyrosine phosphatase SHP-1-regulated VEGFR-2 dephosphorylation through NADPH oxidase-derived ROS is involved in the CML-triggered endothelial cell dysfunction/injury. CML strengthened SHP-1 activity, starting at 5 min, maintaining a peak until 4 h and then dropping down to a lower level at 6 h. In the present work, we further demonstrated that CML aggrandized

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Fig. 8. MKP-3 is located in the ER, and CML, AGEs, and HG enhance the interaction of MKP-3 with Nox4 in HUVECs. (A) HUVECs were treated with CML or AGEs for 4 h for ER subcellular organelle separation. (B) HUVECs were fixed and immunostained with anti-ER tracker–FITC (green) and MKP-3-Rho (red) followed by laser confocal microscopy detection. (C) HUVECs were treated with or without CML, AGEs, or HG for 12 h. MKP-3 was immunoprecipitated by anti-MKP-3 antibody from cell lysates. The immunoblot was probed with antibodies for NADPH oxidase protein subunits. MKP-3 was reprobed by itself. (D) MKP-3 and Nox4 interaction by laser confocal microscopy observation. Results are representative of three independent experiments. (E) Quantification of the intensity shown in (D). (F) HUVECs were transfected with siRNA for Nox4, MKP-3, and scramble. The protein levels of ERK phosphorylation, Nox4, and MKP-3 were detected by immunoblot analysis.

MKP-3 activity in HUVECs or SVECs starting at 8 h, topping at 16– 20 h, and then downregulated it at 26 h. The present results demonstrate for the first time that an MKP-3 pathway might regulate ERK dephosphorylation through NADPH oxidase/Nox4derived ROS during CML-triggered endothelial cell dysfunction/ injury. Taken together, these results suggest that CML initially triggers the activation of NADPH oxidase, especially Nox4, and next generates ROS to activate SHP-1-regulated VEGFR-2 dephosphorylation and MKP-3-regulated ERK dephosphorylation, consequently promoting endothelial cell injury. A previous report indicates that vascular NADPH oxidase may be an important source of ROS [6]. Measuring changes in NADPH oxidase protein subunits induced by CML in HUVECs showed that there is elevated protein expression of membrane bound Nox4, gp91phox (Nox2), gp22phox subunits, cytosolic gp47phox, gp67phox, and small GTPase Rac, but not gp40phox, in the CML-treated human endothelial cells. Such observations are consistent with results of previous reports on HUVECs and

STZ-diabetic mice in which high glucose increased ROS production via NADPH oxidase activity and p22phox expression. Nox4 is a novel isoform of NADPH oxidase expressed in nonphagocytes such as vascular endothelial cells and smooth muscle cells. Nox4 is also a major source of oxidative stress in the failing heart [25]. In the STZ-induced diabetic apolipoprotein E-deficient mice or the db/db mice, Nox4 expression is effectively increased in the isolated aorta and associated with increased inflammation and ROS production. Again, this indicates a pivotal role of Nox4 in diabetic macrovascular disease [26,27]. In this study, Nox4 and MKP-3 expression is increased in CML-activated endothelial cells, and this was reversed by both silencing Nox4 and antioxidant reagents such as NAC, PDTC, and apocynin. Transfection of siRNA targeting Nox4 effectively inhibits MKP-3 protein expression in injured endothelial cells. Kai and colleagues [19] have also shown that Nox4 is localized to the ER and have examined its subcellular localization. In addition, we also investigated whether CML or

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Fig. 9. Immunohistochemical staining of MKP-3 and phospho-ERK in aortas isolated from STZ-induced or high-fat diet-induced diabetic animals. (A) C57BL/6 J male mice were (a) injected with STZ to induce diabetes for 8 weeks or (b) fed a high-fat diet to induce diabetes for 28 weeks. There were increases in endothelial cell sloughing in mouse aortic rings, MKP-3 and CML expression, and phospho-ERK dephosphorylation. The original magnification was 400  for MKP-3, p-ERK, and CML; 50  for CML detection. (B) Proposed mechanism for CML- or AGE-induced endothelial cell dysfunction. CML induced the dephosphorylation of ERK, resulting in endothelial cell dysfunction via Nox4 located in the ER associated with stress-triggered MKP-3 activation.

AGEs induced generation of DCF-sensitive ROS. Treatment with CML or AGEs on HUVECs for 15 min to 36 h was found to increase DCF fluorescence. Antioxidant inhibitor treatment completely suppressed this CML- or AGE-induced ROS generation and MKP3 activity (Supplementary Fig. 9). In conclusion, MKP-3 can be integrated into the transcriptional network that controls endothelial dysfunction under both physiologic and pathologic conditions. CML induces a Nox4/ROSactivated MKP-3 signaling, which in turn initiates ERK dephosphorylation that causes human endothelial cell dysfunction. Pharmacologic inhibitors or the inactivation of MKP-3 may protect against the deleterious effects of CML in the vascular pathogenesis of aging and diabetes. The therapeutic potential of blocking tyrosine phosphatase MKP-3 by small-molecule inhibitors or Nox4/ROS inactivation may be useful for attenuating endothelial cell dysfunction/injury.

Authors’ Contributions W.H.H.S. wrote the manuscript and collected the type 2 DM clinical samples. W.J.L. analyzed the data, and S.H.L. collected the db/db mice. Y.C.Y. and W.C.C. collected human umbilical vein, and S.Y.L. and K.W.L. analyzed the type 2 DM clinical data. C.C.S. and H.R.T. collected the cell biology research data. H.Y.Y., L.Y.L., and Y.C. T collected the animal study data. T.J.Y. analyzed the LC/MS/MS data, and M.R.L. and M.L.S. conducted the LC/MS/MS analysis. M.L. S. collected the research data and wrote/reviewed/edited the manuscript. Acknowledgments The authors thank Dr. Peter Wilds of Taichung Veterans General Hospital, Taiwan, and Gene Alzona Nisperos for proofreading and

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editing the manuscript for English language considerations. This work was supported by research grants from the National Science Council of Taiwan (NSC102-2628-005-001-MY3, NSC1012314-B-075A-006-MY3, NSC102-2314-B-075A-002) and Taichung Veterans General Hospital (TCVGH-1027314C, TCVGH-1017311C, TCVGH-NCHU1017603, TCVGH-NCHU1027602, TCVGH-HK1008001, TCVGH-1017311C, TCVGH-1020102D).

Appendix A.

Supplementary Information

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