The FASEB Journal article fj.15-271627. Published online April 15, 2015.
The FASEB Journal • Research Communication
Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor Koichi Yamamoto,*,1,2 Akemi Kakino,†,‡,§,1 Hikari Takeshita,*,§ Norihiro Hayashi,* Lei Li,† Atsushi Nakano,† Hiroko Hanasaki-Yamamoto,* Yoshiko Fujita,† Yuki Imaizumi,* Serina Toyama-Yokoyama,* Chikako Nakama,* Tatsuo Kawai,* Masao Takeda,* Kazuhiro Hongyo,* Ryosuke Oguro,* Yoshihiro Maekawa,* Norihisa Itoh,* Yoichi Takami,* Miyuki Onishi,* Yasushi Takeya,* Ken Sugimoto,* Kei Kamide,* Hironori Nakagami,{ Mitsuru Ohishi,*,2 Theodore W. Kurtz,k Tatsuya Sawamura,†,‡,§ and Hiromi Rakugi* *Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; †Department of Vascular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan; ‡Department of Molecular Pathophysiology, Osaka University Graduate School of Pharmaceutical Sciences, Suita, Osaka, Japan; §Department of Physiology, Shinshu University School of Medicine, Asahi, Matsumo, Japan; {Division of Vascular Medicine and Epigenetics, Osaka University United Graduate School of Child Development, Suita, Osaka, Japan; and kDepartment of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA The angiotensin II type 1 receptor (AT1) is a 7-transmembrane domain GPCR that when activated by its ligand angiotensin II, generates signaling events promoting vascular dysfunction and the development of cardiovascular disease. Here, we show that the singletransmembrane oxidized LDL (oxLDL) receptor (LOX-1) resides in close proximity to AT1 on cell-surface membranes and that binding of oxLDL to LOX-1 can allosterically activate AT1-dependent signaling events. oxLDLinduced signaling events in human vascular endothelial cells were abolished by knockdown of AT1 and inhibited by AT1 blockade (ARB). oxLDL increased cytosolic G protein by 350% in Chinese hamster ovary (CHO) cells with genetically induced expression of AT1 and LOX-1, whereas little increase was observed in CHO cells expressing only LOX-1. Immunoprecipitation and in situ proximity ligation assay (PLA) assays in CHO cells revealed the presence of cell-surface complexes involving LOX-1 and AT1. Chimeric analysis showed that oxLDL-induced AT1 signaling events are mediated via interactions between the intracellular domain of LOX-1 and AT1 that activate AT1. oxLDL-induced impairment of endothelium-dependent vascular relaxation of vascular ring from mouse thoracic aorta was abolished by ARB or genetic deletion of AT1. These findings reveal a novel pathway for AT1 activation and suggest a new mechanism whereby oxLDL may be promoting risk for cardiovascular disease.— Yamamoto, K., Kakino, A., Takeshita, H., Hayashi, N., Li, L., Nakano,
ABSTRACT
A., Hanasaki-Yamamoto, H., Fujita, Y., Imaizumi, Y., Toyama-Yokoyama, S., Nakama, C., Kawai, T., Takeda, M., Hongyo, K., Oguro, R., Maekawa, Y., Itoh, N., Takami, Y., Onishi, M., Takeya, Y., Sugimoto, K., Kamide, K., Nakagami, H., Ohishi, M., Kurtz, T. W., Sawamura, T., Rakugi, H. Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor. FASEB J. 29, 000–000 (2015). www.fasebj.org Key Words: G protein-coupled receptor • allosteric activation vascular dysfunction • receptor multimerization
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OXIDATION OF LDL PLAYS AN important role in initiating and developing atherosclerosis (1). Accumulating evidence suggests that the type II single-transmembrane protein, lectin-like LOX-1 is a key molecule in oxLDL-induced formation of atherosclerosis (2–4). The proatherosclerotic effects of oxLDL are mediated not only by its tendency to accumulate in macrophages and promote inflammation but also through its ability to bind and activate LOX-1 expressed in vascular endothelial and smooth muscle cells (5). Although oxLDL binding to LOX-1 is known to activate various intracellular signaling cascades (6), the underlying mechanisms, whereby ligand binding to the receptor triggers activation of cell-signaling events, have remained unclear. The GPCR AT1 plays a role in the pathogenesis of cardiovascular disease through activation 1
Abbreviations: ACh, acetylcholine; ARB, angiotensin II type 1 receptor blocker; AT1/2, angiotensin II type 1/2 receptor; AT1D221/222, aa 221 and 222 from FLAG-tagged wild-type AT1 plasmid; BSA, bovine serum albumin; CHO, Chinese hamster ovary; CHO-LOX-1, tetracycline-inducible human lectin-like oxidized LDL receptor (tagged with V5-6 3 His at the C terminus)-expressing Chinese hamster ovary-K1; (continued on next page)
0892-6638/15/0029-0001 © FASEB
These authors contributed equally to this work. Correspondence: Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, 565-0871, Japan. E-mail: kyamamoto@ geriat.med.osaka-u.ac.jp (K.Y.); [email protected] (M. Ohishi) doi: 10.1096/fj.15-271627 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2
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of intracellular signaling events triggered by receptor binding to angiotensin II, the only known endogenous ligand for AT1 (7, 8). LOX-1 and AT1 have a close relationship at the transcriptional level, as angiotensin II induces expression of LOX-1 (9), and oxLDL induces expression of AT1 (10). Furthermore, a recent study suggests that LOX-1 is involved in angiotensin II-induced cardiac angiogenesis and inflammation (11). oxLDL and angiotensin II also share some intracellular signaling effects in common, as both molecules use NADPH oxidase-derived superoxide as a second messenger for the activation of downstream signaling cascades (12, 13). In the current studies, we investigated whether activation of AT1 might be involved in mediating intracellular signaling events triggered by binding of oxLDL to LOX-1. MATERIALS AND METHODS Cell culture HUVECs were cultured in EGM-2 (Clontech, Mountain View, CA, USA). Cells, ,5 passages, were used for the experiments. Transgenic CHO cells were maintained in F-12 Nutrient Mixture with Glutamax-I (Life Technologies, Carlsbad, CA, USA), 10% FBS, and appropriate selection reagents, as described below. COS-7 cells were maintained in DMEM (Wako, Osaka, Japan), supplemented with 10% fetal bovine serum (FBS). All cultures were transferred to serum-free condition, 24 hours before stimulation. Transcription of the genes in CHO cells was induced by adding doxycycline into culture media for 48 hours at a final concentration of 300 ng/ml. Preparation of oxLDL Human plasma LDL (1.019–1.063 g/ml), isolated by sequential ultracentrifugation, was oxidized by use of 20 mM CuSO4 in PBS at 37°C for 24 hours. Oxidation was terminated by adding excess EDTA. Oxidation of LDL was analyzed on agarose gel electrophoresis for migration versus LDL. Labeling of oxLDL with 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate (DiI; Life Technologies) was performed as described previously (14). Detection of phosphorylation of ERK1/2 Cells treated with oxLDL, angiotensin II, or vehicle, were kept in an incubator at 37°C for the indicated period. Subsequently, cells were washed twice with PBS and lysed by use of M-PER mammalian protein extraction reagent (Thermo Scientific, Waltham, MA, USA) with protease inhibitor and phosphatase inhibitor followed by Western blotting analysis, as described below. Western blot analysis Proteins were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes for Western blot analysis. The (continued from previous page) DiI, 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; Fw, forward; Ga, G protein a; HA, hemagglutinin; LOX-1, lectin-like oxidized LDL receptor; oxLDL, oxidized LDL; PGF2a, prostaglandin F2a; PLA, proximity ligation assay, Rv, reverse; siRNA, small interfering RNA, WT, wild-type
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membranes were blocked with 5% nonfat dried milk and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study were anti-phospho-ERK1/2 (Thr202/Tyr204) antibody, anti-total ERK1/2 antibody, antiphospho-epidermal growth factor receptor (EGFR; Tyr 1068) antibody, anti-total EGFR antibody, and anti-pan G protein a (Ga) antibody (Cell Signaling Technology, Danvers, MA, USA); anti-V5 antibody (Nacalai Tesque, Kyoto, Japan); and anti-hemagglutinin (HA) antibody (Roche Applied Science, Panzberg, Upper Bavaria, Germany). Bands were visualized by use of Chemi-Lumi One Super (Nacalai Tesque). Densitometric analysis was performed with a chemiluminescence detection system (LAS-4000 Mini; GE Healthcare, Pittsburgh, PA, USA). Small interfering RNA HUVECs was plated to be 50% confluent on the day of transfection. Silencer Select small interfering RNA (siRNA) for LOX-1, AT1, or EGFR (Life Technologies) was transfected into the cells in media without serum and antibiotics by use of lipofectamine RNAiMAX (Life Technologies), according to the manufacturer’s instruction. Stimulation with angiotensin II or oxLDL was performed 24 hours after transfection.
Quantitative real-time PCR Total RNA was extracted by use of the RNeasy Mini Kit (Qiagen, Venlo, Netherlands), and an equivalent amount of RNA was transcribed to cDNA by the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative real-time PCR was performed and analyzed on a model 7900 sequence detector (Life Technologies) with TaqMan gene-expression assays for LOX-1, AT1, and EGFR (Life Technologies). The expression level of each gene was normalized by 18s rRNA as an internal control. Stable transformants Tetracycline-inducible human LOX-1 (tagged with V5-6 3 His at the C terminus)-expressing CHO-K1 (CHO-LOX-1) cells were maintained as described previously (15). cDNA, encoding human AT1 (GenBank NM_000685), tagged with signal peptide HAFLAG at the N terminus, was subcloned into pTRE2hyg (Clontech). CHO-LOX-1 cells were cotransfected with pTRE2hyg-HAFLAG-human (h) AT1 and pSV2bsr vector (Funakoshi, Tokyo, Japan) by use of Lipofectamine 2000 transfection reagent (Life Technologies), according to the manufacturer’s instructions. The stable transformants were selected with 400 mg/ml hygromycin B (Wako) and 10 mg/ml blasticidin S (Funakoshi). The resistant clones expressing LOX-1 and AT1 in response to doxycycline (Calbiochem, San Diego, CA, USA) were selected for use in experiments (CHO-LOX-1-AT1). Likewise, CHO-LOX-1-AT2 were prepared by cotransfection of HA-FLAG-hAT2 vector and pSV2bsr vector into CHO-LOX-1. CHO-AT1 were prepared by stable transfection of CHO-K1 Tet-On cells (Clontech) with pTRE2hyg-HAFLAG-hAT1, according to the previous report (15). Detection of Dil-labeled oxLDL CHO-LOX-1-AT1 were treated for 60 minutes with Dil-labeled oxLDL at a final concentration of 5 mg/ml on ice. The cells were then washed twice and fixed with neutral-buffered formalin. Nuclei were stained with DAPI (1 mg/ml). Images were acquired by use of a fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany).
The FASEB Journal x www.fasebj.org
YAMAMOTO ET AL.
Detection of cytosolic release of G proteins Cytosolic release of G proteins was detected to verify GPCR activation. Cells were incubated with oxLDL or angiotensin II for 30 minutes at 37°C. The cytosolic fraction was then isolated, separated by SDS-PAGE under reducing conditions, and transferred to PVDF membranes. The blotted membranes were incubated with antibodies against Ga (pan) and Gai (Cell Signaling Technology).
Site-directed mutagenesis of AT1 and transfection of COS-7 cells Primestar Mutagenesis Basal Kit (Takara, Shiga, Japan) was used to delete aa 221 and 222 from FLAG-tagged wild-type (WT) AT1 plasmid (AT1D221/222). V5-Tagged LOX-1 and FLAG-tagged control, WT AT1, or AT1D221/222 were transfected into COS-7 cells by use of Lipofectamine LTX, according to the manufacturer’s instruction (Life Technologies).
Coimmunoprecipitation Luciferase reporter assay Membrane proteins from CHO-LOX-1, CHO-AT1, or CHOLOX-1-AT1 were prepared by use of the Transmembrane Protein Extraction Kit (Merck, Kenilworth, NJ, USA). Immunoprecipitation was performed with anti-FLAG-M2 affinity gel (Sigma-Aldrich, St. Louis, MO, USA), and the FLAG-fusion proteins were eluted with 33 FLAG peptide (Sigma-Aldrich). The purified proteins were then separated by SDS-PAGE under reducing or nonreducing conditions and transferred to PVDF membranes. The blotted membranes were incubated with antibodies against HA and V5. In situ PLA To investigate whether AT1 has the capability to interact with LOX-1, we used an in situ PLA (16), Duolink, from Olink Bioscience (Uppsala, Sweden), according to the manufacturer’s instructions, with slight modifications. In brief, cells were fixed with 10% formalin and incubated with a pair of primary antibodies directed to FLAG tag (clone M2, mouse monoclonal; Sigma-Aldrich) and to V5 tag (rabbit polyclonal; Sigma-Aldrich) in 1% bovine serum albumin (BSA)/PBS for 1 hour at room temperature. This was followed by washing 3 times in PBS and incubation with corresponding PLA probes, secondary antibodies conjugated to oligonucleotides (mouse minus and rabbit plus) in 1% BSA/PBS for 1 hour at room temperature. Thereafter, the cells were washed 3 times in PBS and incubated with the hybridization solution at 37°C for 15 minutes, followed by incubation with the ligation solution for 15 minutes at 37°C. After washing twice in PBS, the cells were treated with polymerase for amplification for 90 minutes at 37°C. The PLA signals were detected with Duolink PLA Detection Kit 563. Nuclei were stained with DAPI (1 mg/ml). Images were acquired by use of a fluorescence microscope (Axiovert 200M; Zeiss). Quantitative fluorescence cell image analysis was performed by use of the IN Cell Analyzer 2000 system (GE Healthcare) (15).
Construction of chimeric plasmids Chimeric plasmids were generated by connecting the extracellular domain of Dectin-1 to the transmembrane and cytoplasmic domains of LOX-1 (Chimera 1) or the extracellular domain of LOX-1 to the transmembrane and cytoplasmic domains of Dectin1 (Chimera 2), with linker sequences (Table 1), as described previously (17). pcDNA6.2/V5/GW/D-TOPO (Life Technologies) constructs of LOX-1 and Dectin-1 were subjected to PCR with a primer pair of N-LOX-1-Fw and L-D linker Rv and a primer pair of L-D linker Fw and C-Dectin-1 Rv for Chimera 1 or a primer pair of D-L-linker Fw and C-LOX-1 Rv and a primer pair of NDectin-1-Fw and D-L-linker Rv for Chimera 2. Obtained Dectin-1 cDNA and LOX-1 cDNA were connected with overlap-extension PCR by use of a primer pair of N-LOX-1-Fw and C-Dectin-1 Rv for Chimera 1 and a primer pair of N-Dectin-1-Fw and C-LOX-1 Rv for Chimera 2. Each product was subcloned into pcDNA6.2/V5/ GW/D-TOPO, and the sequences were confirmed.
OXIDIZED LDL ALLOSTERICALLY ACTIVATES AT1 RECEPTOR
CHO cells, CHO-AT1, CHO-LOX-1, and CHO-LOX-1-AT1 seeded in a 24-well plate were transiently cotransfected with 500 ng pGL3-human LOX-1 promoter Firefly luciferase reporter vector (18) and 10 ng pRL-cytomegalovirus Renilla luciferase control reporter vector (Promega, Madison, WI, USA) by use of Lipofectamine LTX and PLUS Reagent (Life Technologies), according to the manufacturer’s instructions, and cultured for 8 hours. The cells were starved in medium, supplemented with 0.1% FBS, with or without olmesartan for 24 hours. After that, the cells were treated with oxLDL for 20 hours. The luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s protocol. The results were expressed as the intensity relative to the control (incubated with medium only). Ca2+ mobilization Measurement of Ca2+ transients was carried out by use of Fura 2-AM (Dojindo, Kumamoto, Japan), as reported previously (19). In brief, cells plated in 96 wells were incubated with 5 mM Fura 2-AM in recording media (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 13.8 mM glucose, pH 7.4) for 1 hour at 37°C, followed by replacement with recording media without Fura 2-AM. Cells were treated with 80 mg/ml oxLDL, and changes in F340/F380 as an index of intracellular Ca2+ concentration were measured by dualexcitation microfluorometry by use of a digital image analyzer (Aquacosmos; Hamamatsu Photonics, Hamamatsu, Japan). Experimental animals AT1a knockout mice and WT mice on the C57BL/6 background were used in the present study. AT1a knockout mice were a gift from Dr. Masaru Iwai at Ehime University (Matsuyama, Japan). The study protocol was approved by the Animal Care and Use Committee at Osaka University (Permit Number 21-050-3; Osaka, Japan) and was conducted in strict accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were euthanized with a sodium pentobarbital overdose, and every effort was made to minimize suffering. Evaluation of NO production of aortic endothelium in mice Thoracic aortic rings from 12-week-old male mice were incubated with vehicle or 40 mg/ml oxLDL in RPMI with 1% mouse serum for 20 hours at 37°C. For olmesartan treatment, aortic rings were preincubated with 1 mM olmesartan for 1 hour before application of oxLDL. Thereafter, aortic rings were transferred to a Magnus chamber (Labo Support, Osaka, Japan) filled with CO2-supplied buffer at 37°C, and tension was recorded by use of PowerLab software (ADInstruments, Dunedin, New Zealand). Resting tension of the aortic ring was adjusted at 0.3 g. Contraction was
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TABLE 1. PCR primers used for constructing chimeric plasmids Primer
N-LOX-1 L-D linker L-D linker C-Dectin-1 D-L-linker C-LOX-1 N-Dectin-1 D-L-linker
Sequence
Fw 59-caccatgacttttgatgacctaaag-39 Rv 59-ccaaatagccatggttaattgcatgcccagcaccataatg-39 Fw 59-ctgggcatgcaattaaccatggctatttggagatccaatt-39 Rv 59-cattgaaaacttcttctcacaaata-39 Fw 59-gctgtggtcctgggttcccaggtgtctgacctcctaacac-39 Rv 59-ctgtgctcttaggtttgccttcttc-39 Fw 59-caccatggaatatcatcctgattta-39 Rv 59-gtcagacacctgggaacccaggaccacagctatcaccagt-39
induced with 5 mM prostaglandin F2a (PGF2a). After stable contraction, endothelium-dependent vasorelaxation was induced with acetylcholine (ACh; 3 3 1029–1026 M). Immunofluorescence staining CHO cells, CHO-LOX-1, and CHO-LOX-1-AT1 were fixed with 4% (w/v) phosphate-buffered formaldehyde (Wako) at room temperature for 15 minutes. Tagged LOX-1 and AT1 were detected with 2.0 mg/ml rabbit anti-V5 (Sigma-Aldrich) and 1.0 mg/ml mouse anti-FLAG (M2; Sigma-Aldrich) antibodies, respectively. Alexa 488-conjugated anti-rabbit IgG (1.0 mg/ml) and 1.0 mg/ml Alexa 546-conjugated anti-mouse IgG (Life Technologies) were used as secondary antibodies, respectively. All antibody incubations were performed for 1 hour at room temperature. Nuclei were counterstained with DAPI (SigmaAldrich). Images were obtained with a confocal laser microscope (TCS SP5; Leica, Wetzlar, Germany). Statistical analyses All data are presented as means 6 SEM. Percent changes compared with vehicle treatment were analyzed by 1-sample t test, and significant differences among mutiple treatments were determined by 1-way ANOVA and Holm-Sidak testing.
RESULTS
Dependence of oxLDL-induced cellular signaling on LOX-1 and AT1
oxLDL-induced cellular signaling is abolished by inhibition or knockdown of AT1 in vascular endothelial cells oxLDL is known to activate ERK1/2 (p42/44 MAPK) via interaction with LOX-1 in vascular cells (20). In HUVECs, pretreatment with an ARB, olmesartan, for 24 hours inhibited the ability of oxLDL to increase ERK1/2 activity (Fig. 1A). Other ARBs, including telmisartan, valsartan, and losartan, also inhibited oxLDL-induced ERK activation, indicating that the inhibition was a class effect of ARBs (Supplemental Fig. 1A). OxLDL is known to transactivate EGFRs (21), and we found that ARB treatment also inhibited this phenomenon (Fig. 1A). It has been reported that LOX-1 expression is up-regulated by many proatherogenic stimuli, including angiotensin II (9) and oxLDL (22). However, in the current studies conducted in the absence of angiotensin II, ARB treatment did not affect expression of LOX-1, suggesting that the effect of ARB did not result from down-regulation of LOX-1 (Supplemental Fig. 1B). It should be noted that olmesartan 4
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inhibited the up-regulation of LOX-1 promoter activity by oxLDL in HUVECs, suggesting that prolonged oxLDL treatment can up-regulate LOX-1 in an AT1-dependent manner (Fig. 1B). siRNA knockdown of angiotensinogen, a precursor of angiotensin II, or incubation with enalapril, an angiotensin-converting enzyme inhibitor that blocks conversion of angiotensin I to angiotensin II, did not affect oxLDL-induced ERK1/2 activation (Supplemental Fig. 1C). These findings indicate that pharmacologic ARB inhibits oxLDL-induced cell signaling, but the effect is not likely to be mediated by alterations in the endogenous production of angiotensin II or by blockade of angiotensin II binding to AT1. We next investigated whether knockdown of AT1 expression would attenuate the ability of oxLDL to stimulate MAPK activity (Fig. 1C). In HUVECs, siRNA knockdown of LOX-1 or AT1 inhibited the ability of oxLDL to activate ERK1/2 (Fig. 1C, middle), whereas siRNA knockdown of LOX-1 did not affect angiotensin II-induced ERK1/2 activation (Fig. 1C, right). siRNA knockdown of the EGFR also attenuated ERK1/2 activation triggered by oxLDL (Fig. 1C, middle) or by angiotensin II (Fig. 1C, right), suggesting that ERK1/2 activation induced by oxLDL is downstream of EGFR transactivation, similar to the mechanism whereby angiotensin II activates ERK1/2 (23).
August 2015
To investigate signaling actions of oxLDL by use of a knockin system, we developed stable cell lines expressing V5-tagged human LOX-1, HA- and FLAG-tagged human AT1, or both in CHO cells that lack native LOX-1 and AT1 (CHO-LOX-1, CHO-AT1, CHO-LOX-1-AT1, respectively). In CHO-LOX-1-AT1, oxLDL treatment activated ERK1/2 to a much greater extent than in CHO-LOX-1 lacking AT1 receptors, whereas the expression of LOX-1 and binding of oxLDL to LOX-1 were not different between these cells (Fig. 2A, C and Supplemental Fig. 2). Treatment with 40 mg/ml oxLDL modestly activated ERK1/2 to a similar extent in WT CHO cells lacking LOX-1 and AT1, as in CHO cells expressing LOX-1 or AT1, suggesting that oxLDL may cause some minor activation of ERK1/2 through pathways independent of LOX-1 and AT1 (Fig. 2B). In CHO-LOX-1AT1, oxLDL activated ERK1/2 to a much greater degree than the ERK1/2 activation observed in CHO-LOX-1 and CHO-AT1 (Fig. 2B). The ERK activation in response to oxLDL in CHO-LOX-1-AT1 was inhibited by pretreatment with olmesartan for 30 minutes, 6 hours, or 24 hours, with
The FASEB Journal x www.fasebj.org
YAMAMOTO ET AL.
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Olmesartan(-) Olmesartan(+) Phospho/Total ERK (% of vehicle treatment )
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The FASEB Journal • Research Communication
Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor Koichi Yamamoto,*,1,2 Akemi Kakino,†,‡,§,1 Hikari Takeshita,*,§ Norihiro Hayashi,* Lei Li,† Atsushi Nakano,† Hiroko Hanasaki-Yamamoto,* Yoshiko Fujita,† Yuki Imaizumi,* Serina Toyama-Yokoyama,* Chikako Nakama,* Tatsuo Kawai,* Masao Takeda,* Kazuhiro Hongyo,* Ryosuke Oguro,* Yoshihiro Maekawa,* Norihisa Itoh,* Yoichi Takami,* Miyuki Onishi,* Yasushi Takeya,* Ken Sugimoto,* Kei Kamide,* Hironori Nakagami,{ Mitsuru Ohishi,*,2 Theodore W. Kurtz,k Tatsuya Sawamura,†,‡,§ and Hiromi Rakugi* *Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; †Department of Vascular Physiology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan; ‡Department of Molecular Pathophysiology, Osaka University Graduate School of Pharmaceutical Sciences, Suita, Osaka, Japan; §Department of Physiology, Shinshu University School of Medicine, Asahi, Matsumo, Japan; {Division of Vascular Medicine and Epigenetics, Osaka University United Graduate School of Child Development, Suita, Osaka, Japan; and kDepartment of Laboratory Medicine, University of California, San Francisco, San Francisco, California, USA The angiotensin II type 1 receptor (AT1) is a 7-transmembrane domain GPCR that when activated by its ligand angiotensin II, generates signaling events promoting vascular dysfunction and the development of cardiovascular disease. Here, we show that the singletransmembrane oxidized LDL (oxLDL) receptor (LOX-1) resides in close proximity to AT1 on cell-surface membranes and that binding of oxLDL to LOX-1 can allosterically activate AT1-dependent signaling events. oxLDLinduced signaling events in human vascular endothelial cells were abolished by knockdown of AT1 and inhibited by AT1 blockade (ARB). oxLDL increased cytosolic G protein by 350% in Chinese hamster ovary (CHO) cells with genetically induced expression of AT1 and LOX-1, whereas little increase was observed in CHO cells expressing only LOX-1. Immunoprecipitation and in situ proximity ligation assay (PLA) assays in CHO cells revealed the presence of cell-surface complexes involving LOX-1 and AT1. Chimeric analysis showed that oxLDL-induced AT1 signaling events are mediated via interactions between the intracellular domain of LOX-1 and AT1 that activate AT1. oxLDL-induced impairment of endothelium-dependent vascular relaxation of vascular ring from mouse thoracic aorta was abolished by ARB or genetic deletion of AT1. These findings reveal a novel pathway for AT1 activation and suggest a new mechanism whereby oxLDL may be promoting risk for cardiovascular disease.— Yamamoto, K., Kakino, A., Takeshita, H., Hayashi, N., Li, L., Nakano,
ABSTRACT
A., Hanasaki-Yamamoto, H., Fujita, Y., Imaizumi, Y., Toyama-Yokoyama, S., Nakama, C., Kawai, T., Takeda, M., Hongyo, K., Oguro, R., Maekawa, Y., Itoh, N., Takami, Y., Onishi, M., Takeya, Y., Sugimoto, K., Kamide, K., Nakagami, H., Ohishi, M., Kurtz, T. W., Sawamura, T., Rakugi, H. Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor. FASEB J. 29, 000–000 (2015). www.fasebj.org Key Words: G protein-coupled receptor • allosteric activation vascular dysfunction • receptor multimerization
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OXIDATION OF LDL PLAYS AN important role in initiating and developing atherosclerosis (1). Accumulating evidence suggests that the type II single-transmembrane protein, lectin-like LOX-1 is a key molecule in oxLDL-induced formation of atherosclerosis (2–4). The proatherosclerotic effects of oxLDL are mediated not only by its tendency to accumulate in macrophages and promote inflammation but also through its ability to bind and activate LOX-1 expressed in vascular endothelial and smooth muscle cells (5). Although oxLDL binding to LOX-1 is known to activate various intracellular signaling cascades (6), the underlying mechanisms, whereby ligand binding to the receptor triggers activation of cell-signaling events, have remained unclear. The GPCR AT1 plays a role in the pathogenesis of cardiovascular disease through activation 1
Abbreviations: ACh, acetylcholine; ARB, angiotensin II type 1 receptor blocker; AT1/2, angiotensin II type 1/2 receptor; AT1D221/222, aa 221 and 222 from FLAG-tagged wild-type AT1 plasmid; BSA, bovine serum albumin; CHO, Chinese hamster ovary; CHO-LOX-1, tetracycline-inducible human lectin-like oxidized LDL receptor (tagged with V5-6 3 His at the C terminus)-expressing Chinese hamster ovary-K1; (continued on next page)
0892-6638/15/0029-0001 © FASEB
These authors contributed equally to this work. Correspondence: Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, 565-0871, Japan. E-mail: kyamamoto@ geriat.med.osaka-u.ac.jp (K.Y.); [email protected] (M. Ohishi) doi: 10.1096/fj.15-271627 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2
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of intracellular signaling events triggered by receptor binding to angiotensin II, the only known endogenous ligand for AT1 (7, 8). LOX-1 and AT1 have a close relationship at the transcriptional level, as angiotensin II induces expression of LOX-1 (9), and oxLDL induces expression of AT1 (10). Furthermore, a recent study suggests that LOX-1 is involved in angiotensin II-induced cardiac angiogenesis and inflammation (11). oxLDL and angiotensin II also share some intracellular signaling effects in common, as both molecules use NADPH oxidase-derived superoxide as a second messenger for the activation of downstream signaling cascades (12, 13). In the current studies, we investigated whether activation of AT1 might be involved in mediating intracellular signaling events triggered by binding of oxLDL to LOX-1. MATERIALS AND METHODS Cell culture HUVECs were cultured in EGM-2 (Clontech, Mountain View, CA, USA). Cells, ,5 passages, were used for the experiments. Transgenic CHO cells were maintained in F-12 Nutrient Mixture with Glutamax-I (Life Technologies, Carlsbad, CA, USA), 10% FBS, and appropriate selection reagents, as described below. COS-7 cells were maintained in DMEM (Wako, Osaka, Japan), supplemented with 10% fetal bovine serum (FBS). All cultures were transferred to serum-free condition, 24 hours before stimulation. Transcription of the genes in CHO cells was induced by adding doxycycline into culture media for 48 hours at a final concentration of 300 ng/ml. Preparation of oxLDL Human plasma LDL (1.019–1.063 g/ml), isolated by sequential ultracentrifugation, was oxidized by use of 20 mM CuSO4 in PBS at 37°C for 24 hours. Oxidation was terminated by adding excess EDTA. Oxidation of LDL was analyzed on agarose gel electrophoresis for migration versus LDL. Labeling of oxLDL with 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate (DiI; Life Technologies) was performed as described previously (14). Detection of phosphorylation of ERK1/2 Cells treated with oxLDL, angiotensin II, or vehicle, were kept in an incubator at 37°C for the indicated period. Subsequently, cells were washed twice with PBS and lysed by use of M-PER mammalian protein extraction reagent (Thermo Scientific, Waltham, MA, USA) with protease inhibitor and phosphatase inhibitor followed by Western blotting analysis, as described below. Western blot analysis Proteins were separated by SDS-PAGE and electrophoretically transferred to PVDF membranes for Western blot analysis. The (continued from previous page) DiI, 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; Fw, forward; Ga, G protein a; HA, hemagglutinin; LOX-1, lectin-like oxidized LDL receptor; oxLDL, oxidized LDL; PGF2a, prostaglandin F2a; PLA, proximity ligation assay, Rv, reverse; siRNA, small interfering RNA, WT, wild-type
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membranes were blocked with 5% nonfat dried milk and incubated with primary antibodies overnight at 4°C. The primary antibodies used in this study were anti-phospho-ERK1/2 (Thr202/Tyr204) antibody, anti-total ERK1/2 antibody, antiphospho-epidermal growth factor receptor (EGFR; Tyr 1068) antibody, anti-total EGFR antibody, and anti-pan G protein a (Ga) antibody (Cell Signaling Technology, Danvers, MA, USA); anti-V5 antibody (Nacalai Tesque, Kyoto, Japan); and anti-hemagglutinin (HA) antibody (Roche Applied Science, Panzberg, Upper Bavaria, Germany). Bands were visualized by use of Chemi-Lumi One Super (Nacalai Tesque). Densitometric analysis was performed with a chemiluminescence detection system (LAS-4000 Mini; GE Healthcare, Pittsburgh, PA, USA). Small interfering RNA HUVECs was plated to be 50% confluent on the day of transfection. Silencer Select small interfering RNA (siRNA) for LOX-1, AT1, or EGFR (Life Technologies) was transfected into the cells in media without serum and antibiotics by use of lipofectamine RNAiMAX (Life Technologies), according to the manufacturer’s instruction. Stimulation with angiotensin II or oxLDL was performed 24 hours after transfection.
Quantitative real-time PCR Total RNA was extracted by use of the RNeasy Mini Kit (Qiagen, Venlo, Netherlands), and an equivalent amount of RNA was transcribed to cDNA by the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative real-time PCR was performed and analyzed on a model 7900 sequence detector (Life Technologies) with TaqMan gene-expression assays for LOX-1, AT1, and EGFR (Life Technologies). The expression level of each gene was normalized by 18s rRNA as an internal control. Stable transformants Tetracycline-inducible human LOX-1 (tagged with V5-6 3 His at the C terminus)-expressing CHO-K1 (CHO-LOX-1) cells were maintained as described previously (15). cDNA, encoding human AT1 (GenBank NM_000685), tagged with signal peptide HAFLAG at the N terminus, was subcloned into pTRE2hyg (Clontech). CHO-LOX-1 cells were cotransfected with pTRE2hyg-HAFLAG-human (h) AT1 and pSV2bsr vector (Funakoshi, Tokyo, Japan) by use of Lipofectamine 2000 transfection reagent (Life Technologies), according to the manufacturer’s instructions. The stable transformants were selected with 400 mg/ml hygromycin B (Wako) and 10 mg/ml blasticidin S (Funakoshi). The resistant clones expressing LOX-1 and AT1 in response to doxycycline (Calbiochem, San Diego, CA, USA) were selected for use in experiments (CHO-LOX-1-AT1). Likewise, CHO-LOX-1-AT2 were prepared by cotransfection of HA-FLAG-hAT2 vector and pSV2bsr vector into CHO-LOX-1. CHO-AT1 were prepared by stable transfection of CHO-K1 Tet-On cells (Clontech) with pTRE2hyg-HAFLAG-hAT1, according to the previous report (15). Detection of Dil-labeled oxLDL CHO-LOX-1-AT1 were treated for 60 minutes with Dil-labeled oxLDL at a final concentration of 5 mg/ml on ice. The cells were then washed twice and fixed with neutral-buffered formalin. Nuclei were stained with DAPI (1 mg/ml). Images were acquired by use of a fluorescence microscope (Axiovert 200M; Zeiss, Oberkochen, Germany).
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YAMAMOTO ET AL.
Detection of cytosolic release of G proteins Cytosolic release of G proteins was detected to verify GPCR activation. Cells were incubated with oxLDL or angiotensin II for 30 minutes at 37°C. The cytosolic fraction was then isolated, separated by SDS-PAGE under reducing conditions, and transferred to PVDF membranes. The blotted membranes were incubated with antibodies against Ga (pan) and Gai (Cell Signaling Technology).
Site-directed mutagenesis of AT1 and transfection of COS-7 cells Primestar Mutagenesis Basal Kit (Takara, Shiga, Japan) was used to delete aa 221 and 222 from FLAG-tagged wild-type (WT) AT1 plasmid (AT1D221/222). V5-Tagged LOX-1 and FLAG-tagged control, WT AT1, or AT1D221/222 were transfected into COS-7 cells by use of Lipofectamine LTX, according to the manufacturer’s instruction (Life Technologies).
Coimmunoprecipitation Luciferase reporter assay Membrane proteins from CHO-LOX-1, CHO-AT1, or CHOLOX-1-AT1 were prepared by use of the Transmembrane Protein Extraction Kit (Merck, Kenilworth, NJ, USA). Immunoprecipitation was performed with anti-FLAG-M2 affinity gel (Sigma-Aldrich, St. Louis, MO, USA), and the FLAG-fusion proteins were eluted with 33 FLAG peptide (Sigma-Aldrich). The purified proteins were then separated by SDS-PAGE under reducing or nonreducing conditions and transferred to PVDF membranes. The blotted membranes were incubated with antibodies against HA and V5. In situ PLA To investigate whether AT1 has the capability to interact with LOX-1, we used an in situ PLA (16), Duolink, from Olink Bioscience (Uppsala, Sweden), according to the manufacturer’s instructions, with slight modifications. In brief, cells were fixed with 10% formalin and incubated with a pair of primary antibodies directed to FLAG tag (clone M2, mouse monoclonal; Sigma-Aldrich) and to V5 tag (rabbit polyclonal; Sigma-Aldrich) in 1% bovine serum albumin (BSA)/PBS for 1 hour at room temperature. This was followed by washing 3 times in PBS and incubation with corresponding PLA probes, secondary antibodies conjugated to oligonucleotides (mouse minus and rabbit plus) in 1% BSA/PBS for 1 hour at room temperature. Thereafter, the cells were washed 3 times in PBS and incubated with the hybridization solution at 37°C for 15 minutes, followed by incubation with the ligation solution for 15 minutes at 37°C. After washing twice in PBS, the cells were treated with polymerase for amplification for 90 minutes at 37°C. The PLA signals were detected with Duolink PLA Detection Kit 563. Nuclei were stained with DAPI (1 mg/ml). Images were acquired by use of a fluorescence microscope (Axiovert 200M; Zeiss). Quantitative fluorescence cell image analysis was performed by use of the IN Cell Analyzer 2000 system (GE Healthcare) (15).
Construction of chimeric plasmids Chimeric plasmids were generated by connecting the extracellular domain of Dectin-1 to the transmembrane and cytoplasmic domains of LOX-1 (Chimera 1) or the extracellular domain of LOX-1 to the transmembrane and cytoplasmic domains of Dectin1 (Chimera 2), with linker sequences (Table 1), as described previously (17). pcDNA6.2/V5/GW/D-TOPO (Life Technologies) constructs of LOX-1 and Dectin-1 were subjected to PCR with a primer pair of N-LOX-1-Fw and L-D linker Rv and a primer pair of L-D linker Fw and C-Dectin-1 Rv for Chimera 1 or a primer pair of D-L-linker Fw and C-LOX-1 Rv and a primer pair of NDectin-1-Fw and D-L-linker Rv for Chimera 2. Obtained Dectin-1 cDNA and LOX-1 cDNA were connected with overlap-extension PCR by use of a primer pair of N-LOX-1-Fw and C-Dectin-1 Rv for Chimera 1 and a primer pair of N-Dectin-1-Fw and C-LOX-1 Rv for Chimera 2. Each product was subcloned into pcDNA6.2/V5/ GW/D-TOPO, and the sequences were confirmed.
OXIDIZED LDL ALLOSTERICALLY ACTIVATES AT1 RECEPTOR
CHO cells, CHO-AT1, CHO-LOX-1, and CHO-LOX-1-AT1 seeded in a 24-well plate were transiently cotransfected with 500 ng pGL3-human LOX-1 promoter Firefly luciferase reporter vector (18) and 10 ng pRL-cytomegalovirus Renilla luciferase control reporter vector (Promega, Madison, WI, USA) by use of Lipofectamine LTX and PLUS Reagent (Life Technologies), according to the manufacturer’s instructions, and cultured for 8 hours. The cells were starved in medium, supplemented with 0.1% FBS, with or without olmesartan for 24 hours. After that, the cells were treated with oxLDL for 20 hours. The luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s protocol. The results were expressed as the intensity relative to the control (incubated with medium only). Ca2+ mobilization Measurement of Ca2+ transients was carried out by use of Fura 2-AM (Dojindo, Kumamoto, Japan), as reported previously (19). In brief, cells plated in 96 wells were incubated with 5 mM Fura 2-AM in recording media (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 13.8 mM glucose, pH 7.4) for 1 hour at 37°C, followed by replacement with recording media without Fura 2-AM. Cells were treated with 80 mg/ml oxLDL, and changes in F340/F380 as an index of intracellular Ca2+ concentration were measured by dualexcitation microfluorometry by use of a digital image analyzer (Aquacosmos; Hamamatsu Photonics, Hamamatsu, Japan). Experimental animals AT1a knockout mice and WT mice on the C57BL/6 background were used in the present study. AT1a knockout mice were a gift from Dr. Masaru Iwai at Ehime University (Matsuyama, Japan). The study protocol was approved by the Animal Care and Use Committee at Osaka University (Permit Number 21-050-3; Osaka, Japan) and was conducted in strict accordance with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animals were euthanized with a sodium pentobarbital overdose, and every effort was made to minimize suffering. Evaluation of NO production of aortic endothelium in mice Thoracic aortic rings from 12-week-old male mice were incubated with vehicle or 40 mg/ml oxLDL in RPMI with 1% mouse serum for 20 hours at 37°C. For olmesartan treatment, aortic rings were preincubated with 1 mM olmesartan for 1 hour before application of oxLDL. Thereafter, aortic rings were transferred to a Magnus chamber (Labo Support, Osaka, Japan) filled with CO2-supplied buffer at 37°C, and tension was recorded by use of PowerLab software (ADInstruments, Dunedin, New Zealand). Resting tension of the aortic ring was adjusted at 0.3 g. Contraction was
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TABLE 1. PCR primers used for constructing chimeric plasmids Primer
N-LOX-1 L-D linker L-D linker C-Dectin-1 D-L-linker C-LOX-1 N-Dectin-1 D-L-linker
Sequence
Fw 59-caccatgacttttgatgacctaaag-39 Rv 59-ccaaatagccatggttaattgcatgcccagcaccataatg-39 Fw 59-ctgggcatgcaattaaccatggctatttggagatccaatt-39 Rv 59-cattgaaaacttcttctcacaaata-39 Fw 59-gctgtggtcctgggttcccaggtgtctgacctcctaacac-39 Rv 59-ctgtgctcttaggtttgccttcttc-39 Fw 59-caccatggaatatcatcctgattta-39 Rv 59-gtcagacacctgggaacccaggaccacagctatcaccagt-39
induced with 5 mM prostaglandin F2a (PGF2a). After stable contraction, endothelium-dependent vasorelaxation was induced with acetylcholine (ACh; 3 3 1029–1026 M). Immunofluorescence staining CHO cells, CHO-LOX-1, and CHO-LOX-1-AT1 were fixed with 4% (w/v) phosphate-buffered formaldehyde (Wako) at room temperature for 15 minutes. Tagged LOX-1 and AT1 were detected with 2.0 mg/ml rabbit anti-V5 (Sigma-Aldrich) and 1.0 mg/ml mouse anti-FLAG (M2; Sigma-Aldrich) antibodies, respectively. Alexa 488-conjugated anti-rabbit IgG (1.0 mg/ml) and 1.0 mg/ml Alexa 546-conjugated anti-mouse IgG (Life Technologies) were used as secondary antibodies, respectively. All antibody incubations were performed for 1 hour at room temperature. Nuclei were counterstained with DAPI (SigmaAldrich). Images were obtained with a confocal laser microscope (TCS SP5; Leica, Wetzlar, Germany). Statistical analyses All data are presented as means 6 SEM. Percent changes compared with vehicle treatment were analyzed by 1-sample t test, and significant differences among mutiple treatments were determined by 1-way ANOVA and Holm-Sidak testing.
RESULTS
Dependence of oxLDL-induced cellular signaling on LOX-1 and AT1
oxLDL-induced cellular signaling is abolished by inhibition or knockdown of AT1 in vascular endothelial cells oxLDL is known to activate ERK1/2 (p42/44 MAPK) via interaction with LOX-1 in vascular cells (20). In HUVECs, pretreatment with an ARB, olmesartan, for 24 hours inhibited the ability of oxLDL to increase ERK1/2 activity (Fig. 1A). Other ARBs, including telmisartan, valsartan, and losartan, also inhibited oxLDL-induced ERK activation, indicating that the inhibition was a class effect of ARBs (Supplemental Fig. 1A). OxLDL is known to transactivate EGFRs (21), and we found that ARB treatment also inhibited this phenomenon (Fig. 1A). It has been reported that LOX-1 expression is up-regulated by many proatherogenic stimuli, including angiotensin II (9) and oxLDL (22). However, in the current studies conducted in the absence of angiotensin II, ARB treatment did not affect expression of LOX-1, suggesting that the effect of ARB did not result from down-regulation of LOX-1 (Supplemental Fig. 1B). It should be noted that olmesartan 4
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inhibited the up-regulation of LOX-1 promoter activity by oxLDL in HUVECs, suggesting that prolonged oxLDL treatment can up-regulate LOX-1 in an AT1-dependent manner (Fig. 1B). siRNA knockdown of angiotensinogen, a precursor of angiotensin II, or incubation with enalapril, an angiotensin-converting enzyme inhibitor that blocks conversion of angiotensin I to angiotensin II, did not affect oxLDL-induced ERK1/2 activation (Supplemental Fig. 1C). These findings indicate that pharmacologic ARB inhibits oxLDL-induced cell signaling, but the effect is not likely to be mediated by alterations in the endogenous production of angiotensin II or by blockade of angiotensin II binding to AT1. We next investigated whether knockdown of AT1 expression would attenuate the ability of oxLDL to stimulate MAPK activity (Fig. 1C). In HUVECs, siRNA knockdown of LOX-1 or AT1 inhibited the ability of oxLDL to activate ERK1/2 (Fig. 1C, middle), whereas siRNA knockdown of LOX-1 did not affect angiotensin II-induced ERK1/2 activation (Fig. 1C, right). siRNA knockdown of the EGFR also attenuated ERK1/2 activation triggered by oxLDL (Fig. 1C, middle) or by angiotensin II (Fig. 1C, right), suggesting that ERK1/2 activation induced by oxLDL is downstream of EGFR transactivation, similar to the mechanism whereby angiotensin II activates ERK1/2 (23).
August 2015
To investigate signaling actions of oxLDL by use of a knockin system, we developed stable cell lines expressing V5-tagged human LOX-1, HA- and FLAG-tagged human AT1, or both in CHO cells that lack native LOX-1 and AT1 (CHO-LOX-1, CHO-AT1, CHO-LOX-1-AT1, respectively). In CHO-LOX-1-AT1, oxLDL treatment activated ERK1/2 to a much greater extent than in CHO-LOX-1 lacking AT1 receptors, whereas the expression of LOX-1 and binding of oxLDL to LOX-1 were not different between these cells (Fig. 2A, C and Supplemental Fig. 2). Treatment with 40 mg/ml oxLDL modestly activated ERK1/2 to a similar extent in WT CHO cells lacking LOX-1 and AT1, as in CHO cells expressing LOX-1 or AT1, suggesting that oxLDL may cause some minor activation of ERK1/2 through pathways independent of LOX-1 and AT1 (Fig. 2B). In CHO-LOX-1AT1, oxLDL activated ERK1/2 to a much greater degree than the ERK1/2 activation observed in CHO-LOX-1 and CHO-AT1 (Fig. 2B). The ERK activation in response to oxLDL in CHO-LOX-1-AT1 was inhibited by pretreatment with olmesartan for 30 minutes, 6 hours, or 24 hours, with
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YAMAMOTO ET AL.
A
Olmesartan(-) Olmesartan(+) Phospho/Total ERK (% of vehicle treatment )
250
Phospho ERK Total ERK Phospho EGFR
100 50
vehicle
3min
5min
3min
-
5min
Vehicle (10min)
B
+
Phospho/Total EGFR (% of vehicle treatment )
-
+
-
+ 10min
oxLDL
*䯘
200
*#
150 100 50
p
