Mnk1 Is Required for Angiotensin II–Induced Protein Synthesis in Vascular Smooth Muscle Cells
Angiotensin II (Ang II) stimulates protein synthesis in vascular smooth muscle cells (VSMCs), possibly secondary to regulatory changes at the initiation of mRNA translation. Mitogen-activated protein (MAP) kinase signal–integrating kinase-1 (Mnk1), a substrate of ERK and p38 MAP kinase, phosphorylates eukaryotic initiation factor 4E (eIF4E), an important factor in translation. The goal of the present study was to investigate the role of Mnk1 in Ang II–induced protein synthesis and to characterize the molecular mechanisms by which Mnk1 and eIF4E is activated in rat VSMCs. Ang II treatment resulted in increased Mnk1 activity and eIF4E phosphorylation. Expression of a dominant-negative Mnk1 mutant abolished Ang II–induced eIF4E phosphorylation. PD98059 or introduction of kinase-inactive MEK1/MKK1, but not SB202190 or kinase-inactive p38 MAP kinase, inhibited Ang II–induced Mnk1 activation and eIF4E phosphorylation, suggesting that ERK, but not p38 MAP kinase, is required for Ang II–induced Mnk1-eIF4E activation. Further, dominant-negative constructs for Ras, but not for Rho, Rac, or Cdc42, abolished Ang II–induced Mnk1 activation. Finally, treatment of VSMCs with CGP57380, a novel specific kinase inhibitor of Mnk1, resulted in dose-dependent decreases in Ang II–stimulated phosphorylation of eIF4E, protein synthesis, and VSMC hypertrophy. In summary, these data demonstrated that (1) Ang II–induced Mnk1 activation is mediated by the Ras-ERK cascade in VSMCs, and (2) Mnk1 is involved in Ang II–mediated protein synthesis and hypertrophy, presumably through the activation of translation-initiation. The Mnk1-eIF4E pathway may provide new insights into molecular mechanisms involved in vascular hypertrophy and other Ang II–mediated pathological states.
Angiotensin II (Ang II) is a potent stimulus for protein synthesis and cellular hypertrophy in vascular smooth muscle cells (VSMCs) and may contribute to pathological states associated with atherosclerosis, hypertension, and balloon injury. However, the molecular mechanisms involved in Ang II–induced hypertrophy remain largely unknown. In general, changes in protein synthesis results from alterations at the transcriptional and translational levels. The global nature of trophic stimulation by Ang II suggests that alterations in translation-initiation of preexisting mRNA may be involved.
Initiation of mRNA translation is controlled by multiple protein-protein interactions and phosphorylation/dephosphorylation mechanisms. Eukaryotic initiation factor 4E (eIF4E) plays a key role in mRNA translation and its regulation.1,2 eIF4E binds to the 7-methylguanosine triphosphate (“cap”) structure at the 5′-end of cytoplasmic mRNA and interacts with eIF4G, a large scaffold protein that binds to other translation factors, including eIF4A and eIF3. This complex of eIF4E, eIF4G, and eIF4A is termed eIF4F, and functions to translocate mRNA to the proper position, thereby promoting translation.3 Phosphorylation of eIF4E increases its affinity for the 5′-cap4 and facilitates its incorporation into eIF4F complexes.1 Indeed, stimulators of translation often result in enhanced phosphorylation of eIF4E.
A new substrate for extracellular signal-regulated kinase (ERK), called mitogen-activated protein (MAP) kinase signal–integrating kinase-1 (Mnk1), was recently discovered. Mnk1 is a serine/threonine kinase that phosphorylates eIF4E in vitro.5,6 Studies using dominant-negative or activated Mnk1 mutants demonstrated that TPA-induced activation of Mnk1 resulted in direct modulation of eIF4E phosphorylation. Further, activation of Mnk1 by anisomycin, UV, TNF-α, or IL-1β was mediated by p38 MAPK, whereas TPA-induced Mnk1-activation was mediated via ERK. Interestingly, activation of Mnk1 by serum was mediated by both ERK and p38 MAPK.7 Similar data have been reported in terms of eIF4E phosphorylation.2,8,9
In this study, we hypothesized that Ang II–stimulated protein synthesis is mediated via changes in translation-initiation. We assessed changes in Mnk1 activation, eIF4E phosphorylation, and protein synthesis in response to Ang II. Further, we used molecular techniques to characterize signal transduction pathways associated with this phenomenon.
Materials and Methods
Plasmids for GST-Mnk1 (pEBG-Mnk1), dominant-interfering Mnk1 mutant (pEBG-T2A2, Mnk1 containing Thr197Ala/Thr202Ala mutants), and a constitutively active form of Mnk1 (pEBG-T332D, Mnk1 containing Thr332Asp mutant) were kind gifts of Dr Jonathan Cooper (Fred Hutchinson Cancer Research Center, Seattle, Wash). The plasmids for kinase-inactive MEK1/MKK1 (pCMV-HA-MKK1 S222A) and kinase-inactive p38α MAPK (TGY→AGF mutants) were kindly provided by Dr Yukiko Gotoh (Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo) and Dr Jiahuai Han (The Scripps Research Institute, La Jolla, Calif), respectively. Dominant-negative mutants of Ras and Rac were generous gifts from Dr Neil Nathanson (University of Washington, Seattle, Wash) and Dr J. Silvio Gutkind (National Institute of Dental Research, National Institute of Health, Bethesda, Md), respectively.
Polyclonal anti-Mnk1 antiserum was kindly provided by Dr Rikiro Fukunaga (Graduate School of Medicine and Graduate School of Frontier Biosciences, Osaka University) or purchased from Santa Cruz Biotechnology. Monoclonal antibody against HA (12CA5) was purchased from Boehringer-Mannheim. Monoclonal eIF4E antibody was purchased from Transduction Laboratory. Polyclonal anti-phosphospecific Mnk1 antibody was purchased from Cell Signaling Technology.
Cell Culture and Transient Transfection
VSMCs were isolated from the aorta of 200 to 250 g male Sprague-Dawley rats (Charles River, Japan) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, as previously described.10 Animals were maintained in accordance with institutional guidelines for the care and use of laboratory animals. VSMCs at passage 6 to 16 and 70% to 80% confluence were growth-arrested for 48 hours before use by incubation in DMEM supplemented with 0.1% calf serum. For transient transfection, 3×106 cells were split before lipofectamine (Gibco-BRL) or FuGENE 6 (Roche) transfection, and 3 μg of DNA were added to the cells in 100 mm dishes for lipofection. At 24 hours after the addition of DNA, cells were made quiescent by resuspension in serum-free medium. At 48 hours, after the addition of DNA, cells were stimulated with the agonist, harvested, and lysed, as previously described.11,12 As transfection efficiency was approximately 10% with rat VSMCs, a cotransfection method was utilized.
Analysis of Mnk1
Growth-arrested VSMCs were stimulated with Ang II as indicated in each experiment. Cells were lysed in NP-40 buffer (1% NP40, 25 mmol/L Tris, pH 7.5, 50 mmol/L NaF, 10 mmol/L sodium pyrophosphate, 137 mmol/L NaCl, 10% glycerol, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and 10 μg/mL leupeptin). Endogenous Mnk1 was immunoprecipitated with anti-Mnk1 antibody (kindly provided by Dr Rikiro Fukunaga), as described previously.10 Exogenous GST-Mnk1 was isolated using glutathione-Sepharose (Pharmacia). Samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed with anti-phosphospecific Mnk1 antibody.
Isolation and Analysis of eIF4E
eIF4E was analyzed by isoelectric-focusing (IEF), followed by immunoblotting. Samples were prepared in 4E buffer (50 mmol/L β-glycerophosphate, 1 μmol/L microcystine [Calbiochem], 1 mmol/L PMSF, 2 mmol/L sodium vanadate). eIF4E was purified with m7GTP-Sepharose (Pharmacia). Samples were washed 4 times in 4E buffer, eluted in buffer containing urea and ampholytes, and separated on one-dimensional denaturing IEF gel containing equal parts of pH 4 to 6 and pH 6 to 8 ampholytes, as described by Flynn et al.13 Gels were electroblotted onto PVDF membrane (Amersham). Immunoblots were performed either with anti-eIF4E antibody for endogenous eIf4E or with anti-HA antibody (12CA5) for exogenous HA-eIF4E. In some experiments, purified eIF4E was subjected to SDS-PAGE, transferred to nitrocellulose membranes, and analyzed with anti-phosphospecific eIF4E (Ser209) antibody.
Protein Synthesis Measurements
Growth-arrested VSMCs in triplicate wells of 12-well plates were stimulated with 100 nmol/L Ang II in serum-free DMEM medium containing 2 μCi/mL [3H]leucine. After 24 hours, medium was aspirated, and the cells were fixed for 30 minutes with cold 10% TCA. The cells were then washed with 95% ethanol and solubilized in 0.5N NaOH. The radioactivity incorporated into TCA-precipitable material was measured by liquid scintillation counter. CGP57380 was generously provided by Dr Hermann Gram (Novartis Pharma AG, Basel, Switzerland).
Planar Cell Surface Area
VSMCs were sparsely seeded onto 60-mm dishes. Growth arrested VSMCs were pretreated with or without CGP57380 for 1 hour and stimulated with Ang II for 24 hours. After cells were fixed with 1% glutaraldehyde, planar cell surface area was quantified by computer-assisted morphometry. Randomly selected images were analyzed by NIH Image. Measurements were performed in a blind manner. At least 100 cells were counted for each treatment.
Expression of Mnk1 in VSMCs
Expression of Mnk1 has been demonstrated in mouse tissues, including heart, brain, spleen, lung, liver, kidney, testis, and skeletal muscle. To determine if Mnk1 was expressed in VSMCs, Mnk1 was immunoprecipitated from VSMC lysates with anti-Mnk1 antibody. Immunoblotting was then performed with another anti-Mnk1 antibody after SDS-PAGE. A 51-kDa band was visualized in immunoprecipitates from VSMC lysates, but not in immunoprecipitates with preimmune serum or in those from cell-free lysis buffer (Figure 1A). These data are consistent with Mnk1 expression in VSMCs.
Activation of Mnk1 by Ang II
Activation of Mnk1 requires phosphorylation at Thr197 and Thr202.14 To determine whether Mnk1 was activated by Ang II, Mnk1 was immunoprecipitated from the lysate of Ang II–stimulated VSMCs and immunoblotted with phospho-(Thr197/Thr202)-specific Mnk1 antibody. Mnk1 phosphorylation was detected after 5 minutes of Ang II exposure and peaked at 10 to 20 minutes (Figure 1B). Ang II–induced Mnk1 phosphorylation was dose-dependent. Mnk1 phosphorylation by Ang II was induced with as little as 1 nmol/L, and a maximal effect was achieved with 30 to 100 nmol/L (Figure 1C).
Phosphorylation of eIF4E by Ang II
eIF4E is an important eukaryotic translation-initiation factor that is phosphorylated by Mnk1. To examine whether Ang II stimulated the phosphorylation of eIF4E, growth-arrested VSMCs were treated with Ang II, and eIF4E phosphorylation was evaluated with IEF gel electrophoresis, followed by Western blotting. This method resolved two species of endogenous eIF4E: an acidic, phosphorylated form and a basic, nonphosphorylated form. As shown in Figure 2A, eIF4E phosphorylation was detected after 10 minutes of Ang II exposure and peaked at 30 minutes to 2 hours. The phosphorylation decreased gradually and resolved by 48 hours (Figure 2A).
eIF4E is phosphorylated at Ser209 by Mnk1 in response to mitogens.5 To confirm whether eIF4E is phosphorylated at Ser209 by Ang II, purified eIF4E was subjected to SDS-PAGE followed by Western blotting with phosphospecific eIF4E (Ser209) antibody, which specifically detects eIF4E phosphorylated at Ser209. As shown in Figure 2B, eIF4E phosphorylation at Ser209 was present at 10 minutes after Ang II stimulation and peaked at 30 minutes to 2 hours. Overall time course was similar with eIF4E phosphorylation evaluated with IEF.
Mnk1 Activation Is Required for eIF4E Phosphorylation
To test whether Mnk1 regulated eIF4E phosphorylation in VSMCs, we cotransfected VSMCs with HA-tagged eIF4E along with wild-type or mutant GST-Mnk1 or with vector alone. After isolation of eIF4E from cell lysates using m7GTP-Sepharose, phosphorylation status of transfected HA-eIF4E was analyzed by IEF. Western blotting with anti-HA antibody was then performed (Figure 3), revealing two acidic and two basic species, corresponding to phosphorylated and nonphosphorylated forms of HA-eIF4E. It is assumed that the presence of two separate bands for each form of HA-eIF4E was an artifact produced by the HA tag.14 The increase in phosphorylation of HA-eIF4E in vector-transfected VSMCs was small, presumably because endogenous Mnk1 was not abundant enough relative to the large amount of transfected HA-eIF4E. However, a statistically significant increase was detected. Expression of wild-type Mnk1 resulted in a marked increase in Ang II–induced eIF4E phosphorylation, supporting the supposition that endogenous Mnk1 is present in limited amounts relative to transfected HA-eIF4E. Expression of the dominant-interfering mutant of Mnk1, T197A/T202A (T2A2), reduced the presence of the acidic phosphorylated form of eIF4E from 36.1±0.89% to 17.9±6.52% in unstimulated cells and inhibited the response to Ang II. In contrast, T332D, a constitutively active mutant of Mnk1, induced an increase in the phosphorylated form to 80%, even without Ang II stimulation. These data indicate that Ang II enhances phosphorylation of eIF4E in a Mnk1-dependent manner.
Signaling Mechanisms of Mnk1 Activation
Mnk1 was originally characterized as a protein that is phosphorylated by and binds to ERK.5,7 However, subsequent studies demonstrated that Mnk1 was phosphorylated by both ERK and p38 MAPK. Further, an upstream kinase for Mnk1 was dependent on extracellular stimuli.9,13
To characterize the upstream kinase for Ang II–mediated Mnk1 phosphorylation, we first performed pharmacological inhibition studies. Ang II–induced Mnk1 phosphorylation was completely inhibited by the MEK inhibitor, PD098059, whereas the p38 MAPK inhibitor, SB202190, had no effect (Figure 4A). Compatible with ERK dependency of Mnk1 phosphorylation, eIF4E phosphorylation was also inhibited by PD098059, whereas SB202190 had no effect (Figure 4B). These data suggest that Ang II–mediated Mnk1 phosphorylation/activation is dependent on ERK but not p38 MAPK.
To confirm these results, we performed cotransfection experiments with wild-type GST-Mnk1 and the kinase-inactive MEK1/MKK1 mutant (MKK1 S222A) or dominant-negative p38 MAPK mutant (p38αAF). Ang II–induced phosphorylation of GST-Mnk1 was attenuated by the kinase-inactive MEK1/MKK1 mutant, whereas the dominant-negative p38 MAPK mutant did not inhibit Mnk1 phosphorylation (Figure 5). Incomplete inhibition of Mnk1 phosphorylation in MEK1/MKK1 mutant-transfected cells may have resulted from the abundant expression of intrinsic MEK1/MKK1.
We also investigated the role of small G protein(s) in the Ang II–mediated Mnk1/eIF4E phosphorylation pathway. Ang II–induced Mnk1 phosphorylation was completely inhibited by overexpression of dominant-negative Ras (N17Ras), whereas dominant-negative mutants of other small G proteins had no effect (Figure 6). Thus, Ang II–induced Mnk1 activation and eIF4E phosphorylation appear to be mediated via the Ras-ERK pathway, but not by the p38 MAPK pathway.
Significance of Mnk1 Activation
Recently, Knauf et al15 described a novel low-molecular-weight kinase inhibitor of Mnk1, CGP57380. This compound inhibited Mnk1 kinase activity in vitro with a 50% inhibitory concentration (IC50) of 2.2 μmol/L and showed no cellular toxicity at a concentration up to 30 μmol/L. Further, CGP57380 had no inhibitory effect on various other kinases, such as p38 MAPK, JNK1, ERK1, and ERK2, protein kinase C, or c-Src family kinases.15 In an assessment of the specificity of this compound, CGP57380 (10 μmol/L) had no effect on Ang II–induced stimulation of ERK and p70S6 kinase in rat VSMCs (online Figure 1, available in the online data supplement at http://www.circresaha.org). Administration of CGP57380 resulted in reductions in Ang II–induced eIF4E phosphorylation (Figure 7A) and Ang II–stimulated protein synthesis (Figure 7B) in a dose-dependent manner. CGP57380 inhibited the phosphorylation of eIF4E throughout the time course (online Figure 2). Therefore, inhibition of Mnk1 kinase activity and eIF4E phosphorylation correlated with the inhibition of protein synthesis, presumably via inhibition of translation initiation.
To determine whether Mnk1 regulated VSMC hypertrophy, the effects of CGP57380 on Ang II–mediated changes in cell surface area were assessed. Ang II treatment increased cell surface area from 6233±276 to 9918±584 μm2, and CGP57380 dose-dependently inhibited Ang II–induced increases in cell surface area (Figure 8), which is consistent with its effects on protein synthesis. These data indicate that Mnk1 plays a critical role in Ang II–induced translation initiation/protein synthesis and VSMC hypertrophy.
The present study demonstrated that Ang II–induced eIF4E phosphorylation, protein synthesis, and hypertrophy in VSMCs was mediated by activation of Mnk1. Further, the Ras-ERK pathway, but not the p38 MAPK pathway, contributed to this response.
Initiation of mRNA translation is controlled by multiple protein-protein interactions and phosphorylation/dephosphorylation of several proteins. eIF4E is a phosphoprotein that binds to the cap structure at the 5′-end of cytoplasmic mRNA and acts to promote mRNA translation.1,2 eIF4E also interacts with eIF4G, a molecule that binds to other translation factors, including eIF4A and eIF3. Formation of this complex results in translocation of mRNA to the proper position for translation.3 Phosphorylation of eIF4E increases its affinity for the 5′-cap4 and facilitates its incorporation into eIF4F complexes.1 Phosphorylation of eIF4E is generally enhanced by agents that promote translation, such as insulin and growth factors. However, the biological significance of this modification is controversial. In the present study, we demonstrated that Mnk1 inhibition by CGP57380 resulted in decreased Ang II–induced eIF4E phosphorylation and decreased basal and Ang II–induced protein synthesis in VSMCs. Recently, Lachance et al16 reported that introduction of the eIF4ESer251Asp (mimicking constitutive phosphorylation on Ser251 of Drosophila eIF4E) transgene into an eIF4E lethal mutant background in Drosophila can fully rescue the lethality. Further, eIF4ESer251Ala (dominant-negative form of eIF4E in Drosophila) conferred reduced viability, and their escapers had slower development and smaller size, indicating that eIF4E phosphorylation is essential for normal growth and development. On the other hand, Knauf et al15 demonstrated that expression of constitutively active Mnk1 and Mnk2 mutants diminished cap-dependent translation and that Mnk2 overexpression resulted in decreased rates of protein synthesis in 293 cells. The disparity between Knauf’s observations and the present study may be explained by differences in cell types and the stimuli employed. The abundance of Mnk1 and/or the presence of specific adaptor molecules or substrates for Mnk1 may vary among the cell types. Alternatively, expression of constitutively active mutants may cause dramatic shifts in translation initiation mechanisms that are controlled by activation/inactivation mechanisms. Further, kinases upstream of Mnk1 may be stimulus-specific.7 Indeed, Mnk1 activation stimulated by cell stressors such as anisomycin and UV was mediated by p38 MAPK alone, whereas serum-stimulated Mnk1 activation was mediated by both ERK and p38 MAPK.
Although eIF4E phosphorylation can be induced by either ERK1/2 and p38 MAPK in a stimulus-specific manner,8,9 Mnk1 and Mnk2 were originally characterized in vitro as kinases that integrate signals leading from the MAPK pathways to produce eIF4E phosphorylation. Ushio-Fukai et al17,18 reported that the p38 MAPK-Akt pathway as well as ERK was involved in protein synthesis stimulated by Ang II. In the present study, Ang II–mediated Mnk1 activation was regulated by the Ras-ERK pathway and not by p38 MAPK. These data are consistent with observations by previous studies that demonstrated that Ang II–induced protein synthesis was ERK1/2- and Ras-dependent.19 In contrast, Amick et al20 reported that MAP kinases do not directly phosphorylate eIF4E, and Giasson et al21 reported that rapamycin, which does not block the activity of ERK1/2, inhibited Ang II–induced protein synthesis. Thus, it appears that the ERK pathway is necessary but not sufficient for Ang II–induced protein synthesis and hypertrophy.
In translation initiation mechanisms, binding of eIF4E to eIF4G is regulated by eIF4E-binding protein (4E-BP), which interacts with the same region of eIF4E that binds eIF4G. The phosphorylation of 4E-BP blocks its interaction with eIF4E, which, in turn, releases eIF4E to bind to eIF4G.1,2 Several kinases have been shown to phosphorylate 4E-BP, including FKBP 12-rapamycin associated protein/mammalian target of rapamycin (FRAP/mTOR) and ERK1/2.22 In addition, other phosphorylation/dephosphorylation, binding/unbinding mechanisms and negative feedback loops23 regulate translation initiation.24 Thus, it appears that the Ras-ERK to Mnk1/eIF4E pathway may be only one of the mechanisms that regulate translation-initiation in Ang II–induced protein synthesis. Further studies concerning the phosphorylation of eIF4E and the interacting proteins may help to resolve the relative importance of eIF4E and Mnk1.
Because of the low transfection efficiency of VSMCs and the difficulty associated with establishing stably transfected VSMC lines, a pharmacological approach to investigate the role of Mnk1 in protein synthesis and hypertrophy was used in this study. CGP57380 inhibited Mnk1 kinase activity in vitro with a 50% inhibitory concentration (IC50) of 2.2 μmol/L and showed no cellular toxicity at a concentration up to 30 μmol/L.15 Further, the compound had no inhibitory effect on various other kinases, such as p38 MAPK, JNK1, ERK1/2, protein kinase C, or c-Src family kinases.15 In fact, CGP57380 had no effect on Ang II–induced stimulation of ERK and p70S6 kinase in rat VSMCs up to 10 μmol/L. The present study demonstrated that CGP57380 inhibited Ang II–induced eIF4E phosphorylation, protein synthesis, and the increase in cell size in a dose-dependent manner. These data suggest that the Mnk-eIF4E axis plays a critical role in Ang II–induced protein synthesis and hypertrophy in VSMCs.
Mnk2 is also phosphorylated and activated by ERK and p38 MAPK and can phosphorylate eIF4E in vitro and in vivo.25 Whereas Mnk1 binds both ERK2 and p38 MAPK, Mnk2 interacts strongly only with ERK2. Further, Mnk2 has a high basal activity that is minimally affected by ERK and p38 MAPK inhibitors. Scheper et al25 speculated that the high levels of Mnk2 activity are secondary to efficient activation by basal activity of upstream kinases. We were unable to characterize the presence or activity of Mnk2 in the present study because the specific antibody for Mnk2 is not yet available. Because Ang II–mediated eIF4E phosphorylation was ERK-dependent, further investigation to determine whether Mnk2 plays a role in this process would be of benefit.
Although these data are the first to implicate Mnk1 in the process of cellular hypertrophy, much is still unclear regarding the role of Mnk1. For example, signal transduction pathways stimulated by mitogens overlap with those stimulated by hypertrophic stimuli; thus, an investigation of the role of Mnk1/eIF4E signaling in cell proliferation would be of interest. In addition, stressors such as heat shock, sorbitol, and H2O2 activate Mnk1 without increasing phosphorylation of eIF4E, presumably through increased binding of eIF4E to 4E-BP.9 Other biological properties of Mnk1 are suggested by studies that demonstrate phosphorylation of cPLA2 in platelets by Mnk1 in vivo26 and translocation of stimulated Mnk1 to the nucleus.14 Therefore, further investigation into the role of Mnk1 in protein synthesis, hypertrophy, and other biological functions is recommended.
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.I. and T.I.) and Japan Heart Foundation/Zeria Pharmaceutical Grant for Research on Molecular Cardiology (to M.I. and T.I.). We thank Dr Akihiko Muto, Dr Satoshi Tashiro, and Noriko Kuwaba for technical assistance and Dr Oren Traub for reading this manuscript.
Original received June 3, 2003; revision received October 6, 2003; accepted October 28, 2003.
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