We previously demonstrated that proto-oncogene Fyn decreased energy expenditure and increased metabolic phenotypes. Also Fyn decreased autophagy-mediated muscle mass by directly inhibiting LKB1 and stimulating STAT3 activities, respectively. AMPK, a downstream target of LKB1, was recently identified as a key molecule controlling autophagy. Here we identified that Fyn phosphorylates the α subunit of AMPK on Y436 and inhibits AMPK enzymatic activity without altering the assembly state of the AMPK heterotrimeric complex. As pro-inflammatory mediators are reported modulators of the autophagy processes, treatment with the pro-inflammatory cytokine TNFα resulted in 1) increased Fyn activity 2) stimulated Fyn-dependent AMPKα tyrosine phosphorylation and 3) decreased AICAR-dependent AMPK activation. Importantly, TNFα induced inhibition of autophagy was not observed when AMPKα was mutated on Y436. 4) These data demonstrate that Fyn plays an important role in relaying the effects of TNFα on autophagy and apoptosis via phosphorylation and inhibition of AMPK. Although autophagy is originally described as an evolutionarily conserved cellular recycling program , 2, rece2+/calmodulin-dependent protein kinase kinase (CaMKK2) in the brain and Liver Kinase B1 (LKB1) in peripheral organs , one of the major energy sensors of the cell , 12, 13.l organs , 18, resl organs .Although the link between AMPK and autophagy is established, upstream regulators of AMPK in the context of autophagy response are still largely unknown. Previously, we reported that Fyn knockout (FynKO) mice display increased AMPK activity as a result of Fyn-dependent LKB1 regulation in peripheral tissues , 21. AnoIn this study, we demonstrate that 1) Fyn specifically phosphorylates AMPKα on Y436, resulting in AMPK activity inhibition and thereby suppressing autophagy, 2) TNFα activates Fyn kinase, 3) Fyn deficiency blocks TNFα inhibition of autophagy by preventing AMPK α subunit Y436 phosphorylation and 4) Fyn deficiency protects against TNFα activation of apoptosis.Having recently identified LKB1 and STAT3 as direct substrates of Fyn in the respective regulation of metabolism  and musctyrosine phosphorylation of AMPK, gastrocnemius muscle tissue extracts were prepared from Fyn knockout (FynKO), wild type (WT), skeletal muscle-specific over expressing FynB and FynT mice. The extracts were immunoblotted for AMPK α subunit and GADPH as a loading control  cDNA with or without an epitope tagged constitutively active Fyn (V5-Fyn-CA) cDNA in NIH-3T3 cells Figure . Expressg Figure . We recen Figure . These dTo demonstrate that AMPKα is a direct Fyn substrate, we incubated human purified AMPKα2 protein with purified Fyn-CA Figure . In the To evaluate whether tyrosine phosphorylation on Y436 has an effect on AMPK activity, we silenced the endogenous AMPK α1 and α2 subunits in HEK293T cells using specific human siRNA. As shown in Figure Recently, studies have demonstrated that AMPK kinase activity is regulated by modification of the AMPK α, β, γ subunit trimeric complex assembly state , 15, 17.Although a number of studies have shown that the pro-inflammatory cytokine TNFα regulates AMPK activity , 29, theShort term (12 h) of TNFα treatment enhanced both basal and AICAR-stimulated T172 AMPKα phosphorylation levels Figure . In contTo further investigate the role of Fyn in relaying the effects of TNFα on AMPKα phosphorylation, we next silenced Fyn protein expression by siRNA in HEK293T cells. As shown in Figure As AMPK plays an important regulatory function in the control of autophagy , 36, we To confirm these results, we utilized the GFP-LC3 puncta formation assay. As shown Figure Since prolonged exposure of inflammatory mediators inhibit autophagy resulting in severe cell damage and apoptosis , we nextAMPK is a master energy sensor in cells and it is activated in various conditions where cellular ATP is decreased. AMPK also has a major role in pathological conditions  and therapeutic activation of AMPK is believed to have beneficial advantages for several disease processes. For example, metformin increases AMPK activity and is the most commonly prescribed oral anti-diabetic agent , 35 and AMPK is a heterotrimeric complex composed of three different subunits α, β, γ and currently only two AMPK kinases (LKB1 and CaMKK2) and two AMPK phosphatases (PP2A and PP2C) are known to regulate AMPK activity , 38, 39.nd part of αRIM and interacting with AID. This suggested that Y436 phosphorylation could affect AMPK subunits assembly. However, we found that Y436-dependent inhibition of AMPK activity was not due to the assembly state of the AMPK heterotrimeric complex  and a regulatory C-terminus. The regulatory C-terminus has an auto-inhibitory domain (AID), a sensor (αRIM) loop and a β-subunit interaction domain (β-ID). Additionally, a recent publication has implicated that AMP binding to CBS-3 of γ subunit induces the interaction of AID-αRIM2 and γ unit resulting in AMPK activation by releasing AID inhibition of the kinase domain . Interesx Figure  and sincin vivo patho-physiological conditions. As expected, prolonged incubation of TNFα suppressed AICAR stimulated T172 α subunit phosphorylation and subsequent ACC phosphorylation, indicating that prolonged TNFα desensitized AMPK activation. Additionally, we demonstrated that under these conditions Fyn was activated and AMPK was phosphorylated on Y436, suggesting that this desensitization directly results from the Fyn-dependent tyrosine phosphorylation of the AMPK α subunit. Moreover we found that Fyn also regulated AICAR-dependent AMPK activation under prolonged TNFα stimulation.Utilizing TNFα, a pro-inflammatory cytokine, we next examined the role of Fyn regulation of AMPK under metabolic stress conditions. Recent studies have reported that TNFα infusion into lean animals increases PP2C expression, resulting in a reduction in AMPK signaling, fatty acid oxidation, and the development of skeletal muscle insulin resistance , 30, butRecent studies have implicated that AMPK regulates autophagy through a direct AMPK-dependent phosphorylation of ULK1 , 13 therSince autophagy induction by AMPK activation rescues cells from cell death , we examIn summary, we found that Fyn directly phosphorylates the α subunit of AMPK on Y436 and this phosphorylation suppresses AMPK activity, subsequently resulting in increased ACC and ULK1 phosphorylation. Prolonged exposure to pro-inflammatory cytokines such as TNFα decreased AICAR-dependent AMPK activation through activation of Fyn. These modulatory events manifest in the inhibition of AMPK-dependent autophagy activation and subsequent apoptosis, suggesting that targeting Fyn activity may have positive therapeutic potential as a novel pathway to activate AMPK.Flag antibody was from Sigma-Aldrich , the 4G10 and GST antibodies were from Millipore , GAPDH and V5 antibodies were purchased from MBL , Fyn antibody was from Santa Cruz Biotechnology , LC3 antibody was from Novus Biologicals  and p62 antibody was from Enzo Life sciences . All other antibodies were purchased from Cell Signaling . Bafilomycin A1 and AICAR were from Sigma-Aldrich . TNFα and Metformin were from EMD Millipore , Toronto Research Chemicals  and Cayman Chemical  respectively. Leupeptin Hemisulfate was from Fisher Scientific . DharmaFECT Duo Transfection Reagent and siRNAs for AMPKα1, α2 and Fyn were purchased from GE Healthcare . X-tremeGENE HP DNA Transfection Reagent were from Roche . All other reagents were purchased from Sigma-Aldrich.The pCMV6 Entry Myc-DDK tagged ORF clone of Homo sapiens AMPK α2 construct was obtained from Origene  and used to generate the pCMV6-3Myc-2-AMPKα2-Myc DDK construct. The gene encoding AMPK α2 was amplified with the oligonucleotides: 5′- GAATTCGCCCGGGCGGGATCCCCCG GGCTGCAGATGGCTGAGAAGCAGAAGCA-3′ and 5′- AAGCTTGGTACCGGGCCCCCCCTCGAGGTCGA CGGTATCGATAACCTTATCGTCGTCATCCT-3′ and cloned to pCR-BluntII-TOPO . This construct and pCMV-3Myc-2  were digested with EcoRI and HindIII to obtain pCMV-3Myc-2-AMPKα2-Myc-DDK. GST-AMPKα1(r), pcDNA3, pcDNA3-Fyn-CA, pcDNA3-LKB1 and eGFP-hLC3 construct were constructed or obtained before . Human Yfyn knockout mice and their controls were obtained from The Jackson Laboratory . Animals were housed in a facility equipped with a 12 h light/dark cycle and fed ad libitum a standard chow diet  containing 67%  carbohydrates, 19% protein, and 4% fat. All studies were approved by and performed in compliance with the guidelines of the Albert Einstein College of Medicine Institutional Animal Care and Use Committee (IACUC).Mice over expressing the murine FynB and FynT cDNA were established as previously described . pp59fyn2. DNA Transfection was performed using X-tremeGENE HP DNA Transfection Reagent and siRNA transfection was performed using DharmaFECT Duo Transfection Reagent according to the manufacture's instructions. siGENOME Human AMPK α1 and α2 and FYN siRNA SMART pool and Non-target siRNA were from GE Healthcare. We saw the effect of AICAR mediating AMPK phosphorylation and activity was fully regulated under confluent condition in HEK293T cells, so every experiments with HEK293T cells were performed under confluent cells condition.HEK293T, NIH3T3 and HeLa cells  were cultured in Dulbecco's Modified Eagle's Medium  with 10% Fetal Bovine Serum  and 1% Penicillin-Streptomycin . Cells were incubated at 37°C moisturized incubator with 5% COTM Protein Assay . Protein lysates (40 μg) were separated on 8 to 15% reducing polyacrylamide gels and transferred onto Immobilon-P polyvinylidene difluoride membranes. Immunoblots were blocked with 5% milk in Tris-buffered saline or Odyssey Bocking Buffer  for 60 min at room temperature and incubated overnight at 4°C with the indicated antibodies in Tris-buffered saline and 0.05% Tween 20 (TBST) containing 1% BSA. Blots were washed in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies  for 30 min at room temperature. Membranes were washed in TBST, and antigen-antibody complexes were visualized by chemiluminescence using an ECL kit (Pierce). Alternatively, immunoblots were incubated with IRDye800CW Goat Anti Mouse (H+L) or IRDye680 Goat Anti Rabbit (H+L) secondary antibodies and signal was detected with the Odyssey Infrared Imaging System (Li-COR Biotechnology).Tissues and cell extracts were prepared as described before . BrieflyTM Protein Assay. Lysates (5-6 mg) were immunoprecipitated with indicated antibodies followed by incubation with ProteinA/G plus. Samples were washed 3 times and boiled and separated onto 10% SDS-PAGE followed by an overnight transfer. Western blot analysis was performed with the indicated antibodies. Alternatively, immunoprecipitation was performed with Catch and Release® v2.0 Reversible Immunoprecipitation System (EMD Millipore) according to manufacturer's protocol, followed by immunoblotting with the indicated antibodies.Protein from cells and tissues were prepared in a buffer containing 50mM Tris (pH 7.4), 1% glycerol and 1% NP40 supplemented with protease inhibitor . Homogenates were centrifuged for 30 min at 14,000 rpm at 4°C, and supernatants were collected. Protein concentration was determined with the BCAAMPKα2 subunit fusion protein and FynT was purchased from Abnova and BPS bioscience, respectively. AMPKα2 protein (1 μg) was incubated with the recombinant FynT kinase active (1.8U) in presence of ATP and kinase buffer  and kinase reaction was performed for 1 h at 35°C. Samples were separated on 10% SDS-polyacrylamide gels and immunoblotting was performed with 4G10 monoclonal antibody.® v2.0 Reversible Immunoprecipitation System (EMD Millipore). Samples were washed three times with NP-40 lysis buffer and once with the kinase reaction buffer. Immuno-complexes were diluted 2.5 fold and 40 μl samples and 10 μl ATP solution were incubated in an ELISA plate provided. Measurement of the kinase activity was performed according to the manufacturer's protocol.Fyn activity assay was performed with Universal Tyrosine Kinase Assay kit  as described previously . BrieflyHEK293T cells were transfected with GFP-human LC3 construct. Transfected cells were washed with PBS and fixed for 10 min in PBS containing 4% PFA. Cells were imaged using a confocal fluorescence microscope .p < 0.05 using Student's t test or ANOVA.All data are presented as mean ± s.e.m. Significant data were defined as


The hormone prolactin promotes lactational differentiation of mammary epithelial cells (MECs) via its cognate receptor and the downstream JAK2-STAT5a signalling pathway. In turn this regulates transcription of milk protein genes. Prolactin signalling depends on a cross-talk with basement membrane extracellular matrix (ECM) via β1 integrins which activate both ILK and Rac1 and are required for STAT5a activation and lactational differentiation. Endocytosis is an important regulator of signalling. It can both enhance and suppress cytokine signalling, although the role of endocytosis for prolactin signalling is not known. Here we show that clathrin-mediated endocytosis is required for ECM-dependent STAT5 activation. In the presence of ECM, prolactin is internalised via a clathrin-dependent, but caveolin-independent, route. This occurs independently from JAK2 and Rac signalling, but is required for full phosphorylation and activation of STAT5. Prolactin is internalised into early endosomes, where the master early endosome regulator Rab5b promotes STAT5 phosphorylation. These data reveal a novel role for ECM-driven endocytosis in the positive regulation of cytokine signalling. In breast, joint signalling between extracellular matrix (ECM) and cytokines is critical for lactational differentiation. Whilst the mechanisms controlling signal integration are still being determined, an emerging theme is the involvement of endocytic trafficking pathways2.Cells within the tissues of multicellular organisms respond to positional signals from the extracellular matrix (ECM) and cell-cell adhesions, and to temporal signals from hormones, cytokines and growth factors3, however the mechanisms linking ECM and Prl signalling are not fully understood.Alveoli, which make milk proteins, are comprised of a layer of polarised mammary epithelial cells (MECs) surrounded by myoepithelial cells, and subtended by a laminin-rich basement membrane (LrBM). The engagement of LrBM ECM is crucial for MECs to respond to the lactation-inducing cytokine prolactin (Prl)4. STAT5a then becomes phosphorylated by JAK2 on tyrosine 694 to induce dimerisation and nuclear translocation5. There, the STAT5a dimer causes transcription of milk protein genes, such as that encoding β-casein. Despite this detailed understanding to the molecular signalling pathway, the mechanism through which class I cytokine receptors are activated by ligand , and the localisation of downstream signalling within the cell , remain enigmatic7.Prl binds to its cognate type I cytokine receptor (PrlR) at the plasma membrane (PM) of MECs to activate Janus kinase 2 (JAK2), resulting in phosphorylation of tyrosine residues on PrlR and recruitment of signal transducer and activator of transcription 5a (STAT5a)8. Endocytic trafficking can enhance signalling downstream of cytokine receptors and is implicated in the activation and nuclear translocation of STATs14. For example, mutations which retain granulocyte colony stimulating factor receptor (G-CSFR) in early endosomes of myeloid progenitor cells promote sustained STAT5 activity15. Furthermore, clathrin-mediated endocytosis (CME) is required for INFα receptor (a type II cytokine receptor) internalisation and STAT1/2 activation16. Signalling downstream of type I cytokine receptors is also facilitated by endocytosis. Caveolar endocytosis of growth hormone receptor  is implicated in STAT-mediated transcription, and JAK2 associates with GHR following internalisation, suggesting that signalling continues on endosomes18. Interleukin-5 (IL-5) receptor is internalised by CME and caveolar endocytosis, both of which are required for optimal downstream signalling via JAK219. CME has been implicated in the endocytosis and degradation of PrlR20, and in breast cancer cells can enhance downstream signalling to ERK but not STATs21.Endocytosis, mediated by clathrin-dependent and -independent mechanisms, has an important role in regulating receptor-mediated signalling both positively and negatively22, suggesting a link between endocytic regulators and Prl signalling. However, the role of endocytosis in Prl signalling in MECs has not been investigated.In MECs, Caveolin-1 (Cav1) associates with JAK2, and Cav1-null mice exhibit hyper-phosphorylation of STAT5 and precocious milk production23. Primary MECs isolated from β1-integrin conditional knockout mice do not synthesise β-casein, and β1-integrin-ILK-Rac signalling blocks the activation of SHP2, a phosphatase that inhibits JAK227. Integrins are cargoes of endocytic trafficking pathways, but can also regulate endocytosis28. For example β1-ILK signalling promotes dynamin-dependent endocytosis of apical proteins from the basolateral membrane, thereby establishing polarity29. Similarly, in keratinocytes lacking β1 integrin or ILK, caveolae are absent from the PM due to a defect in microtubule targeting and stability30.MECs interact with basement membrane ECM through β1-integrins, which are critical for Prl-stimulated PrlR-JAK2-STAT5 signallingIn this study we examine the hypothesis that LrBM impacts upon Prl signalling by controlling endocytosis and downstream signalling to STAT5. We show that in the presence of LrBM, Prl promotes STAT5 phosphorylation in a clathrin-dependent, caveolin-independent manner that requires Rab5b.27, and this is the only cell line model which robustly responds to ECM engagement and Prl in the way that primary cells do. When cultured on plastic dishes, they show little basal STAT5 phosphorylation, and only a modest increase upon stimulation with Prl , there were only low levels of STAT5 phosphorylation in response to Prl and ECM Fig. . EndocytInhibition of CME with Pitstop-2 also prevented Prl/ECM-induced STAT5 activation to levels similar to those seen with only Prl, without ECM Fig. . Equival31, we also targeted the major clathrin adaptor AP2, depleting the AP2M1 subunit by RNAi. AP2M1 knockdown significantly reduced STAT5 phosphorylation in response to LrBM and Prl  was used to deplete clathrin heavy chain (CHC). Efficient CHC knockdown, using a SmartPool designed to reduce off-target effects, significantly reduced the phosphorylation of STAT5 in response to Prl stimulation Fig. . IndividThese results show that, clathrin- but not caveolar-, mediated endocytosis is required for Prl/ECM signalling and STAT5 phosphorylation.Because CME is implicated in the endocytosis of cargoes that include cytokine receptors, we determined whether Prl was internalised during lactational differentiation. We monitored Prl internalisation as a surrogate for PrlR, because of the lack of reagents available to detect mouse PrlR. Cells cultured in the presence or absence of LrBM overlay were stimulated with Prl, and acid washed to remove surface Prl, leaving only internalised Prl Fig. . Prl labUsing siRNA-mediated knockdown, we investigated the route of endocytic entry of Prl. In accordance with the requirement for CME in STAT5 activation, knockdown of CHC using two individual oligos significantly reduced Prl internalisation Fig. . BecauseThese results show that LrBM ECM induces Prl internalisation via a clathrin-dependent, but caveolin-independent route.32, we elucidated other mechanisms that could be involved with Prl internalisation. However, inhibition of JAK2 with its specific inhibitor AZD-1480 had no effect on Prl internalisation in cells cultured with ECM overlay, despite a 60% reduction in the phosphorylation of STAT5 on Y694 . Together these drive STAT5 phosphorylation and activation.To image internalised Prl, we fluorescently labelled Prl with Alexafluor-594. We confirmed that labelled Prl (i.e. Prl-594) could induce STAT5 phosphorylation to the same extent as unlabelled-Prl Fig. . MECs cu8 (consistent with the peak in STAT5 phosphorylation and activation), we investigated the localisation of internalised Prl with respect to the early endosome marker, early endosome antigen 1 (EEA1). Cells showed little Prl endocytosis in the absence of ECM or at 4 °C, and little overlap between any internal Prl structures and EEA1. However, ECM promoted significant colocalisation of internalised Prl with EEA1-postive early endosomes at physiological temperature  does not completely abrogate STAT5 phosphorylation, suggesting that while clathrin-dependent endocytosis is of important, clathrin-independent pathways could also play a role.GHR is closely related to PrlR, and caveolar endocytosis has been implicated in its downstream signalling36. In this cancer context, JAK2 activation is implicated in the control of internalisation, but JAK2-STAT5 signalling does not require endocytosis. Moreover, whilst Prl elicits JAK2-STAT5 signalling and ERK signalling in cancer cells, normal MECs do not produce an ERK response, suggesting that important differences in the regulatory mechanisms controlling PrlR signalling exist in different contexts. Our results therefore reveal distinctions between Prl internalisation and signalling in tumorigenic versus non-tumorigenic cells, which could be exploited for therapeutic benefit.Interestingly in cancer cell lines, PrlR signalling and internalisation is not dependent upon ECM, and CME is involved in down-regulation of PrlR38. Integrin signalling can also control endocytosis and signalling of PDGF and BMP receptors40. The Rac signalling axis is not required for Prl endocytosis, and likely acts via phosphatases that control JAK225, suggesting that ECM impacts on Prl endocytosis via an alternate route (perhaps through β1-integrins or other LrBM receptors). For example a Rac1-independent, β1-ILK-microtubule pathway controls tight junction protein internalisation in polarising MECs29, and in keratinocytes a similar pathway controls plasma membrane distribution of caveolae30. Alternate integrin-dependent signalling pathways may therefore regulate Prl endocytosis downstream of LrBM ECM.Integrins elicit a plethora of signalling responses upon engagement of their ECM ligands, and we and others have previously shown that they are involved in a wide variety of signalling responses initiated by growth factorsIn summary, this study demonstrates that basement membrane ECM influences Prl internalisation through a clathrin-mediated endocytic route. Delivery to an early endosomal platform promotes downstream signalling to STAT5, the master transcriptional regulator of lactational differentiation. We deliver critical new insight into the ECM-mediated regulation of hormonal signalling in the mammary gland, and reveal the Prl/PrlR/JAK2/STAT5 signalling axis as a beneficiary of endosomal signalling.Endocytosis is central for Prl/ECM signalling.Clathrin- but not caveolar-, mediated endocytosis is required for Prl/ECM signalling.Internalisation of prolactin into early endosomes via a Rab5b pathway is required for STAT5 phosphorylation.Prl and ECM are both required for tissue-specific gene expression in MECs, and we reveal that their signalling pathways communicate via endocytosis and early endosomes.41 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) foetal calf serum (FCS), 5 μg/ml insulin and antibiotics. Two rounds of knockdown were performed to achieve protein suppression: 100 pmol siRNA  was used to transfect a single well of a six well plate of EpH4 cells and cells were reseeded 24 hours later for a second round of knockdown at 48 hours, and experiments were performed and/or cells harvested at 72 hours. Primary MECs were isolated and cultured as described25.Low passage EpH4 cellsFor differentiation assays, EpH4 cells were cultured in DMEM-F12 supplemented with 5 µg/ml insulin, 1  μg/ml hydrocortisone and antibiotics overnight with 2% Matrigel  added to the culture medium (LrBM overlay) as required. Cells were stimulated with 3 µg/ml sterile-filtered sheep pituitary Prl  for 15 minutes. EpH4 cells were treated with inhibitors  or an equal volume of DMSO (where appropriate) diluted in differentiation medium for 15 minutes at 37 °C before addition of Prl.Sample buffer  glycerol, 4% (w/v) SDS, 0.004% (w/v) bromophenol blue) with 10% (v/v) 2-mercaptoethanol (Sigma-Aldrich) was added to samples, which were then incubated at 95 °C for 10 minutes. The samples were run alongside Precision Plus Protein All Blue Standards (Bio-Rad Laboratories) on 4–12% SDS polyacrylamide gels (Life Technologies) at 100 V. The gels were transferred to nitrocellulose membrane at 30 V for 75 minutes. Membranes were incubated with a casein-based blocking buffer (Sigma-Aldrich) or 5% BSA in Tris-buffered saline with 0.1% Tween-20  for 45 minutes. The membranes were incubated with primary antibodies or Alexa Fluor 680–conjugated streptavidin  in blocking buffer overnight at 4 °C.Proteins levels were analysed by western blotting using the following primary antibodies: pSTAT5 ; pERK1/2 , ERK1/2 , ERK2 (sc-154) STAT5a (sc-1081) and Rab5b ; Prl ; Caveolin 1 ; Clathrin heavy chain (ab172958), α-tubulin  and AP2M1 . Secondary antibodies used were: anti-rabbit Alexa Fluor 680 (A-21109), anti-mouse Alexa Fluor 680 (A-21058), and anti-rat Alexa Fluor 680 ; anti-mouse DyLight 800 ; anti-rat Alexa Fluor 800 . Fluorescent secondary antibodies were detected by Odyssey scanner (Li-Cor), and intensity of bands quantified using ImageJ.3 and incubated with 1 mg Alexa Fluor 488 or 594 NHS Ester  for 1 h at room temperature. The labelled Prl was then dialysed into PBS.10 μg/ml Prl  was dialysed into 0.1 M NaHCOEpH4 cells cultured with a BM overlay were stimulated with 3 μg/ml Prl or Alexa Fluor-labelled Prl for 15 minutes at 37 °C to allow internalisation or at 4 °C to prevent internalisation. The cells were washed three times for three minutes each with PBS  or with PBS-HCl pH 4.0 to remove the surface Prl . The cells were then washed three times with PBS and lysed or fixed.Cells were fixed in 4% paraformaldehyde (PFA) before permeabilisation and blocked with 1% BSA in PBS for 30 minutes. Primary antibody (EEA1 #3230 CST) was diluted PBS/1% BSA and detected with fluorophore-conjugated secondary antibodies, actin using fluorophore labelled Phalloidin (Invitrogen) and nuclei with DAPI. Z-stacks were obtained using the 64x objective of a Leica TCS SP5 inverted confocal microscope. Images were processed using ImageJ.For quantification of internalisation, background was subtracted using a radius of 100 pixels, and the threshold set to 100 pixels to produce a binary (black and white) image. The number of particles was counted automatically using ImageJ, and normalised to the area selected. For quantification of co-localisation, the background was subtracted and threshold set as above, and an image of the overlap between the two binary images was produced using Image Calculator. The number of particles was counted automatically using ImageJ, and calculated as a percentage of the number of particles in the Prl image.Statistical analysis was performed using GraphPad Prism. Analysis of variance (ANOVA) and post-hoc analysis was used to assess significance of data. Data are presented in box and whisker plots (Tukey) or bar graphs as mean −/+ standard error of the mean (SEM).The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information


Lipid raft is an important element for the cellular entry of some viruses, including coronavirus infectious bronchitis virus (IBV). However, the exact role of lipid rafts in the cellular membrane during the entry of IBV into host cells is still unknown. In this study, we biochemically fractionated IBV-infected cells via sucrose density gradient centrifugation after depleting plasma membrane cholesterol with methyl-β-cyclodextrin or Mevastatin. Our results demonstrated that unlike IBV non-structural proteins, IBV structural proteins co-localized with lipid raft marker caveolin-1. Infectivity assay results of Vero cells illustrated that the drug-induced disruption of lipid rafts significantly suppressed IBV infection. Further studies revealed that lipid rafts were not required for IBV genome replication or virion release at later stages. However, the drug-mediated depletion of lipid rafts in Vero cells before IBV attachment significantly reduced the expression of viral structural proteins, suggesting that drug treatment impaired the attachment of IBV to the cell surface. Our results indicated that lipid rafts serve as attachment factors during the early stages of IBV infection, especially during the attachment stage. Coronaviridae family, which includes many human and animal pathogens of global concern, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome virus (MERS-CoV), and mouse hepatitis virus(MHV). Total RNA was extracted, and the cDNA template was synthesized with PrimeScriptVero cell monolayers that adhered to coverslips were transfected with plasmids encoding IBV E protein and were then incubated at 37°C for 8, 16, and 24 h. The cells were fixed with 4% paraformaldehyde (PFA) and treated with 0.1% Triton X-100. The cells were incubated with anti-IBV E protein or GM1 antibodies at 37°C for 1 h. Immunofluorescence confocal microscopy images were captured with LSM510 META microscope .t test. Values of p < 0.05 were considered statistically significant.The relative intensities of protein bands were analyzed with ImageJ software. Data were presented as mean ± standard deviation (SD). Results from different groups were compared by the Student’s Beaudette strain, and membrane flotation was performed by sucrose density gradient centrifugation after depleting plasma membrane cholesterol with MβCD. As shown in Depending on their membrane locations, lipid rafts are mainly involved in viral entry and release. This study investigated the relationship between lipid rafts and IBV infection. Vero cells were infected with IBV To further investigate the association of IBV structural proteins with lipid rafts, IBV E protein was overexpressed in Vero cells. The total cell lysates in sucrose gradients were analyzed by membrane flotation assay. The flotation gradient fractionations were monitored by Western blot analysis with specific antibodies against IBV E protein and assessed by dot blot analysis with the lipid raft marker ganglioside GM1 antibody. As shown in To directly observe the dynamic changes of co-localization and to verify the results of the flotation gradients, IFA was performed to investigate the interaction between IBV E protein and lipid rafts via confocal microscopy. Vero cells overexpressing IBV E protein were harvested for IFA detection at 8, 16, and 24 h post-transfection. IFA staining was performed with anti-IBV E and FITC-conjugated GM1 antibodies. As shown in The co-translocation of IBV E protein with lipid rafts on the cellular membrane suggested that lipid rafts are involved in IBV infection. To investigate whether lipid rafts are involved in IBV release, cell supernatants and total lysates after IBV infection with or without MβCD treatment were collected to detect viral proteins with specific antibodies. As shown in Our previous study found that IBV localized with GM1 protein at different infection stages. To determine the effect of lipid rafts on IBV genome replication, Vero cells treated with MβCD or mevastatin after IBV infection were collected at different time points to evaluate the level of viral RNA copies. IBV RNA copies were determined by semi-quantitative RT-PCR with specific primers. The RNA level represents the IBV genome copies after IBV replication. Lipid rafts are required for IBV infection, but are not involved in viral genome replication and particle release. To further study the participation of lipid rafts in the early stages of viral infection, as well as to identify the specific involvement of lipid rafts in viral endocytosis, lipid rafts were inactivated with MβCD. To determine the role of lipid rafts in IBV attachment, host cells were pre-treated with MβCD for 1 h at 4°C, incubated with IBV for 1 h at 4°C, and then incubated at 37°C for 2, 4, 6, and 8 h. To determine the role of lipid rafts in IBV entry, host cells were pre-incubated with IBV for 1 h at 4°C, treated with MβCD for 1 h at 4°C, and then cultured at 37°C for 2, 4, 6, and 8 h. Vero cells without the MβCD treatment were set as the control. To assess viral replication, IBV N protein was subjected to Western blot analysis. As shown in Viral infection comprises several steps, and viral attachment to host cells is the first step that determines tissue tropism, host specificity, and viral pathogenicity . AlthougGamma- coronavirus. It is an enveloped, positive-strand RNA virus. Its envelope contains the major attachment spike protein (S), the membrane protein (M), and the minor envelope protein (E). The spikes are composed of S protein trimers, which are necessary for viral attachment and subsequent fusion of viral and cellular membranes. Therefore, the IBV S protein and other structural proteins likely interact with lipid rafts. These direct or indirect interactions can affect the infection cycle of the virus. To test this hypothesis, we performed several experiments to determine the function of lipid rafts in IBV infection. Our data showed that IBV structural proteins co-localized with lipid rafts in the plasma membrane and that IBV structural proteins remain localized with the GM1 protein. In addition, the drug-induced disruption of lipid rafts in Vero cells inhibited IBV infection, which likely occurred because lipid rafts are involved in viral attachment. These results indicated that lipid rafts in the cellular surface may mediate viral adhesion to facilitate IBV endocytosis.IBV is a prototype avian coronavirus that belongs to the genus Although IBV receptor(s) remain unidentified, the ass


The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5’TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5’TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5’ and 3’UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5’UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3’UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.DOI:http://dx.doi.org/10.7554/eLife.25237.001 Mechanistic target of rapamycin complex 1 (mTORC1) functions as a positive regulator of translation initiation and protein synthesis to promote cell growth and proliferation . Short-tThe 4EBP family of proteins have been proposed to play a key role in suppressing the translation of PES-containing mRNAs . HoweverIn this report, we investigated the molecular mechanisms of LARP1 function in the mTORC1-mediated translation of RP mRNAs. We first identified mRNAs and sequences directly bound by endogenous LARP1 in vivo under normal growing and mTORC1-inhibited conditions using photoactivatable ribonucleoside–enhanced crosslinking and immunoprecipitation (PAR-CLIP) . As predWhile several recent studies have indicated that LARP1 associates with 5’TOP mRNAs through their TOP sequences or polyA tails , the comGene ontology (GO) term enrichment analysis of genes bound by LARP1 in growing conditions revealed enrichment for terms related to translation. LARP1 was bound to 137 translation-related genes, including 42 genes encoding ribosomal proteins (RP), as well as genes involved in cellular differentiation and development . Under mTo identify where LARP1 binds across a transcript, we summed LARP1 binding coverage across the 5’UTR, CDS, and 3’UTR regions of its targets, which were separated into non-translation-related (non-TR) genes, translation-related (TR) genes, and the subset of TR genes encoding RPs . Strikint-test: p=1.4e-15) and mTOR-inactive (p=1.2e-18) conditions , indicating that pyrimidine richness at LARP1-interacting regions of 5’UTRs is correlated with strong decreases in TE upon mTOR inactivation. Together, these results suggest that the relationship between LARP1 binding and decreased TE is conserved from mouse to human.The relationship between LARP1 binding and decreases in translational efficiency (TE) upon mTOR inactivation are paralleled in mouse embryonic fibroblasts. We obtained measurements of changes in mouse transcript TE upon treatment of cells with Torin1 . Thirty-Of the 88 annotated human ribosomal proteins, mRNAs encoding 84 were expressed in our mRNA-seq libraries under both conditions. Summing LARP1 binding site coverage of these genes confirms increased LARP1 binding at 5’UTRs and slightly decreased LARP1 binding at CDS and 3’UTR upon mTOR inactivation . We veriTo investigate the mechanisms by which site-specific LARP1 interaction with RP mRNAs is regulated by the activity of mTOR, we examined the roles of post-translational modifications of LARP1 regulated by mTOR activity. Previous genome-wide phospho-mass spectrometry analyses showed that LARP1 is a highly phosphorylated protein and has Torin1-sensitive phosphorylation sites , suggestUnder the steady state growth condition, inhibition of S6K1 with rapamycin or PF 4708671, a S6K inhibitor, equally and significantly decreased LARP1 phosphorylation, and Torin1 and MK-2206 further blocked these phosphorylations. Taken together, these observations indicate that Akt is a physiologically relevant primary kinase for S770/S979 phosphorylation of LARP1 especially under acute stimulatory conditions. Under steady state conditions, S6K plays a major role while Akt also partially contributes to the phosphorylation of LARP1 .Our PAR-CLIP data indicated that while LARP1 primarily interacts with 3’UTRs of RP mRNAs under growth conditions, it also binds to 5’UTRs of RP mRNAs upon mTOR inactivation . To inveImportantly, phosphorylation of LARP1 (S689/T692 and S770/S979) identified in To examine the configuration of LARP1 binding to the 5’UTR and 3’UTR of RpL32 mRNA, we performed PAR-CLIP assays using endogenous LARP1 with the reporter mRNAs containing both the 5’ and 3’UTR of RpL32 mRNA. Interestingly, under cross-linking conditions, LARP1 can bind the wild type (5’/3’) or the 5’UTR-mutated (5’conv/3’) reporter mRNA equally well in the presence or absence of Torin1 treatment, respectively . These oTo examine the significance of LARP1 interaction with the 5’ or 3’UTR in the translation of RpL32 mRNA, we measured luciferase production from the indicated reporters . LuciferTo stimulate the translation of 5’TOP mRNAs, previous studies have proposed that LARP1 directly interacts with initiation factors and polyA-binding protein (PABP) in response to mTORC1 activity . In contLARP1 also co-IPs PABP1, a polyA tail and eIF4G1 binding protein, as previously reported . HoweverImportantly, the activity of mTORC1 plays a key role in LARP1’s binding to mTORC1 but not to PABP1. LARP1 failed to interact with mTORC1 but maintained its interaction with PABP1 subjected to mTOR inhibitors, suggesting that LARP1 associates with mRNAs regardless of cellular mTOR activity , consistTo investigate the mechanisms underlying the formation of the LARP1-mTORC1 complex, we determined regions of LARP1 necessary to associate with mTORC1. Interestingly, serial truncations of the LARP1 carboxyl terminus revealed that the DM15 motif and an N-terminal region adjacent to the DM15 motif , where tTo examine the role of LARP1 phosphorylation in the regulation of interaction between LARP1 and mTORC1, we first assessed mTORC1 binding with a series of phospho-defective LARP1 mutants . While the replacement of LARP1 Akt/S6K1 phosphorylation sites with alanines only slightly reduced LARP1 interaction with mTORC1, mutations in the LARP1 mTOR phosphorylation sites largely abrogated its interaction with mTORC1 . ImportaTo further investigate the roles of LARP1 in mTORC1-dependet phosphorylation of its substrates, endogenous LARP1 was knocked down in mammalian cells with varying degrees of insulin sensitivity. In HEK293T cells (insulin insensitive), ablation of LARP1 slightly decreased phosphorylation of S6K1, S6, and 4EBP1 compared to those in control cells under normal growth culture conditions . HoweverImmunostaining of LARP1 and phosphorylated forms of 4EBP1 and S6 revealed that endogenous LARP1 was predominantly expressed in the cytoplasm where 4EBP1 and S6 phosphorylation occurs in response to growth factor and nutrient stimulation . In addi7GTP pull down assays showed that the interaction of 4EBP1 with eIF4E was increased in LARP1 knockdown cells  LARP1 is a direct substrate of mTOR and S6K1/Akt; (2) the phosphorylation of LARP1 by the mTORC1 pathway triggers the dissociation of LARP1 from the 5’UTR of the RP mRNAs; (3) concomitantly, phosphorylation of LARP1 induces its stable interaction with mTORC1 and recruits mTORC1 to the 3’UTR of RP mRNAs; (4) LARP1 facilitates mTORC1-dependent phosphorylation of 4EBP1 and S6K1, a process essential for inducing translation initiation of RP mRNAs; and (5) while LARP1 is necessary for the blockade of RP mRNA translation in response to starvation, it is required for efficient translation elongation of RP mRNAs. Thus, LARP1 is a unique mTORC1 substrate and regulator, and functions as a molecular switch for turning off or on the translation of RP mRNAs .Our data reveal that LARP1 mainly associates with the 3’UTR of mRNAs including RP mRNAs under high mTOR activity conditions, while it also directly binds to PES regions of RP 5’UTRs when mTOR activity is inhibited. Intriguingly, recent reports demonstrate that recombinant LARP1 binds to the TOP sequence of RpL32 and RpS6 in vitro . HoweverImportantly, our data also demonstrate that phosphorylated LARP1 serves as a nucleation site onto which mTORC1 can associate with translationally-competent mRNAs. mTORC1-dependent LARP1 phosphorylation plays a key role in scaffolding mTORC1 on LARP1-interacting RpL32 mRNA . FurtherGiven that mTORC1-dependent LARP1 phosphorylation to scaffold mTORC1 is important for mTORC1 phosphorylation of S6K1, mTORC1 may phosphorylate LARP1 prior to S6K1-dependent LARP1 phosphorylation. In support of this model, formation of the LARP1-mTORC1 complex precedes full S6K1 activity/phosphorylation by growth factor/nutrient stimulation . AlternaTcherkezian et al. recently proposed that upon mTOR activation, LARP1 associates with the initiation complex as well as polysomes through PABP . In addiCircularization of linear mRNAs through the interaction between the 5’Cap-binding translation initiation factors and PABPs has been recognized as the active conformation for their translation . mTORC1-AB_2105622 for western blotting and immunostaining), Raptor , pT389 S6K1 , S6K , pS473 Akt , Akt , phospho-Akt Substrate (RXXS/T) , pT37/46 4EBP1 , 4EBP1 , S6 , pS235/236 S6 , pS240/244 S6 , RpL13a , PABP1 , mLST8 , PRAS40 , pULK1 , and ULK1  from Cell Signaling Technology; antibodies to LARP1 , Rictor , eIF4G , eIF3D , and eIF3B  from Bethyl Laboratories; antibodies to eIF4E  and mTOR/FRAP  from Santa Cruz Biotechnology; antibodies to β-Actin , and Flag M2 antibodies  from Sigma-Aldrich; antibodies to HA tag , and Myc tag  from Covance; antibody to LAMP2  from the Developmental Studies Hybridoma Bank at the University of Iowa; HRP-conjugated mouse secondary antibody  and HRP-conjugate rabbit secondary antibody  from GE Healthcare; Alexa fluor 594 goat anti-rabbit IgG  and Alexa fluor 488 goat anti-mouse IgG  from Invitrogen.Antibodies were purchased from the following sources: Antibodies to mTOR  cells  were culCells were harvested with CHAPS lysis buffer  by incubating on ice for 15 min. The soluble fractions were isolated by centrifugation at 14,000 rpm for 15 min at 4°C. For immunoprecipitation, 1 µg of antibodies was added and incubated with gentle rocking for 3–6 hr at 4°C. 20 µl of 50% slurry of protein G sepharose  was added and incubated for additional 1 hr. Immunoprecipitates were washed with CHAPS lysis buffer for five times and then denatured at 100°C for 5 min in 1X SDS sample buffer. For RNase A treatment, lysates were incubated with 20 μg of RNase A (Affymetrix) for 1 hr at room temperature with gentle rocking. After spinning at 14,000 rpm at 4°C for 15 min to remove protein aggregates, remaining supernatant was applied for further analysis. For immunostaining, cells on round cover slips (Fisher Scientific) were fixed with 4% paraformaldehyde for 20 min and washed with PBS three times. Fixed cells were permeabilized with 0.2% TX-100 for 10 min at room temperature and blocked in 2% BSA in PBS for 1 hr. After washing with PBS three times, cells were incubated with indicated primary antibodies for three hours at room temperature and washed with PBS three times. Cells were incubated with Alexa fluor 488/594 goat anti-mouse/rabbit IgG for 1 hr at room temperature. Cells were washed with PBS three times and once with water. The cover slips were mounted on the glass slides using ProLong Gold antifade reagent with DAPI (Life technologies) and imaged with 63X oil-immersion objective by using a Leica TCS SP5 confocal microscope.2. Cross-linked cell pellets were collected by scraping and lysed with 600 μl PAR-CLIP lysis buffer  on ice for 10 min. To remove DNA, 10 μl of RQ1 DNAse was added into each tube and tubes were incubated at 37°C for 10 min with gentle rocking. Afterward, 1 U/µl of RNase T1 (Fermentas) was added and the lysates were incubated at room temperature for 15 min with gentle rocking. Lysates were spun at 4°C for 10 min at 14,000 rpm. The soluble fractions were incubated with LARP1 antibody-Dynabeads Protein A for 1 hr at 4°C. Immunoprecipitates were collected on a magnetic stand and were then washed three times with IP-wash buffer . Immunoprecipitates were resuspended in 40 μl of IP wash buffer containing 50 U/μl RNase T1 (Fermentas) and incubated at room temperature with gentle rocking for 15 min followed by incubation on ice for 5 min. Immunoprecipitates were washed with high-salt wash buffer , three times with the PAR-CLIP lysis buffer, and twice with high-salt wash buffer  followed by washing twice with PNK (polynucleotide kinase) buffer . For visualization of crosslinked RNAs, immunoprecipitates were incubated in 40 μl of the PNK mixture  for 30 min at 37°C. Labeled immunoprecipitates were washed three times with PNK buffer and resuspended with 30 μl of 2X NuPAGE LDS sample buffer (15 μl of 1 X PNK with 15 μl of Novex LDS sample buffer). Denatured samples were resolved in 4–12% NuPAGE Bis-Tris gel and transferred to nitrocellulose membrane at 30 V for 1 hr using NuPAGE transfer buffer. The membrane was exposed to X-ray film at −80°C for 4 hr to visualize crosslinked RNAs. Molecular biology procedures for cloning LARP1-bound RNA fragments was described previously (To label cells with 4-thiouridine (4-SU), cells were seeded in two 15 cm plates to grow overnight to reach 70% confluency. On the next day, cells were incubated with 100 μM 4-SU for 14 hr. After washing with cold PBS, cells were irradiated in the CL-1000 ultraviolet Crosslinker (UVP) on ice with 150 mJ/cmeviously .2 = 0.99975), so transcript RPKM values were averaged from the two libraries. PAR-CLIP-seq reads with 0–2 T-to-C mismatches were clustered into peaks with at least one overlapping nucleotide. Clusters were smoothed with a Gaussian smoothing technique as described in  for 10 min on ice. The soluble fractions isolated by centrifugation were transferred into new tubes and the Click-IT reaction was performed in 1 X Click-IT reaction buffer according to the manufacturer’s guide. The Click-IT reaction was stopped by addition of SDS sample buffer and denatured by boiling. Samples were resolved by SDS-PAGE and transferred to PVDF membrane for immunoblot analysis. Labeled proteins were probed with Avidin-HRP (BioRad) and membranes were visualized by ECL on x-ray film. Intensity of signal was quantified by Image J.Cells were seeded in 6-well plates a day before labeling to reach 70% confluency on the day of labeling. Cells were washed twice with warm methionine/cysteine free DMEM with 1 x L-glutamine (Invitrogen) and incubated with methionine/cysteine free DMEM with 1 X L-glutamine and 10% dialyzed FBS (Invitrogen) for 1 hr. Cells were labeled with 25 μM Click-IT AHA  for 4 hr in a CO2, 1.5 mM KCl, 0.3% CHAPS, 20 mM glycerophosphate, 10 mM NaF, 10 mM sodium pyrophosphate and EDTA-free protease inhibitor) containing 100 µg/mL CHX, 2 mM DTT, and 200 U/ml RNasin (Promega) by vortexing for 5 s. Supernatants were collected by centrifugation for 10 min at 14,000 rpm, 4°C, and the A260 of lysates was measured to normalize RNA levels. Lysates of 30 O.D.260 were loaded on 10–50% sucrose density gradients  and centrifuged at 35,000 rpm for 3 hr at 4°C. Gradients were fractionated and the optical density at 254 nm was recorded using an ISCO fractionator (Teledyne ISCO). For immunoblotting, the fractions were concentrated by Vivaspin concentrator (Sartorius). For quantitative PCR normalization, 5 ng of luciferase control mRNA (Promega) was added to each fraction. RNA was extracted using TRIzol and cDNA was synthesized by SuperScript III First-Strand Synthesis System (Invitrogen) following the manufacturer’s instructions.Prior to lysis, cells were treated with 100 µg/mL cycloheximide (CHX) for 5 min and washed with cold PBS containing 100 µg/mL CHX. Cells were harvested in 1 ml of cold PBS containing 100 µg/mL CHX. Cell pellets were resuspended in hypotonic buffer  using 2 µg of GST-LARP1 fragment (654–731 aa) purified from bacteria. For 4EBP1 phosphorylation, 200 ng of GST-4EBP1 (full length) was used as a substrate for the kinase reaction without using 32PγATP, and the phosphorylation of 4EBP1 was detected by the indicated phospho-specific 4EBP1 antibody. For AKT and S6K1 kinase assays, AKT-HA, AKT-HA KD, HA-S6K1 F5A 3DE (constitutive active), or HA-S6K1 KD was immunoprecipitated from HEK293T cells and kinase reactions were performed in the AKT/S6K1 kinase reaction buffer  using 2 µg of GST-LARP1 fragments (722–822 aa or 929–1019 aa).For mTOR in vitro kinase assays, HEK293T cells were lysed with CHAPS lysis buffer and endogenous mTOR, Flag-mTOR Wt (wild type), or Flag-mTOR KD (kinase defective) was immunoprecipitated and subjected to in vitro kinase assays. For LARP1 phosphorylation, the kinase reaction was performed in kinase reaction buffer  against LARP1 or eIF4G1 were cloned into the lentivirus plasmid pLKO.1-puro. HEK293T cells were transfected with pLKO.1-puro cloned with shRNA, psPAX2 (packaging plasmid) and pMD2 (envelope plasmid) using the calcium phosphate method. Lentivirus-containing supernatants were collected and spun at 23,000 rpm for 90 min. Virus pellets were resuspended with Opti-MEM (Invitrogen). Cells were infected for 24 hr and selected with 2 μg/ml puromycin for additional 24 hr. 72 hr post-infection, stably knocked-down cells were harvested and processed for further analysis.shRNA scramble (control):Sense:5’CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTC3’Antisense:5’AATTGAAAAACCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG3’ shRNA LARP1 UTR-2:Sense:5’CCGGGGTGAGGACTTCATCTCAACACTCGAGTGTTGAGATGAAGTCCTCACCTTTTTC3’Antisense:5’AATTGAAAAAGGTGAGGACTTCATCTCAACACTCGAGTGTTGAGATGAAGTCCTCACC3’ shRNA LARP1-3Sense:5’CCGGGCCAGTCTCAGGAGATGAACACTCGAGTGTTCATCTCCTGAGACTGGCTTTTTC3’Antisense:5’AATGAAAAAGCCAGTCTCAGGAGATGAACACTCGAGTGTTCATCTCCTGAGACTGGC3’ shRNA eIF4G1-1Sense:5’CCGGGGATCCCACTAGACTACAAGGCTCGAGCCTTGTAGTCTAGTGGGATCCTTTTTC3’Antisense:5’AATTGAAAAAGGATCCCACTAGACTACAAGGCTCGAGCCTTGTAGTCTAGTGGGATCC3’RpS6:Forward: 5’TGTCCGCCTGCTACTGAGTAA3’Reverse: 5’GCAACCACGAACTGATTTTCTC3’RpS3A:Forward: 5’AGGGTCGTGTGTTTGAAGTGA3’Reverse: 5’CATGGAAGTTAGTCAGGCAGTTT3’RpS18:Forward: 5’GCGGGAGAACTCACTGAGG3’Reverse: 5’CGTGGATTCTGCATAATGGTGAT3’RpL26:Forward: 5’GACTTCCGACCGAAGCAAGAA3’Reverse: 5’TGCACCCGTTCAATGTAGATAAC3’RpL32:Forward: 5’GCCCAAGATCGTCAAAAAGAGA3’Reverse: 5’TCCGCCAGTTACGCTTAATTT3’Actin:Forward: 5’CATGTACGTTGCTATCCAGGC3’Reverse: 5’CTCCTTAATGTCACGCACGAT3’GAPDH:Forward: 5’TTGCCATCAACGACCCCTTC3’Reverse: 5’TTGTCATGGATGACCTTGGC3’Firefly luciferase:Forward: 5’CTCACTGAGACTACATCAGC3’Reverse: 5’TCCAGATCCACAACCTTCGC3’GGTAGtccttctcggcatc3’Forward: 5’gcctacggaggtggcagCTACCctgccacctccgtaggc3’Reverse: 5’gatgccgagaaggaData are representative of at least two independent experiments. All values were given as means±SEM from three independent biological replicates. Comparisons were performed using Student’s t-test or one-way factorial ANOVA followed by Bonferroni’s post-hoc analysis. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Tony Hunter (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this elegant study, the authors characterize the role of LARP1 in the regulation of mRNA translation, specifically assessing TOP mRNAs. The role of LARP1 in the translation of TOP mRNAs has been a contentious issue, and the authors bring novel data suggesting a dual function for LARP1, which they describe as a phosphorylation-sensitive molecular switch for mRNA translation and ribosome biogenesis. Previous reports have shown both positive and negative functions for LARP1 in TOP mRNA translation. Hong et al. suggest that LARP1 binds the 5'UTR of translation-related mRNAs to repress their translation during nutrient starvation. However, LARP1 phosphorylation by mTOR/S6K decreases this binding to promote 3'UTR binding and enhance translation. They show that LARP1 acts as an mTORC1 scaffold to facilitate mTORC1-dependent phosphorylation of S6K and 4E-BP1. Together, these data suggest that LARP1 functions as a molecular switch that senses mTORC1 activity and act as an effector.This manuscript provides very insightful results that increase our understanding of the function of LARP1.Some modifications and/or clarifications would be required prior to publication:Essential revisions:1) Although the manuscript is well done the main conclusion that LARP1 inhibits or facilitates translation depending on where it binds on the mRNA has not been fully demonstrated. The experiments shown in 2) The authors repeatedly state that LARP1 is a substrate for both S6K1 and Akt. However, Akt as a kinase for LARP1 is only shown by in vitro kinase assays and overexpression of a constitutively active Akt in cells – both are artifact-prone. In fact, Rap or PP242 abolishes pS979-LARP1 in cells , which s3) It is not clear why the authors used Torin instead of rapamycin in the reporter assays , and tot4) What is the proposed mechanism by which LARP1 phosphorylation decreases 5'UTR binding and increases mTORC1 and 3'UTR binding? Have the authors tested phospho-mimetic mutants? Related to this point: The model in  Essential revisions:1) Although the manuscript is well done the main conclusion that LARP1 inhibits or facilitates translation depending on where it binds on the mRNA has not been fully demonstrated. The experiments shown in In response to the reviewer’s suggestions, we examined the roles of LARP1 phosphorylation on its mRNA binding and in the regulation of mRNA translation using the wild type or mutant RpL32 mRNA reporters containing both 5’ and 3’ UTRs. Although our RIP assays using either the 5’ or 3’ UTR reporter of RpL32 mRNA showed dynamic changes in the affinity for LARP1 binding , and E, resist or Myc-LARP1resist 4A mutant was co-transfected with the indicated reporters in HEK293T cells. Levels of luciferase activity were monitored under growth cell culture conditions. As performed in the original To further address the critical point raised by the reviewer, we performed luciferase assays using the RpL32 reporter, which contains full length 5’/3’ or 5’conv/3’ UTRs in cells exclusively expressing the wild type or 4A LARP1 mutant. For this experiment, endogenous LARP1 was first ablated by shRNAs, and the shRNA-resistant Myc-LARP1resist has some advantages in stimulating translation of the 5’conv/3’ reporter compared to the wild type 5’/3’ reporter. In contrast, the phopho-defective Myc- LARP1resist 4A mutant significantly and equally inhibits the translation of both reporters, regardless of the existence (5’/3’) or absence (5’conv/3’) of the LARP1 binding site in the 5’UTR. These observations indicate that the dissociation of LARP1 from the PES motif in the 5’UTR and the phosphorylation of LARP1, which enhances both LARP1 binding to the 3’UTR and to mTORC1, are important processes in stimulating RpL32 mRNA translation. Note that the translation of the 5’conv/3’ mutant reporter was slightly, but significantly, enhanced compared to the 5’/3’ wild type reporter in cells expressing ectopic Myc-LARP1resist, which was not seen in the cells expressing just endogenous LARP1 (The data demonstrated that phosphorylatable wild type Myc-LARP1us LARP1 . We spec2) The authors repeatedly state that LARP1 is a substrate for both S6K1 and Akt. However, Akt as a kinase for LARP1 is only shown by in vitro kinase assays and overexpression of a constitutively active Akt in cells – both are artifact-prone. In fact, Rap or PP242 abolishes pS979-LARP1 in cells , which sThe reviewer raises an important question. Our original data suggested that Akt and S6K1 may function redundantly to phosphorylate the S770/S979 sites of LARP1 that are required for its dissociation from the 5’UTR and subsequent tight association with the 3’UTR of RpL32 mRNA. We showed that mTORC1 associates with the 3’UTR of RpL32 mRNA in a manner dependent on phosphorylated LARP1  and propTo demonstrate that Akt is responsible for phosphorylating S770/S979 of LARP1 in a physiological in vivo context, we examined the phosphorylation status of LARP1 under acutely stimulated  or steady state growth conditions with or without the addition of a specific Akt inhibitor, MK-2206.As shown in the left panel of the new Under the steady state growth condition (new 3) It is not clear why the authors used Torin instead of rapamycin in the reporter assays , and totIn response to the reviewer’s question, we examined the effect of rapamycin or PF4708671 on the interaction between endogenous LARP1 and the 5’ or 3’ UTR of RpL32 mRNA reporter. As expected, rapamycin or Torin1 treatment produced the same effect on the interaction of LARP1 with the 5’ and 3’UTRs of RpL32 mRNA: rapamycin enhanced the affinity of LARP1 for the 5’UTR but suppressed LARP1’s binding to the 3’UTR under steady state growth conditions.However, PF4708671, a specific S6K inhibitor, had little effect on LARP1’s binding to the 3’UTR, although PF4708671 did enhance LARP1’s binding to the 5’UTR, as does rapamycin, Torin1, and amino starvation. These data are consistent with the previous observations in our original Figure 3E4) What is the proposed mechanism by which LARP1 phosphorylation decreases 5'UTR binding and increases mTORC1 and 3'UTR binding? Have the authors tested phospho-mimetic mutants? Related to this point: The model in eLife 2017, Fonseca et al. JBC 2015). Additional studies will obviously be required to understand the precise molecular mechanisms underlying phosphorylation-dependent alterations of LARP1-RP mRNA configuration.How LARP1 phosphorylation by Akt/S6K1 and mTOR triggers its dissociation from the 5’UTR but keeps the LARP1-mTORC1 complex on the 3’UTR of mRNA remains unclear. LARP1 possesses two RNA binding motifs (La and RRM-like). La protein (LARP3), which also contains La and two RRMs, has been shown to interact with 5’TOP mRNAs , suggesting that one or both of these RNA binding motifs may play a role in LARP1 association with the 5’TOP structure. However, recent structural studies demonstrated that the LARP1 DM15 motif, which is specific to LARP1/2  and is pIn response to the reviewer’s questions, we examined whether LARP1 dimerizes and found that HA-LARP1 interacts with MYC-LARP1 in vivoin a manner independent of LARP1’s RNA binding (lane 4) or cellular mTOR activity (lane 3), indicating that LARP1 indeed forms at least a homodimer .10.7554/In addition, our new biochemical data new  suggest both the 5’ and 3’ of one RpL32 mRNA and “bridge” these two ends of the mRNA. In our luciferase assay, both the 5’ and 3’ UTR of the reporter would be bound by LARP1 in low mTOR conditions. In the Bridging Model, we would predict that the ratio of luciferase mRNA bound to LARP1 homodimer to be 1-to-1. In the alternative, “non-Bridging Model,” LARP1 homodimer would bind either the 5’ or 3’ UTR of RpL32, such that a single RpL32 mRNA would be bound by one LARP1 homodimer at its 5’UTR and another homodimer at its 3’UTR. Here, we would predict that the ratio of luciferase mRNA and LARP1 to be 0.5-to-1, i.e. for every mRNA there would be two LARP1 homodimers bound.Taken together, we considered two models of LARP1 interaction with its substrate mRNA. First, in the “Bridging Model,” one LARP1 molecule of the homodimer binds the 5’ UTR, while the other LARP1 molecule of the homodimer binds the 3’UTR of a single RpL32 mRNA . However, the phospho-mimetic mutant (3DE) behaved the same way as the phospho-defective mutant (4A): the LARP1 3DE mutant displayed reduced binding to mTORC1 much like the LARP1 4A mutant . This is


Moreover, febuxostat was revealed to be the most promising candidate of all the potential ABCG2 inhibitors based on its potent inhibition at clinical concentrations; the half-maximal inhibitory concentration of febuxostat was lower than its maximum plasma unbound concentrations reported. Indeed, our in vivo study demonstrated that orally administered febuxostat inhibited the intestinal Abcg2 and, thereby, increased the intestinal absorption of an ABCG2 substrate sulfasalazine in wild-type mice, but not in Abcg2 knockout mice. These results suggest that febuxostat might inhibit human ABCG2 at a clinical dose. Furthermore, the results of this study lead to a proposed new application of febuxostat for enhancing the bioavailability of ABCG2 substrate drugs, named febuxostat-boosted therapy, and also imply the potential risk of adverse effects by drug-drug interactions that could occur between febuxostat and ABCG2 substrate drugs.ATP-binding cassette transporter G2 (ABCG2) is a plasma membrane protein that regulates the pharmacokinetics of a variety of drugs and serum uric acid (SUA) levels in humans. Despite the pharmacological and physiological importance of this transporter, there is no clinically available drug that modulates ABCG2 function. Therefore, to identify such drugs, we investigated the effect of drugs that affect SUA levels on ABCG2 function. This strategy was based on the hypothesis that the changes of SUA levels might caused by interaction with ABCG2 since it is a physiologically important urate transporter. The results of the  ATP-binding cassette transporter G2 (ABCG2) is a member of ABC transporter superfamily and is recognized as one of the most important drug efflux transporters . FollowiABCG2 in humans. ABCG2 often lowers the bioavailability of other drugs such as rosuvastatin ; atorvastatin, chlorothiazide, febuxostat, mizoribine, pyrazinecarboxylic acid, ribavirin, tacrolimus, xylitol ; ethambutol, losartan ; fenofibrate, probenecid, sulfasalazine, Ko143, ATP, AMP, creatine phosphate disodium salt tetrahydrate, creatine phosphokinase type I from rabbit muscle ; pyrazinamide ; theophylline ; and topiroxostat . The [8-14C]-uric acid (53 mCi/mmol) was from American Radiolabeled Chemicals . All other chemicals used were commercially available and of analytical grade.The following compounds were purchased commercially from the sources indicated: allopurinol, benzbromarone, cyclosporine, L-glutamine , and 1 × Non-Essential Amino Acid (Life Technologies) at 37°C in an atmosphere of 5% CO2 as described previously  and cultured in DMEM  supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin, 2 mM eviously . All expeviously , respectAbcg2 KO mice . The  KO mice  had been KO mice . In the 14C]-urate transport assay with the ABCG2/Abcg2-expressing membrane vesicles was performed using a rapid filtration technique . Then, the calculated values were fitted to the following formula using the least-squares methods  was calculated by subtracting the ratio of bound drugs from total drugs. The maximum unbound concentration of each drug in human plasma (fuCmax) was calculated by multiplying fu and the reported Cmax together.where, E5 cells/well. Twenty-four hours after seeding, the cells were transiently transfected with the FLAG-URAT1 pcDNA3.1(+) vector or empty vector using PEI-MAX (0.4 μg plasmid/4 μL PEI-MAX/100 μL serum free DMEM/2 × 105 cells) as described previously  and preincubated in Buffer T2 for 15 min at 37°C. Then, the buffer was exchanged to fresh Buffer T2 containing 2 μM [8-14C]-urate, and the cells were further incubated for the indicated periods. Subsequently, the cells were washed with ice-cold Buffer T2 twice and then lysed with 500 μL 0.2 M NaOH on ice with gently shaking for 1 h. The resulting lysates were transferred to 1.5-mL tubes, neutralized with 100 μL 1 M HCl, and then the radioactivity were measured using a liquid scintillator. The protein concentrations were determined using a bicinchoninic acid assay kit (Life Technologies) according to the manufacturer’s instruction. The urate transport activity was calculated as the incorporated clearance (μL/mg protein/min): (incorporated level of urate [DPM/mg protein/min]/urate level in the incubation mixture [DPM/μL]). URAT1-dependent urate transport activity was calculated by subtracting the urate transport activity of mock cells from that of the URAT1-expressing cells.The 293A cells were seeded on a 12-well plate  at 2 × 10eviously . Forty-e2+ and Mg2+ and then dissolved in RIPA buffer  containing protease inhibitor cOmplete, EDTA-free . After centrifugation at 15,000 × g for 10 min at 4°C, the resulting supernatants were collected as the cell lysates. The membrane vesicles prepared from the adenovirus-infected 293A cells or cell lysates of vector-transfected 293A cells were subjected to 8.5% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes . After blocking with 3% bovine serum albumin in 0.05% Tween 20 containing Tris-buffered saline for 1 h at room temperature, the membranes were treated with following antibodies as described previously (+/K+-ATPase antibody (Santa Cruz Biotechnology) (1:1000), anti-mouse IgG or anti-rabbit IgG antibody labeled with horseradish peroxidase  (1:2500). The immunoblotted membranes were treated with ECL Prime  and analyzed using a Chemidoc XRS .The expression of Myc-ABCG2 on membrane vesicles and FLAG-URAT1 in transiently transfected-293A cells was confirmed using western blot analysis. To obtain the cell lysate samples of URAT1-expressing 293A cells, 48 h after the transfection, the cells were washed twice with phosphate-buffered saline without Caeviously ; mouse ain vivo Abcg2 inhibition test to examine the effect of febuxostat on the intestinal absorption of sulfasalazine, an ABCG2 substrate. Febuxostat was dissolved in 100 mM sodium bicarbonate buffer, pH 10. Prior to the experiment, the mice were fasted overnight, and then they were orally administered febuxostat by gavage at a dose of 150 mg/kg body weight (b.w.) with the expectation of complete inhibition of intestinal Abcg2. Twenty minutes later, sulfasalazine (20 mg/kg b.w.) was orally administered by gavage. At the indicated periods, the mice were anesthetized with diethyl ether and blood was collected from the jugular veins using heparinized syringes, followed by centrifugation at 3,000 × g for 10 min. The resulting supernatant (plasma) was collected and stored at -80°C until the LC/MS/MS analysis was performed.We conducted an g for 15 min at 4°C. Then, the supernatants were analyzed using the LC/MS/MS technique.The collected plasma was deproteinized with a fourfold volume of methanol containing 2 μg/mL 4-hydroxy chalcone as an internal standard. After vortexing for 10 min, the samples were centrifuged at 20,000 × The LC/MS/MS analysis was conducted using an ultra-performance LC system connected to a Xevo TQ-S mass spectrometer . The samples were separated using a 1.7-μm particle ACQUITY C18 column , maintained at 50°C, under gradient mobile phase conditions with a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile as solvents  with a flow rate of 0.3 mL/min. The separated samples were introduced into an MS in the positive and negative electrospray ionization mode for sulfasalazine and 4-hydroxy chalcone, respectively. Each compound was quantified in the multiple reactions monitoring mode .The AUC of sulfasalazine was calculated as the total area of the trapezoids formed by the points of the concentration and time in the concentration-time plots.post hoc test. The values were considered significant when P < 0.05.All statistical analyses were performed using the Excel 2007 program. Significant differences were identified using a two-way ANOVA followed by Tukey-Kramer Table 1) on ABCG2 function, we measured the urate transport activity of ABCG2 in the presence of each SUA-affecting drug. Expression of ABCG2 in the plasma membrane vesicles was confirmed using western blot analysis . According to our previous reports . The urate transport activities of ABCG2 were higher than those of the mock (EGFP), and linearly increased for 20 min. Therefore, we examined the effect of SUA-affecting drugs on ABCG2 for 10 min at the indicated concentrations in Table 1. In this experiment, the concentrations of each drug (3–1000 μM) were determined as the maximum soluble levels in the transport buffer.To examine the effects of 25 SUA-affecting drugs . Although 12 drugs tended to increase the urate transport activity of ABCG2, their effects were slight compared to the drastic inhibitory effects of the 10 drugs. Therefore, we focused on the most active 10 drugs in the further analyses.The in vitro screening was conducted in the presence of maximum concentrations of each drug, we subsequently estimated the clinical effects of the selected 10 drugs. First, to determine the IC50 values of each drug against the urate transport activity of ABCG2, we measured the urate transport activities in the presence of several concentrations of each drug . Then, the IC50 values were calculated from the results obtained using the least-squares methods (Table 2). Among the 10 drugs we examined, three  potently inhibited ABCG2 with IC50 values at submicromolar concentrations . Indeed, these values were considerably lower than those of four drugs that have been reported to interact with ABCG2, atorvastatin (4.3 μM), cyclosporine (3.2 μM), tacrolimus (3.1 μM) and rosuvastatin (2.3 μM).Since the To further characterize the potent inhibition of ABCG2 by febuxostat, we compared the activity of ABCG2 in the presence of febuxostat and two well-known ABCG2 inhibitors; Ko143, a fumitremorgin C analog, and elacridar (GF120918). In the presence of 100 nM of each compound, the urate transport activities of ABCG2 were 20 ± 3% with febuxostat, 75 ± 10% with Ko143 and 92 ± 10% with elacridar compared with the vehicle. These results indicate that the inhibitory effect of febuxostat against ABCG2 is stronger than those of these well-known ABCG2 inhibitors.uCmax based on the general information from pharmaceutical industries as described in Section “Materials and Methods” (Table 2), and calculated the ratio of fuCmax/IC50, as an indicator of the possible in vivo clinical inhibition of ABCG2 by each drug. A high fuCmax/IC50 value suggests that the drug has the potential to inhibit ABCG2 in humans. Among the 10 drugs we examined, benzbromarone and febuxostat had a relatively high fuCmax/IC50 value . Since febuxostat had the highest fuCmax/IC50 value and was expected to inhibit ABCG2 in humans, we proceeded to further analyze this drug.Next, we estimated the fin vitro findings strongly suggested that febuxostat would inhibit ABCG2 in vivo. To examine this possibility, we investigated the inhibitory effect of febuxostat on the intestinal absorption of an ABCG2 substrate drug in WT and Abcg2 KO mice. Prior to the in vivo analyses, we confirmed that febuxostat inhibited mouse Abcg2 in vitro in a similar manner to human ABCG2 as described above. The IC50 value of febuxostat against the urate transport activity of mouse Abcg2 was 35 nM , which was comparable to that of human ABCG2 , indicating that febuxostat inhibited mouse Abcg2 as strongly as it did human ABCG2.Our in vivo, we focused on its effect on the absorption of sulfasalazine, an ABCG2 substrate, in the intestine where ABCG2 acts as a gatekeeper for xenobiotics. It is noteworthy that the intestinal absorption of sulfasalazine majorly depends on the ABCG2 function in both humans and mice .To examine whether febuxostat inhibited Abcg2 and mice . First, Abcg2 KO mice and calculated the AUC of sulfasalazine. In WT mice, the administration of febuxostat increased the AUC of sulfasalazine . On the other hand, in Abcg2 KO mice, febuxostat did not increase the AUC of sulfasalazine. Moreover, there was no significant difference in the AUC of sulfasalazine among three groups; febuxostat-treated WT mice, vehicle-treated Abcg2 KO mice and febuxostat-treated Abcg2 KO mice. These results suggest that febuxostat enhanced the absorption of orally administered sulfasalazine by inhibiting Abcg2 in vivo.We then performed similar experiments using Figure 1C), while their effects on other urate transporters remained unclear. Therefore, we focused on URAT1, another physiologically important urate transporter recognized as a pharmacological target of SUA-lowering drugs, and compared the effects of the drugs on ABCG2 and URAT1.Some of the SUA-affecting drugs we investigated hardly affected ABCG2 function . Then, we measured the amount of [8-14C]-urate incorporated into URAT1-expressing 293A cells for 0–120 s . The result showed that the urate transport activity in URAT1-expressing cells was more than fivefold higher than that in the mock cells, and the amount of incorporated urate increased in a time-dependent manner. Then, we evaluated the effect of drugs on the URAT1 activity for 20 s in subsequent investigations.To evaluate the effect of SUA-affecting drugs on the urate transport activity of URAT1, we established an Figure 4C; Supplementary Figure S1). Among the 25 SUA-affecting drugs, only benzbromarone strongly inhibited both ABCG2 and URAT1. The urate transport activities of ABCG2 and URAT1 in the presence of 3 μM benzbromarone were 13 ± 2% and 12 ± 5%, respectively, compared to the vehicle control. On the other hand, febuxostat and topiroxostat did not affect the urate transport activity of URAT1, although they potently inhibited ABCG2. The rest of the investigated drugs had little effect on both ABCG2 and URAT1 at 3 μM.We compared the effect of each SUA-affecting drug  on the urate transport activity of ABCG2 and URAT1  and in vivo in the murine intestine . Based on these in vitro and in vivo findings, it is possible that febuxostat has potential to inhibit ABCG2 in humans. It is worth noting that the inhibitory effect of febuxostat against ABCG2 was more potent than those of two well-known ABCG2 inhibitors, Ko143 and elacridar. This means that febuxostat has advantages not only in the safety but also in the inhibitory ability against ABCG2 as compared with those two ABCG2 inhibitors. Accordingly, in our opinion, febuxostat would be the most promising ABCG2 inhibitor in humans.In the present study, we examined the effects of 25 SUA-affecting drugs on the transport activity of ABCG2 in an attempt to identify a new chemical modulator of ABCG2 function from drugs on the market. The results of the Figure 5A). Subjects with ABCG2 SNPs, which lower ABCG2 function, reportedly exhibit higher bioavailability of ABCG2 substrates such as sulfasalazine . In pharmaceutical fields, taking advantages of inhibitory effect of ritonavir on CYP3A4, enhanced bioavailability of CYP3A4 substrate drugs is achieved by co-administration of ritonavir, which is known as ritonavir-boosting . Since ABCG2 is one of the genes responsible for the development of MDR in cancer cells, the inhibition of ABCG2 could enhance the efficacy of chemotherapy, leading to an efficient accumulation of anticancer agents. In addition, ABCG2 is involved in the efflux of photosensitizers in 5-aminolevulinic acid-based photodynamic therapy (PDT) and photodynamic diagnosis (PDD)  . ConsideFigure 5A). For instance, while the efficacy of rosuvastatin would be enhanced by ABCG2 inhibition as described above, excess administration of rosuvastatin could induce rhabdomyolysis, a well-known severe adverse event  in Europe and Japan. TLS is an oncogenic emergency resulting in metabolic disturbances including drastic increases in SUA levels. Considering the risk of TLS in chemotherapy with sunitinib , the co-The clinical prospects of febuxostat as an ABCG2 inhibitor should be confirmed by clinical investigations in future to compensate the limitation in the translational implication of the current study. Considering the diversity of substrate specificity in ABC transporters that is responsible for the multidrug efflux , the inhFigure 5B). Therefore, the SUA-lowering effects of these drugs might be weaker than expected because of their inhibition of ABCG2-mediated urate excretion. A similar inhibition pattern is also the case with benzbromarone, a URAT1 inhibitor . Since allopurinol, febuxostat, topiroxostat and benzbromarone are the major SUA-lowering drugs presently known, the development of SUA-lowering drugs that do not inhibit ABCG2 would be the next strategy for developing a more effective hyperuricemia therapy.Focusing on urate kinetics, our results suggest that existing SUA-lowering drugs should have a room for improvement in efficacy. To develop more effective SUA-lowering drugs, the biological effect of the candidates should be carefully assessed not only on their primary molecular targets but also on other urate transporters or metabolic enzymes. Approved SUA-lowering drugs function as inhibitors of either xanthine oxidase (XO), a urate production enzyme, or URAT1. Allopurinol, the oldest XO inhibitor, has been used for patients with hyperuricemia over the last half-century , even thin vitro and in vivo, suggesting that it could be the first clinical and safe ABCG2 inhibitor in humans. The possible drug–drug interaction between febuxostat and ABCG2 substrate drugs would have both beneficial and harmful effects. Our successful demonstration is an example of the promising beneficial effects, which provided the proof of concept for the potential of febuxostat-boosting. To ensure the safe clinical application of febuxostat, further investigations in humans would be important.We demonstrated that febuxostat potently inhibited ABCG2 both HsM, TT, and YT designed the research. HsM and YT conducted the all experiments. HsM, TT, and YT performed data analysis. All authors contributed to the writing and final approval of the manuscript.TT, HtM, KI, and HS have a patent pending. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The matricellular protein thrombospondin-1 (TSP1) is a potent inhibitor of angiogenesis. Specifically, TSP1 has been experimentally shown to inhibit signaling downstream of vascular endothelial growth factor (VEGF). The molecular mechanism of this inhibition is not entirely clear. We developed a detailed computational model of VEGF signaling to Akt–endothelial nitric oxide synthase (eNOS) to investigate the quantitative molecular mechanism of TSP1 inhibition. The model demonstrated that TSP1 acceleration of VEGFR2 degradation is sufficient to explain the inhibition of VEGFR2 and eNOS phosphorylation. However, Akt inhibition requires TSP1-induced phosphatase recruitment to VEGFR2. The model was then utilized to test various strategies for the rescue of VEGF signaling to Akt and eNOS. Inhibiting TSP1 was predicted to be not as effective as CD47 depletion in rescuing signaling to Akt. The model further predicts that combination strategy involving depletion of CD47 and inhibition of TSP1 binding to CD47 is necessary for effective recovery of signaling to eNOS. In all, computational modeling offers insight to molecular mechanisms involving TSP1 interaction with VEGF signaling and provides strategies for rescuing angiogenesis by targeting TSP1–CD47 axis. Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis in physiological and pathophysiological states. The VEGF family consists of number of secreted proteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF), with VEGF-A being the most widely studied of the group. VEGF plays a crucial role in vasculogenesis and developmental angiogenesis  and adulPhysiological VEGF signaling is tightly regulated by a balance of promoters and inhibitors of angiogenesis . Among tTSP1–CD47 interaction has been demonstrated to potently inhibit VEGFR2 phosphorylation and Akt activation . Indeed,Considering the complexity of receptor-level interactions and downstream signaling, computational models should prove expedient in elucidating and clarifying molecular mechanisms involved in TSP1–CD47 interaction with other receptors and suggest new avenues for experimental investigation. Moreover, computational models may inform and discriminate between various therapeutic modalities targeting TSP1–CD47 signaling.Recently, computational modeling has been applied to investigate the mechanism of inhibition of VEGF signaling by TSP1–CD47 interaction and also test several therapeutic interventions . A detaiExtending this to look at a different arm of VEGF signaling pathway and angiogenesis, our aim in this study is to build upon previous models of VEGFR2 signaling and present a fundamentally novel computational model of VEGF signaling to PI3K/Akt that takes into account recent experimental data in endothelial cells demonstrating the critical role of the receptor Axl-1 and Src in transducing VEGFR2 activation signal to PI3K/Akt . We alsoClinically, TSP1–CD47 signaling axis might offer an attractive target for pro-angiogenic interventions in diseases such as PAD  and impaa priori knowledge of all the intermediate reaction steps. The model is simulated within MATLAB 2015b  using the Sundials solver suite  format. Western blot data from the literature were quantified using ImageJ  for the receptor and a single binding site for the coreceptor NRP1 (NRP1bd). VEGFR2 has a ligand-binding site (L), a tyrosine phosphorylation site (represented as Y1175), and a ligand independent coupling site (c).Detailed receptor interaction module was constructed using a rule-based methodology utilizing BioNetGen similar to previous studies . As showFigure 1B. TSP1 binds to VEGFR2-coupled CD47 . Biologically, TSP1 is a tetramer with potentially three different binding sites for CD47, but to simplify the model, we assume that TSP1 binding to CD47 is effectively a one-to-one interaction. VEGFR1 and NRP1 interact constitutively as shown in Figure 1D. VEGFR1/VEGFR2 heterodimerization is also included along with constitutive binding of VEGFR2 to CD47 . For completeness, model here includes ligand-independent dimerization as shown in Figure 1E . Furthermore, VEGFR2 receptors internalize after the homodimerization induced by the binding of VEGF . The model includes a multi-compartmental receptor cycling model that comprises two compartments as signaling endosomes to more accurately fit the experimental data . The transition from the signaling endosome to the recycling endosome is dependent on NRP1 consistent with experimental data . Consistent with the experimental evidence, only unphosphorylated receptors cycle back to the plasma membrane from the recycling compartment  and another for NRP1 (NRP1bd), and a ligand-independent coupling domain (c). NRP1 has a single-binding site for both the ligand and VEGFR1 (R1bd/L) . The maiigure 1E . Engagemtal data  and may partment . The sigThe signal transduction pathway is constructed by taking into account recent experimental evidence demonstrating that Axl-1 and Src are crucial for the transduction of the signal from phosphorylated receptor to Akt . We alsoFigure 2B). Ca activates CaM which then binds eNOS–caveolin-1 complex and results in the dissociation of caveolin-1 and partial activation of eNOS  as shown in dynamics  and surfdynamics . The inidynamics , 2014. Idynamics .Figure 3C . The model is also fitted to the data in the presence or absence of cycloheximide (CHX), a potent inhibitor of protein synthesis. This is to estimate the effective rate of protein synthesis in the model, namely the parameter ksingleR2syn. The model adequately fits the data under control condition  as in Figure 3D. In the absence of CHX, the VEGFR2 levels drop considerably due to inhibition of receptor synthesis . With both VEGF and CHX present, the drop in VEGFR2 levels is even faster, fitting the available experimental data .The model correctly captures this as shown in Figure 1F) was necessary for better fitting of the model to the set of data on receptor dynamics. The value of ksingleR2syn is computed to be about 1.4e-4 s-1 (this translates into a synthesis rate of ∼0.84 receptor/s). The normalized phosphorylated VEGFR2 versus time is fitted to the data in Figure 3G).While constructing the model we determined that having two signaling internalized compartments . The model predicts that the majority of the signal comes from the internalized receptors (red line) while the signal from surface receptors is short lived (blue). Note that this is an emergent property of the model and is not an a priori assumption. In fact, this is consistent with recent experimental evidence demonstrating that in many receptor tyrosine kinases including VEGFR2 the bulk of signaling comes from the internalized receptors . Additionally, the cytoplasmic Ca trace from the model and the data from Figure 3J) to constrain the parameters for the Ca cycling module. For the signaling to PI3K/AKT the data in Figure 3K. Also shown in Figure 3L, is the Src phosphorylated Axl-1 dynamics fitted to the data points. The traces for the phosphorylation of S473 and T308 residues on Akt are shown in Figures 3M,N, respectively; the data points are taken from Figure 3O along with the experimental data from To fit the Ca regulation arm of the pathway, phosphorylated PLCγ trace from the model is fitted to the experimental data in Figure 4. Both positively and negatively correlated parameters are included. The sensitive positively and negatively correlated parameters determining the activation of S473 on Akt are shown in Figures 4A,B. The top positively correlated parameters  are the catalytic rate of PI3K (kcatPI3KPIP2), PIP3 concentration for half activation of PTEN (kmPIP3PTEN), the total level of PI3K (PI3K0), and several parameters determining activation of Axl-1 . Notably, synthesis rate of VEGFR2 (ksingleR2syn) and the on-rate for the binding of VEGF to NRP1 (kVEGFNRP1on) are also significant parameters. The top negatively correlated parameters include the total level of PTEN (PTEN0), the catalytic rate of PTEN (kcatPTENPIP3), the PIP3 concentration for half-maximal activation of PI3K (kmPIP2PI3K), the inactivation rate of PI3K (koffPI3KAxl), and the dephosphorylation rate of Axl-1 (kdpautoAxl and kdpSrcAxl). Other notable parameters include the catalytic rate of PLCγ (kcatPLCγDAG), the off-rate of VEGF-VEGFR2 unbinding from NRP1 (kVEGFR2NRP1off), and the internalization rate of VEGFR2 in the absence of NRP1 (kr2si).Global sensitivity analysis is then carried out using the partial rank correlation coefficient (PRCC) method described in Axlauto), and the rate of PI3K activation by Axl-1 (konPI3KAxl). The total level of Axl-1 (Axl0) and Src (Src0) and the phosphorylation rate of VEGFR2 are significant. Other notable parameters are VEGFR2 synthesis rate, the on-rate of VEGF binding to NRP1 (kVEGFNRP1on), and ligand-independent decoupling rate between receptors (kdRR).For the doubly phosphorylated Akt (phosphorylated on both S473 and T308), the top positively correlated parameters are the total level of PI3K, the autophosphorylation rate of Axl-1 (kps and kdpi), and the degradation rate of the receptor (kdegi0noP and kdegi0). The top positively correlated parameters determining phosphorylated eNOS on S1177 are the amplitude of the CRAC channel current (ICRACamp), the total level of PIP2 (PIP20), the on-rate of Ca/CaM binding to eNOS–caveolin complex, and the amplitude of current through the IP3-sensitive receptors (Iip3Ramp). The synthesis rate of VEGFR2 and catalytic rate of PLCγ are also notable positively correlated parameters. Top negatively correlated parameters are the current through the SERCA pump (vSERCA), the degradation rate of IP3 (kdegip3), and the plasma membrane Ca pump rate (IPMCAamp).The top negatively correlated parameters are the dephosphorylation rate of autophosphorylated Axl-1, the rate of PI3K inactivation, and the dephosphorylation rate of the site on Axl-1 phosphorylated by Src. Other notable parameters include the dephosphorylation rate of VEGFR2  and 1 (control). As demonstrated by the simulations in Figures D, total block of receptor transport from signaling endosomes to the recycling endosomes only slightly reduces VEGFR2 phosphorylation , Akt activation , or eNOS phosphorylation .The first possibility is that TSP1 inhibits the transport of receptors from the signaling endosomes to the recycling endosome as illustrated in Figure 5E, also see Figure 1F). This is modeled by the parameter fTSP1deg . As shown in Figure 5F, this mechanism is sufficient to explain inhibition of VEGFR2 phosphorylation as a fivefold increase in degradation represses pR2. Figure 5G illustrates the changes in maximum pR2 as a function of enhanced degradation. Increasing degradation ∼20-fold is predicted to be sufficient to lead to near maximal inhibition. The degradation from signaling endosomes also reduces Akt activation as shown in Figure 5H, however, the inhibition plateaus .The second mechanism for TSP1 inhibition of VEGFR2 signaling considered here is that TSP1 enhances degradation from the signaling endosomes . Interestingly, max peNOS is linearly correlated with max CaCaM  predicting that the inhibition is mainly through the inhibition of Ca/CaM. In other words, TSP1 effect on VEGFR2 potently inhibits Ca/CaM elevation downstream of VEGFR2 thereby blocking the initial activation of eNOS by Ca/CaM, the necessary step in the activation of eNOS . Traces in Figure 6B demonstrate the effectiveness of this mechanism in inhibiting Akt activation. Figure 6C further shows that 20-fold increase in dephosphorylation rate potently inhibits Akt activation. In reality, it is plausible for both enhanced degradation and phosphatase recruitment mechanisms to work in concert as shown in Figure 6D that shows the effect of changing both parameters on Akt activation. What the simulation shows is that a 10-fold increase in dephosphorylation and degradation each would be sufficient to explain TSP1 inhibition. Phosphatase recruitment would also augment enhanced degradation in the inhibition of eNOS activation as shown in Figures 6E,F. What these simulations show is that when both mechanisms work in concert, the inhibitory effect of TSP1 may be very potent.The simulations should be viewed as test of hypothesis that should motivate and guide additional experimental investigation. The proposed mechanism is illustrated in In the next section, we utilize this knowledge to perform simulations for the inhibition of TSP1–CD47 signaling as pro-angiogenic intervention.in vivo conditions such depletion could be achieved by administration of a TSP1-specific antibody. As shown in Figures 7A,B, 80% depletion of TSP1 is necessary for the Akt activation signal to begin to recover  and for effective rescue of Akt signal, a 98% inhibition is necessary . However, this is unlikely to be achievable physiologically. The rescue of eNOS activation with this intervention is even less efficient as shown in Figures 7C,D. We next postulate that depleting CD47, the receptor for TSP1, might be more efficient in the restoration of VEGF signaling to Akt and eNOS. Indeed, Akt activation is restored in a linear fashion after ∼30% of CD47 were depleted . The reason for the initial delay is that assumed total CD47 level of 10,000 receptors is higher than the total number of VEGFR2 at 6000. Because VEGFR2 and CD47 are constitutively associated with each other, initial depletion is necessary to reduce the number of CD47 to a comparable level as VEGFR2. The traces in Figure 7F show the gradual restoration in Akt activation.Previous simulations illustrate that TSP1–CD47 inhibition is very effective in shutting down VEGF signaling to Akt and eNOS via enhancing the degradation of VEGFR2 from signaling endosome coupled with phosphatase recruitment to the plasma membrane. Simulations indicated that a 10-fold increase in both phosphatase recruitment and VEGFR2 degradation would effectively block both Akt and eNOS activation. Here we aim at utilizing the model to test different interventions to best rescue VEGF signaling to Akt and eNOS in the presence of TSP1. This may be relevant to disease conditions such as wound healing and PAD where angiogenesis mediated by VEGF may be compromised. We initially simulate the effect of depleting TSP1 in the extracellular space; for example, under Figure 7G versus Figure 7C), the simulations still predict difficulty in the plausibility of targeting CD47 to rescue eNOS. The recovery curve still has a strong switching behavior with minimum 80% depletion necessary for peNOS recovery. The traces for peNOS in Figure 7H also indicate that 96% inhibition may be required to sufficiently restore eNOS activation.While eNOS activation in response to CD47 inhibition is less steep than the case of TSP1 depletion . Considering a different scenario, suppose CD47 is depleted by 50%, then a 23-fold reduction in TSP1 binding to CD47 would result in near complete recovery of Akt . Similarly, the recovery in maximum eNOS is much improved if both interventions are combined as in Figure 7K. As illustrated in Figure 7K, a 70-fold decrease in the binding of TSP1 to CD47 (a 70-fold increase in Kd) by a hypothetical compound would significantly improve the recovery of eNOS activation in response to CD47 depletion .The model predicts that neither CD47 nor TSP1 depletion is efficient in eNOS activation rescue. We therefore hypothesized that combining CD47 depletion with a hypothetical therapy that inhibits TSP1 binding to CD47 (modeled by increasing the Kd of TSP1 binding to CD47) may produce a better recovery curve for both Akt and eNOS. Indeed, simulations predict that a hypothetical therapy, such as a TSP1-specific antibody, that sequesters TSP1 and reduces TSP1 binding to CD47 by 10-fold (increasing Kd by 10-fold), improves the anti-CD47 recovery curve for Akt . Simulations here predict that while Akt activation may be rescued efficiently by targeting CD47 alone (rather than TSP1), to effectively restore eNOS activation, combination therapies that target both CD47 and another mechanism such as the binding of TSP1 to CD47 may be necessary. Overall, computational modeling suggests that targeting TSP1–CD47 axis has potential as a pro-angiogenic intervention. Modeling results also suggest that inhibiting CD47 might be more advantageous than TSP1 alone. However, a dual approach that captures both TSP1 and CD47 is predicted to be optimal.Suppose again that 50% of CD47 has been depleted, a 52-fold reduction in the binding of TSP1 to CD47 would lead to a significant restoration of eNOS activation signal  and has equity interests in the same and Tioma Therapeutics  which have licensed CD47 technologies. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.