Influenza virus infection causes significant morbidity and mortality and has marked social and economic impacts throughout the world. The influenza surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), act cooperatively to support efficient influenza A virus replication and provide the most important targets for anti-influenza chemotherapy. In this study, povidone-iodine (PVP-I), which has a broad-spectrum microbicidal property, was examined for its inhibitory effects against influenza virus infection in MDCK cells and the mechanisms of PVP-I action on HA and NA were revealed.50 values of 9.5–212.1 μg/ml by a mixed-type inhibition mechanism. Receptor binding inhibition and hemagglutinin inhibition assays indicated that PVP-I affected viral hemagglutinin rather than host-specific sialic acid receptors.Results obtained using a novel fluorescence- and chromogenic-based plaque inhibition assay showed that 1.56 mg/ml PVP-I inhibited infections in MDCK cells of human (8 strains) and avian (5 strains) influenza A viruses, including H1N1, H3N2, H5N3 and H9N2, from 23.0–97.5%. A sialidase inhibition assay revealed that PVP-I inhibited N1, N2 and N3 neuraminidases with ICMechanisms of reduction of viral growth in MDCK cells by PVP-I involve blockade of viral attachment to cellular receptors and inhibition of viral release and spread from infected cells. Therefore, PVP-I is useful to prevent infection and limit spread of human and avian influenza viruses. Among the three types  of influenza viruses, A type is the most virulent, infecting various avian and mammalian species and causing human pandemics as a consequence of antigenic change (antigenic shift) in their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) . Sixteen®), which is administered by inhalation, and oseltamivir phosphate  as a substrate. In brief, confluent monolayers of MDCK cells in a 96-well plate were washed with serum-free EMEM and incubated with PVP-I at a final concentration ranging from 0–25 mg/ml for 24 h at 37°C in 5% CO5 fluorescent forming units, which corresponds to 250 PFU/well equivalent to a multiplicity of infection (MOI) of 0.025 PFU/cell, in the absence of an inhibitor [2. After 45 min of incubation at 37°C, the reaction was stopped by addition of 100 mM sodium carbonate buffer (pH 10.6). Fluorescent intensity of released 4-methylumbelliferone (MU) was measured by excitation at 336 nm and emission at 490 nm (Ex355/Em460) using a fluorescence microplate reader (Fluorescence staining step).Inhibitory effects on virus development in MDCK cells were determined as shown in Figure nhibitor ) was preFor virus quantitation, a 5-μl aliquot of virus sample serially diluted in PBS was incubated with 50 μl of 0.5% suspension of guinea pig erythrocytes for 2 h at 4°C. Virus titers expressed as HAU were determined by visual reading.For determination of inhibition of HA binding, a 25-μl aliquot of fetuin or PVP-I serially diluted in PBS was preincubated with 25 μl of virus (4 HAU) in 96-well U-bottom plates at 4°C for 1 h. Hemagglutination was determined as described above.14 HAU) to obtain a final HAU of about 400 [To visualize agglutination directly, 100 mg/ml of PVP-I was added to a well of a 24-well flat-bottom plate to give a final concentration of 1.25, 2.50, 3.75 or 5.00 mg/ml, followed by addition of 5 μl of virus (2bout 400 . To avoio-phenylenediamine (OPD) and H2O2 in 100 mM citrate buffer (pH 6.0), prepared according to the instruction manual , terminated by adding H2SO4, and assessed by measuring optical densities at 492 and 620 nm.Specific binding of virus to α2,3 and α2,6 sialoglycopolymers was performed in 96-well plates coated with 0–2,000 ng/ml of sialoglycopolymers as described previously . BrieflyA receptor inhibition experiment was performed as described above, but each virus (12 HAU) was preincubated with PVP-I (0–780 μg/ml) or fetuin  in PBST at 4°C for 2 h before the virus mixture was added to the plate coated with 50 ng/ml of sialoglycopolymer.m value of each virus. After 15 min at 37°C, the reaction was terminated by adding 200 μl of 100 mM sodium carbonate buffer (pH 10.6). The released MU products were measured at Ex355/Em460.Five μl of 2 viral enzyme units (one unit being the amount required to liberate 1 nmole of product per min) was preincubated with 5 μl of an inhibitor serially diluted in 20 mM sodium acetate buffer (pH 6.0) at 37°C for 15 min. Enzymatic reaction was started by addition of 5 μl of MUNA to a final concentration equivalent to a Km values and then transformed and plotted as Lineweaver-Burk plots using GraphPad Prism software. Inhibition constant of each inhibitor was calculated according to their inhibition type as follows:To determine the inhibition mechanism, 5 μl of 2 viral enzyme units was incubated with 5 μl of 280–1000 μM MUNA either alone or in the presence of 5 μl of OC, DANA or PVP-I for 15 min at 37°C. The MU products were measured as described above. Data were fitted to the Michaelis-Menten equation for determination of apparent Vmax and Kmapperance = Km(1+I/Ki).For competitive inhibitor, Kmaxapperance = Vmax/(1+(I/Kis)) and Kmapperance = Km(1+I/Ki)/(1+I/Kis).For mixed-type inhibitor, Vi and Kis are dissociation constants for free enzyme-inhibitor and substrate-bound enzyme-inhibitor complex, respectively.KThe authors declare that they have no competing interests.Conceived and designed the experiments: NS, YS, PW, MT. Performed the experiments: NS, TT. Analyzed the data: NS, YS, TT, TS. Contributed reagents/materials/analysis tools: YS, MT, HH, MI, YI. Wrote the paper: NS, YS, PW. All authors have read and approved the final manuscript.


In the present study, we investigated the mechanism of the induction of waf1p21 gene by vesnarinone in TYS cells. We constructed several reporter plasmids containing the waf1p21 promoter, and attempted to identify vesnarinone-responsive elements in the waf1p21 promoter. By the luciferase reporter assay, we identified the minimal vesnarinone-responsive element in the waf1p21 promoter at −124 to −61 relative to the transcription start site. Moreover, we demonstrated by electrophoretic mobility shift assay that Sp1 and Sp3 transcription factors bound to the vesnarinone-responsive element. Furthermore, we found that vesnarinone induced the histone hyperacetylation in TYS cells. These results suggest that vesnarinone directly activates waf1p21 promoter via the activation of Sp1 and Sp3 transcription factors and the histone hyperacetylation in TYS cells.We previously demonstrated that a differentiation inducing drug, vesnarinone induced the growth arrest and British Journal of Cancer (2002) 87, 1042–1046. doi:10.1038/sj.bjc.6600592www.bjcancer.comCancer Research UK© 2002  Vesnarinone  was dissolved in dimethyl sulphoxide  at a concentration of 10 mg ml−1 as the first stock solution, and the first stock solution was diluted with the complete culture medium described above. Trichostatin A  was dissolved in ethanol at a concentration of 1 mg ml−1, and diluted with the complete culture medium at 10 μg ml−1.TYS cells  site of the luciferase reporter vector, pGL3-Basic  to generate pGL3-WWP  and BglII , and re-ligated to generate pGL3-WWP-0.2 (pWP124 and pWPdel-SmaI (The human wild-type 5 cells dish−1) were seeded in 35 mm culture dish  in DMEM supplemented with 10% FCS. Twenty-four hours later, the cells were transfected with 5 μg of reporter plasmid DNA by using Superfect reagent . Fifteen hours after transfection, vesnarinone (50 μg ml−1) was added, and 20 h later, cell lysates were collected. Luciferase activities were measured by Promega luciferase assay Kit (Promega). The luciferase activities were normalised by the amount of protein. Each experiment was repeated at least three times.TYS cells (5×106 cells dish−1) were seeded in 100 mm culture dish  in DMEM supplemented with 10% FCS. Twenty-four hours later, cells were treated with vesnarinone (50 μg ml−1) for 15, 30 and 45 min. Cell lysates were prepared according to the method described by M HEPES-KOH (pH 7.9) buffer containing 400 mM NaCl, 0.2% NP-40, 10% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM sodium orthovanadate, 0.5 mM phenylmethanesulphonyl fluoride (PMSF), 1 μg ml−1 of aprotinin, 1 μg ml−1 of leupeptin, and 1 μg ml−1 of pepstatin A. The protein concentrations of samples were determined with a Bio-Rad protein assay kit . Double stranded oligonucleotides,   by T4 polynucleotide kinase (Promega), and purified by a spin column system (Amersham Pharmacia Biotech.). Sp1-A contains two Sp1 sites, and Sp1-B contains three Sp1 sites (4 c.p.m.) in a binding buffer of 10 mM HEPES-KOH (pH 7.9), 0.1 mM EDTA, 0.01% NP-40, 100 μg ml−1 of poly (dI-dC) (Amersham Pharmacia Biotech.), and 5% glycerol. The reaction was allowed to proceed for 20 min at room temperature before loading on 6% polyacrylamide gel at a low-ionic-strength buffer (0.5×TBE). The gels were run at 100 V on ice for approximately 1 h and dried. The dried gels were exposed to X-ray film. For supershift experiments, anti-Sp1 and/or anti-Sp3 antibody  was added to the reaction mixture, and the mixture was incubated for 20 min at room temperature before addition of the radiolabelled oligonucleotide.TYS cells (1.5×10−1) or TSA (10 μg ml−1) was added to the medium. Sixteen hours later, the cells were collected and the nuclear extracts were prepared as follows; cells were suspended in 400 μl of hypotonic buffer (20 mM HEPES-KOH (pH 7.9) containing 1 mM EDTA, 1 mM DTT, 20 mM NaF, 1 mM sodium orthovanadate, 0.5 mM PMSF, 0.2% NP-40, 1 μg ml−1 leupeptin, 10 units ml−1 aprotinin, and 1 μg ml−1 pepstatin A). Samples were centrifuged at 15 000 r.p.m. and the pellets were resuspended in 200 μl of hypertonic buffer (20 mM HEPES-KOH (pH 7.9) containing 1 mM EDTA, 1 mM DTT, 20 mM NaF, 1 mM sodium orthovanadate, 0.5 mM PMSF, 0.2% NP-40, 420 mM NaCl, 20% glycerol, 1 μg ml−1 leupeptin, 10 units ml−1 aprotinin, and 1 μg ml−1 pepstatin A). Samples were incubated on ice for 20 min and were centrifuged at 15 000 r.p.m. for 15 min. The supernatants were used as nuclear extracts. The protein concentrations of samples were determined with a Bio-Rad protein assay. Samples were electrophoresed on SDS-polyacrylamide gel. Proteins from gels were transferred to nitrocellulose (Bio-Rad) and were detected with an anti-acetylated Histone H3 antibody  and an Amersham ECL kit (Amersham Pharmacia Biotech.).TYS cells were seeded in 100 mm culture dishes. Twenty-four hours later, vesnarinone .According to the results from the luciferase assay, the vesnarinone-responsive element exists within 77 bp region relative to the TATA element. This 77 bp region harbours four independent and two overlapping nearly consensus binding sites for transcription factor Sp1. They are tentatively termed upstream . To dete-6 sites . After tTo elucidate whether the retarded bands represent the binding of Sp1 or Sp3, supershift assay was performed by the nuclear extracts pre-incubated with anti-Sp1 or anti-Sp3 antibody . In the We investigated whether or not vesnarinone induced histone acetylation in TYS cells. Vesnarinone clearly induced histone acetylation in TYS cells like as TSA did  and two overlapping Sp1 binding sites   . We idenofactors . Sp1 andVesnarinone induced histone acetylation in TYS cells. Recent studies demonstrated that there were various kinds of histone acetyltransferase (HAT) and histone deacetylase (HDAC) in mammalian cells, and the level of histone acetylation was controlled by equilibrium of the activities of HAT and HDAC . The trawaf1p21 gene with the DNA-damaging therapy, such as radiation and the administration of conventional chemotherapeutic drugs. These informations are useful for creating new strategy for differentiation-inducing-therapy.Several histone acetylation inducing drugs show the growth-inhibitory effect or differentiation-inducing effect, and are used as a chemotherapeutic agent on several human malignancies (


Growth factor-induced receptor dimerization and cross-phosphorylation are hallmarks of signal transduction via receptor tyrosine kinases (RTKs). G protein-coupled receptors (GPCRs) can activate RTKs through a process known as transactivation. The prototypical model of RTK transactivation involves ligand-mediated RTK dimerization and cross-phosphorylation. Here, we show that the platelet-derived growth factor receptor β (PDGFRβ) transactivation by the dopamine receptor D4 (DRD4) is not dependent on ligands for PDGFRβ. Furthermore, when PDGFRβ dimerization is inhibited and receptor phosphorylation is suppressed to near basal levels, the receptor maintains its ability to be transactivated and is still effective in signaling to ERK1/2. Hence, the DRD4-PDGFRβ-ERK1/2 pathway can occur independently of a PDGF-like ligand, PDGFRβ cross-phosphorylation and dimerization, which is distinct from other known forms of transactivation of RTKs by GPCRs. Receptor tyrosine kinases (RTKs) consist of a large family of receptors whose members serve a wide range of physiological functions including growth, differentiation and synaptic modulation. The members of this receptor family generally feature an extracellular ligand-binding domain, linked by a transmembrane domain to an intracellular tyrosine kinase domain, as well as several SH2 domain-binding sites. It is generally believed that the mechanism of RTK signaling involves ligand-induced dimerization of the RTK followed by cross-phosphorylation of the tyrosine-containing motifs, which subsequently interact with SH2 domain-containing molecules such as the PI3-kinase, PLC-γ, Src, SHP-2, Grb-2 and RasGAP, to effect downstream responses .2 adrenergic receptor [1 muscarinic receptor [2 adrenergic receptor is also characterized by increased dimerization of EGFR [The large family of G protein-coupled receptors (GPCRs) activates heterotrimeric G proteins and can mediate several cellular processes, including proliferation, differentiation and survival. The ERK1/2 signaling pathway is among the major effector pathways through which GPCRs mediate their responses . Many GPreceptor , M1 muscreceptor , angiotereceptor , lysophoreceptor , ET1 recreceptor  and throreceptor  have bee of EGFR . In manyPrevious work from our laboratory and our collaborators has demonstrated the DRD4-mediated transactivation of PDGFRβ in hippocampal neurons  as well Recombinant human PDGF-BB was purchased from R&D Systems . Dopamine, wortmannin and tyrphostin A9 were obtained from Sigma-RBI . AG1295 and GM6001 were purchased from Calbiochem . CRM197 was purchased from List Biochemical Laboratories . Antibodies raised against β-tubulin, phospho-Shc and the carboxy terminal region of human PDGFRβ from residues 958 to 1106 were obtained from Santa Cruz Biotechnology . Antibodies raised against the extracellular domain of human PDGFRβ were obtained in a biotinylated form from R&D Systems . Antibodies specific to different phosphorylation sites on PDGFRβ were obtained from two different sources. Anti-phospho-PDGFRβ-Tyr716 was from Upstate Biotechnology , and phosphospecific PDGFRβ antibodies directed against Tyr740, 751, 857, and 1021 were purchased from Santa Cruz Biotechnology . General phosphotyrosine antibodies in an unconjugated form (4G10) and in a horseradish peroxidase-conjugated form (PY20) were purchased from Upstate Biotechnology  and BD Transduction Laboratories , respectively. Antibodies to ERK1/2 and phospho-ERK1/2 (Thr202/Tyr204) (E10) were obtained from Cell Signaling Technology . Anti-FLAG antibody was purchased from Sigma . Peroxidase-conjugated antibodies to mouse and rabbit IgG were purchased from Sigma  and Cell Signaling Technology , respectively. Lipofectamine, G418, zeocin, fetal bovine serum, and horse serum were purchased from Invitrogen Life Technologies . Media used in cell cultures were obtained from either Invitrogen Life Technologies  or Sigma .et al. [EcoRI and XbaI restriction endonuclease sites in pcDNA3.1-Myc-His-A vector (Invitrogen) for C-terminal tagging of the protein and expression in mammalian cells. To construct the GST-Ig4β, the fourth immunoglobulin domain of the mouse PDGFRβ was taken as the region from amino acid residues 314 to 413 [BamHI and EcoRI restriction endonuclease sites in pGEX-3X . The sequences of the inserts in all the plasmids used in the present study were confirmed by automatic sequencing .Expression vectors for epitope-tagged DRD4 and PDGFRβ have been described by us previously . The plaet al.  for the et al. . The ins4 to 413 , based o4 to 413 , and cloCHO-K1 cells were maintained in α-MEM supplemented with 2.5% fetal bovine serum and 2.5% horse serum [6 cells per 100 mm. Transfection was allowed to take place for 5 h, after which time an equal volume of 2 × serum-containing α-MEM was added. On the following day, cells intended for assays were replated at 60-80% confluency or at 0.4-1% for establishing stable cell lines. To establish cell lines expressing the desired constructs stably, the transfected cells were allowed to form isolated colonies. Positive colonies were selected by their resistance to G418 or zeocin. The expression of the desired proteins in the isolated colonies was determined by western blotting.For assays on cell lines not subject to further transfection, the cells were seeded and grown to 70-90% confluency prior to serum-deprivation. Transfection was performed on 100 mm plates with a mixture containing 36 μL lipofectamine and 8 μg DNA. The mixture was prepared in 0.6 mL OPTI-MEM (Invitrogen) and incubated for 45 minutes at room temperature before another 6.4 mL of OPTI-MEM was added to yield the final transfection mixture. One day prior to transfection, cells were seeded at a density of 2 × 10The two double-stranded RNA constructs containing the PDGFRβ-specific sequences used were PDGFRβ silencer 1: (sense) 5'-GCAUCUUCAACAGCCUCUAtt-3' and (antisense) 5'-CAGAGGCUGUUGAAGAUGCtt-3' and PDGFRβ silencer 2: (sense) 5'-GUGGACUCCGAUACUUACUtt-3' and (antisense) 5'-AGUAAGUAUCGGAGUCCACtt-3'. These oligonucleotides, as well as the scrambled siRNA (cat. # 4611), were purchased from Ambion . To suppress PDGFRβ expression, CHO/DRD4 cells were transfected with 30 or 100 nM dsRNA using siPORT transfection reagent  according to the manufacturer's instructions. After 72 h, the cells were used for pharmacological manipulation and harvested for western analysis.E. coli. Purification from bacterial lysates was carried out according to the manufacturer's protocol (Amersham). Elution from glutathione-Sepharose beads (Amersham) was performed as described by Omura et al. [GST and GST-Ig4β fusion proteins were expressed in BL21 a et al. . For cel2HPO4, 1.8 mM KH2PO4, pH 7.4). Cells were scraped on ice in RIPA buffer supplemented with protease and phosphatase inhibitors . Sodium orthovanadate was activated using the method described by Gordon et al. [CHO-K1 cells were serum-deprived for 24 h prior to assays with either PDGF-BB (10 ng/mL) or dopamine (1 μM) for 5 minutes. In experiments using kinase inhibitors, the cells were pre-incubated with the inhibitor or the appropriate vehicle control solution for 1 h before stimulation with the agonists. Following incubation, cells were chilled immediately on ice and washed two times with ice-cold PBS .Immunoprecipitation was performed by mixing 1-2 μg antibody with RIPA cell lysates. The mixture was shaken for a minimum of 1 h to overnight at 4°C. After addition of 25 μL Protein A/G Plus beads (Santa Cruz), the incubation was allowed to continue for a total of 17-19 h at 4°C. Immunoprecipitates were collected by centrifugation after washing three times with the NP-40 buffer . Following incubation with antibodies, the membranes were washed twice with the buffer B , once with blocking buffer B supplemented with 0.3% Tween 20, and twice more with the blocking buffer B. Each wash was carried out for 5 minutes on a rocking platform. Blocking buffer C  was used in all associated procedures for immunoblotting with biotinylated antibodies, and blotto  was used for immunoblotting with other antibodies. Working dilutions of antibodies were prepared as recommended by manufacturers.Immunoblotting with phosphotyrosine antibody was performed with a procedure adapted from Klapper et al.  to reducDensitometry was performed on Storm 860 phosphorimager (Amersham) with ECL Plus chemiluminescent reagent (Amersham). Quantitation was done using ImageQuant 5.0 software (Molecular Dynamics). Curve fitting was performed in GraphPad Prism 3.0 . Details pertaining to specific experiments are provided in the legends to the figures.Growth factor-activated PDGFRβ results in receptor cross-phosphorylation of tyrosine residues. We have previously shown that, in CHO-K1 cells stably expressing DRD4 (CHO/DRD4), dopamine stimulates the phosphorylation of ERK1/2 in a manner sensitive to the inhibition of the PDGFRβ kinase . TherefoUsing the CHO/DRD4-PR cells, we compared the pattern of PDGFRβ phosphorylation after stimulation with either 1 μM dopamine or 10 ng/mL PDGF-BB. As shown in Figure Since the receptor kinase activity is not enhanced through Tyr857 phosphorylation, the dopamine-induced PDGFRβ phosphorylation may be a result of the basal kinase activity of the PDGFRβ. The phospho-specific antibody against Tyr1021 of the PDGFRβ does recognize the endogenously expressed hamster receptor; therefore, we used this antibody to measure PDGFRβ phosphorylation in CHO/DRD4 cells. As shown in Figure To provide direct evidence for a role of the PDGFRβ in dopamine-stimulated ERK1/2 activation, we used siRNA to suppress endogenous PDGFRβ expression in the CHO/DRD4 cells. In these cells, the PDGFRβ exists as two isoforms that can be detected by western blotting: a maturely glycosylated receptor (180 kDa) and a 140 kDa, immaturely glycosylated, isoform ,23. In oWe then tried to examine the existence of PDGFRβ-specific ligand PDGF-B in the CHO-K1 cells. Previous reports have shown that HB-EGF acts as a paracrine factor in the GPCR-induced transactivation of EGFR . In addiThe metalloproteinase inhibitor GM6001 is known to block LPA-mediated Shc phosphorylation in COS-7 cells . Thus, wleft hand side) when cultured alone. Interestingly, when the two cell populations were co-cultured at a high density in a 1:1 ratio, dopamine-induced ERK1/2 phosphorylation was approximately half as that observed with the CHO/DRD4 monoculture, unlike the PDGF-BB-mediated ERK1/2 response, which was similar in the monocultures and co-culture. We speculated that the reduced dopamine-mediated ERK1/2 phosphorylation in the co-culture is due to the decrease of paracrine mediator, as only half of the cells in this culture express DRD4. Furthermore, immunoprecipitation of the FLAG-tagged PDGFRβ in the CHO/PR cells from the co-culture, followed by immunoblotting of phospho-tyrosine residues revealed an enhanced phosphorylation in response to PDGF-BB, but not dopamine . This further suggests that a paracrine mediator of PDGFRβ activation is not released by the CHO/DRD4 cells within the co-culture, in response to dopamine. Taken together, our results from the co-culture and metalloproteinase inhibitor studies suggest that the mechanism of DRD4-mediated transactivation of PDGFRβ does not involve a paracrine factor or it involves a paracrine factor that does not induce transactivation.Although the RT-PCR experiments showed the absence of PDGFRβ-specific ligands, we sought to exclude the possible activation of PDGFRβ by a yet undiscovered ligand. To identify any potential PDGFRβ ligand released upon dopamine stimulation, a co-culture strategy ,26 in whEC50 values were determined by fitting to a sigmoidal dose-response equation in GraphPad Prism. The log EC50 values for dopamine were: CHO/DRD4, -8.56 ± 0.11 (n = 4); CHO/DRD4-PR, -8.44 ± 0.23 (n = 3). The log EC50 values for PDGF-BB were: CHO/DRD4, -10.13 ± 0.16 (n = 4); CHO/DRD4-PR, -10.41 ± 0.15 (n = 3). Assays were carried out at or near EC50 concentrations of dopamine and PDGF-BB so that a false negative inhibition due to over-stimulation of the signaling pathway could be avoided  perturbs PDGFRα dimerization [S-transferase-PDGFRβ-Ig4 (GST-Ig4β) fusion protein was constructed. Pre-incubation of CHO/DRD4-PR cells with GST alone had little effect on the ability of dopamine or PDGF-BB to elicit an ERK1/2 response (Figure The lack of the need for PDGFRβ cross-phosphorylation in DRD4-mediated ERK1/2 activation suggests that PDGFRβ dimerization is also not required. We investigated the need for receptor dimerization, by utilizing a glutathione rization . The extrization ,18,27,28e Figure . HoweverPrevious data from our laboratory  suggesteThe present study has demonstrated a novel mechanism for PDGFRβ signaling, in which DRD4-mediated transactivation of PDGFRβ and the subsequent activation of ERK1/2 does not involve mechanisms that are characteristic of RTK activation. This new scheme breaks away from the prototypical model, where GPCR-mediated RTK transactivation is ligand-dependent, and so occurs similarly to classical RTK signaling involving receptor dimerization and cross-phosphorylation.The use of RT-PCR failed to detect any of the known endogenous PDGFRβ ligands within our CHO-K1 cells Figure . AdditioFurthermore, dimerization and subsequent cross-phosphorylation of PDGFR are also not required for DRD4-mediated transactivation. DRD4 stimulation led to increased general tyrosine phosphorylation of the PDGFRβ Figure . HoweverIn the context of transactivation, the PDGFRβ tyrosine phosphorylation that follows DRD4 stimulation appears to be unrelated to the ERK1/2 signaling pathway. It is not clear how the transactivated PDGFRβ mediates signaling in the absence of enhanced tyrosine phosphorylation. The sensitivity of DRD4-mediated ERK1/2 signaling to PDGFRβ kinase inhibitors suggests that a certain level of basal kinase activity is required. An alternative explanation is that through a tyrosine phosphorylation-independent conformational change, the PDGFRβ may act as a scaffold to mediate DRD4-PDGFRβ-ERK1/2 signaling. In fact, growth factor-activated PDGFRβ is known to undergo conformational changes that can be either enhanced by mutations that result in constitutive activity  or thoseThe present study shows that unlike the prototypical mechanism underlying GPCR-RTK transactivation, DRD4-PDGFRβ-ERK1/2 signaling does not involve a paracrine component, nor does it require PDGFRβ cross-phosphorylation and dimerization. This suggests that PDGFRβ can act as a monomeric scaffold to transmit DRD4-mediated signals, in a tyrosine phosphorylation-independent manner. We have recently demonstrated that DRD4 is able to transactivate immaturely glycosylated PDGFRβ, which is intracellularly localized . These fThe authors declare that they have no competing interests.CSS did the RT-PCR, dose-response curves, and c-truncated PDGF receptor experiments. SMV carried out the GST fusion protein work. RSG performed the siRNA studies. MSH carried out the immunoprecipitation and Western blotting experiments for PDGF receptor phosphorylation. Both SMV and CSS participated in the experimental design and writing of the manuscript. FL contributed to important discussions and manuscript writing. The project was initially under the supervision of the late Dr. Van Tol who passed away in April 2006. All authors read and approved the final manuscript.


Release of ASK1 activates its kinase activity thus stimulating                        the p38 MAPK pathway.  The level of p38 MAPK activity is,                        therefore, regulated by the balance of free vs. bound ASK1.                        Longevity of Ames dwarf mice is attributed to their resistance                        to oxidative stress.  The levels of (SH)2 Trx-ASK1 are more abundant                        in young and old dwarf mice compared to their age-matched controls                        suggesting that the levels of this complex may play a role in                        their resistance to oxidative stress.  In these studies we demonstrate                        that dermal fibroblasts from these long-lived mice exhibit (a)                        higher levels of (SH)2Trx-ASK1 that correlate with their                        resistance to ROS generated by inhibitors of electron transport                        chain complexes CI (rotenone), CII (3-nitropropionic acid),                        CIII, (antimycin A), and H2O2-mediated activation of p38 MAPK,                        and (b) maintain their in vivo resistance to ROS generated by                        3NPA.  We propose that elevated levels of (SH)2Trx-ASK1 play a                        role in conferring resistance to mitochondrial generated oxidative                        stress and decreased endogenous ROS which are characteristics of                        longevity determination.                Activation of p38 MAPK by ROS involves dissociation of                        an inactive, reduced thioredoxin-ASK1 complex [(SH) The Free Radical Theory of Aging proposes                        that endogenously produced oxygen radicals (ROS) are a basic cause of                        the progressive age-associated declines in tissue functions, and that oxidative                        stress generated by extrinsic environmental factors accelerate these declines                        -6.  Some2)Trx-ASK1] 2 by certain oxidants including                        mitochondrial generated ROS mediates the dissociation of the complex and                        activation, by phosphorylation, of the free ASK1 [2O2, TNF, and Fas to                        activation of the p38 MAPK and SAPK/JNK stress response pathways [ 2Trx-ASK1                        may be altered by ROS generated by mitochondrial electron transport chain (ETC)                        dysfunction.  Thus, the association and dissociation of the (SH) 2Trx-ASK1                        complex may be part of the molecular mechanism that elevates the endogenous                        activity of the p38 MAPK pathway in aged mice [                                2Trx-ASK1 complex level is dramatically decreased in response to                        mitochondrial ROS generated by rotenone (ROT), an inhibitor of electron transport                        chain complex 1 (CI) [The mechanism that links ROS generated by                        dysfunctional mitochondria to the activation of the p38 MAPK and SAPK/JNK                        pathways involves regulation of the level of a reduced thioredoxin-ASK1 complex                        [ ,25, and x 1 (CI) .       e.g.,                        CI,  can activate the p38 MAPK stress signaling cascade via the dissociation of                        the (SH)2Trx-ASK1complex.  Studies with nematodes [Drosophila [2O2, paraquat and heat shock [in vivo levels of the (SH)2Trx-ASK1                        complex in young vs. old controls to those in age-matched long-lived Snell                        dwarf mice and shown that the complex levels are significantly elevated in the                        dwarf livers and that the activities of the p38 MAPK pathway are significantly                        down regulated in vivo [2Trx-ASK1                        complex have been reported [ 2Trx-ASK1 levels is dependent upon the redox status of Trx                         embryonic                        fibroblasts by H2O2 and TNF is abolished in these cells                        which exhibit resistance to these ROS producing stress factors [2O2,                        paraquat and heat [ 2Trx-ASK1 complex, which is redox                        sensitive, play a role in their resistance to oxidative stress and survival.                         Mechanistically, these studies suggest that the activity of uncomplexed ASK1                        may be required for the sustained activities of p38 MAPK and SAPK/JNK [These studies suggest that ROS generated by the                        dysfunction of a specific component of the electron transport chain (ETC), ematodes -29,    osophila -33, and osophila -40 suggeosophila ,37,38.  at shock ,42.     in vivo .  Similareported -21,23. Wand heat ,37,41,42SAPK/JNK ,20,21,23 2Trx-ASK1                        complex and activation of the p38 MAPK pathway in fibroblasts from young (3-4                        mos), middle aged (10-12 mos) and old (21-24 mos) wild type and Ames dwarf mice                        and whether this in vivo redox sensitive regulatory process is                        maintained in the fibroblast cell cultures.  Our studies address the potential                        role of the regulation of the (SH)2Trx-ASK1 complex levels in the                        mechanism of response of stress pathways to ROS generated by specific                        mitochondrial electron transport (ETC) dysfunction, which is a major                        physiological source of endogenous oxidative stress in aging tissues.  We                        propose that the mechanism by which long-lived mice exhibit characteristics of                        resistance to oxidative stress may involve the intracellular balance between                        free ASK1 vs. (SH)2Trx-ASK1 complex, that this mediates the level of                        activity of the stress response p38 MAPK and SAPK/JNK pathways, and is a basic                        difference between wild type and long lived mice.  To test our hypothesis, we                        correlate the levels of (SH)2Trx-ASK1 complex formation to the                        activity of the downstream p38 MAPK pathway, and resistance to oxidative stress                        in the Ames dwarf fibroblasts treated with rotenone (ROT), a specific inhibitor                        of ETC CI, 3-nitropropionic acid (3-NPA), a specific inhibitor of CII,                        antimycin A (AA), a specific inhibitor of CIII, and H2O2,                        a product of metabolism and inducer of oxidative stress, all of which mimic the                        generation of ROS by mitochondrial dysfunction [In these studies we focus upon the role                        of ETC generated ROS on determination of the levels of (SH)d old 21- mos wildfunction ,43.    2-ASK1complex                            and lower p38 MAPK pathway activities than their age matched wild-type controls                            [2-ASK1                            complex as a part of the mechanism of the redox regulation of p38 MAPK activity                            and resistance to oxidative stress and in development of age associated                            characteristics we established fibroblast cultures from the tails of young (4-6                            mos), middle aged (10-12 mos) and old (20-24 mos) wild-type and Ames mouse tail                            fibroblasts  are significantly lower than those of the                            confluent middle aged (30 x 104) and old cell cultures (~40 x 104).                             At the same time, a major difference in the                            growth of the Ames cells involves a                            lag period of ~ 3 days in both young and old cells after which time the young cells                            begin to replicate to attain a confluence of ~20 x 104 at day 8.                             The middle aged cells, however, exhibit a 6 day lag period after which they                            replicate and reach a stationary phase (12 x 104) which is                            considerably lower than the age-matched wild-type cells  which is                            considerably lower than the corresponding wild type cells (~40 x 104).                             In general, the growth characteristics of the Ames dwarf fibroblasts include an                            extended lag period, and a lower cell population at stationary phase.  There                            are no indications that any of these cultured cells, wild-type or dwarf,                            developed characteristics of transformation.                                        Our previous studies have shown that the livers from                            young (3-6 mos) and old 20-23 mos) long-lived Snell dwarf male mice exhibit                            significantly higher endogenous levels of the Trx(SH)         .  We int         ,34-39.  0-23 mos s Figure -2C.  Thes Figure .  The wis Figure .  The ol2Trx-ASK1 complex [i.e.,                            dissociation of the (SH)2Trx-ASK1 complex.  To address this we                            examined whether inhibitors of CI (ROT), CII ( 3-NPA), CIII (Antimycin A) and H2O2                            stimulate the dissociation of this complex and p38 MAPK activity in wild-type                            vs. dwarf fibroblasts.  To examine the                            stability of the (SH)2Trx-complex in response to these factors we measured the                            levels of the intact complex by co-immunoprecipitation.  The data in Figure 2O2                             complex .  These   Figure .  These 2Trx-ASK1 complex in the dwarf cells.                             To demonstrate that the dwarf fibroblasts are in a more reduced state than                            those from wild type mice, we treated the extracts of young wild type and dwarf                            fibroblasts with a thiol-reactive probe,                            4-acetamido-4'-maleimidyl-stilbene-2,2'-disulfonic acid (AMS), to measure the                            level of Trx in these fibroblasts.  The binding of the thiol probe to reduced                            Trx causes a decreased mobility of the modified Trx in PAGE gels.  The results                            show the dwarf fibroblasts have an approximately 5-fold higher level of the                            reduced form of Trx as in the wild-type fibroblasts  cells after both 5 μM and 20 μM treatment p38 MAPK .  These 71 and that this                            activity is significantly lower in both young and aged Snell dwarf liver nuclei                            [in vitro phosphorylation                            of Thr71 of  recombinant ATF-2 in wild-type and dwarf cell                            extracts.  The data in Figure 71                            is induced by treatment of the wild-type and dwarf fibroblasts with 5 μM and 20 μM ROT for 60 min.  Although the                            endogenous nuclear levels of p38 MAPK phosphorylation of the ATF-2 Thr71                            are similar in the wild-type and dwarf fibroblasts, treatment of the wild type                            fibroblasts with 5 μM ROT resulted in ~4-5-fold induction of the Thr71                            phosphorylation and a ~10-fold induction by 20 μM ROT.  These results showed that the kinase                            activity in ROT treated dwarf fibroblasts is significantly lower than their                            age-matched wild-type controls.  The attenuated activity of nuclear P-p38 MAPK                            correlates with the decrease in ATF-2 activation and with the elevated levels                            of (SH)2Trx-ASK1complex in the dwarf fibroblasts.  Our data suggest                            that the attenuated targeting and activation of ATF-2 in the dwarf fibroblast                            is indicative of their resistance to oxidative stress.                                        Our past studies have                            indicated that the elevated basal level of p38 MAPK kinase activity in nuclei                            of old mouse livers mediates an increased activation of ATF-2 via the                            phosphorylation of its catalytic site at Thr         . Since Ai.e., phosphorylation of p38 MAPK catalytic site                            residues by the fibroblasts from young and old wild type and dwarf mice are                            shown in Figure i.e., at                            60 minutes of treatment whereas the response by the old wild-type fibroblasts                            which also peaked at 30 min. was sustained for up to 120 minutes, twice the                            time of the recovery of the young fibroblasts.  We interpret this to indicate                            that the Ames fibroblasts, young and old exhibit a similar level of resistance                            to CII (succinic dehydrogenase) dysfunction.                                        In recent studies we demonstrated that                            3-NPA, an inhibitor of succinic dehydrogenase at CII of the mitochondrial ETC                            generates ROS at a site between the ubiquinol pool and the 3-NPA block in CII                            .  Furthe0 center,                            oriented toward the intermembrane space and the Qi center, located                            in the inner membrane and facing the mitochondrial membrane.  Antimycin A (AA)                            inhibits CIII at the Qi center and increases superoxide generation                            at the Q0 center.  In order to analyze the effects of ROS generated                            by CIII dysfunction we treated the fibroblasts with 50 μM AA for 120 minutes.  The data in Figure CIII, which has been identified as a key                            site for ROS generation , has two2O2 has been used as a measure                            of CI and CIII as well as extra-mitochondrial sites of ROS production.  The                            data in Figure 2O2 treatment for 30                            minutes mediates almost complete dissociation of the (SH)2Trx-ASK1                            complex in the young wild-type fibroblasts.  On the other hand the dwarf cells                            show a significant resistance to the same treatment.  Thus, although the basal                            levels of the complex are similar in wild-type and dwarf cells, its                            dissociation is significant-ly attenuated.                             These results suggest that the H2O2-mediated induction                            of phosphorylation of the p38 MAPK catalytic site may involve dissociation of                            the complex and that this response to H2O2 is attenuated                            in the dwarf.  To address this question we measured the levels of activation of                            p38 MAPK by varying concentrations of H2O2.  The data in                            Figure 2O2  and                            increases dramatically from 0.6 mM to 1 mM H2O2.                             On the other hand, similar treatment of the young and old dwarf cells show that                            the response to all H2O2 concentrations is significantly                            attenuated.  The responses of wild-type and dwarf fibroblasts to H2O2                            correlate with the levels of dissociation of the (SH)2Trx-ASK1                            complex.   Our data suggest that the dwarf fibroblasts are resistant to H2O2mediated oxidative stress.                                        HThe data in Figure Approximately                        90% of age-associated ROS production is of mitochondrial origin due to ETC                        dysfunction .  This e2O2may activate the p38 MAPK stress response pathway, and may be a chronic                        physiological characteristic that plays an important role in the development of                        physiological characteristics of aging [i.e., CI, CII                        and CIII, and H2O2                        activate the p38 MAPK stress response pathway. We propose that the mechanism                        of this activation involves the ROS mediated release of ASK1 from an                        (SH)2Trx-ASK1 complex thereby enabling ASK1 to activate the p38 MAPK                        and SAPK/JNK pathways 2Trx-ASK1                        may be a physiological characteristic of  longevity.  This hypothesis is                        strongly supported by the studies presented here, which have shown that in the                        dwarf fibroblasts (a) the levels of the (SH)2Trx-ASK1 complex and                        reduced Trx are significantly higher; (b) the dissociation of the complex by                        ROT is attenuated in both young and old Ames dwarf cells.  These mice also                        exhibit an extended life span and resistance to oxidative stress.  Our proposal                        that the level of the (SH)2Trx-ASK1 complex is indicative of and                        part of the mechanism of resistance to oxidative stress is supported by the                        fact that the dissociation of the complex is much more complete in response to                        the specific inhibitors of CI, CII, and CIII                        activity and to H2O2  in the wild-type cells than in age-matched                        dwarf cells.                                Our past studies have                        demonstrated that the levels of the (SH)in vivo mechanism of activation of the p38 MAPK pathway is not                        altered by their in vitro environment (tissue culture).  Thus, the                        regulation of the level of (SH)2Trx-ASK1 complex formation and                        dissociation, and resistance to the oxidative stress generated by the ETC                        complexes is retained in the cultured dwarf fibroblasts.  Furthermore, this in                                vivo characteristic is maintained in the fibroblast for 50 passages as                        indicated by their continued resistance to H2O2.  We                        propose, therefore that the mechanism of resistance to oxidative stress in the                        dwarf fibroblasts may be an epigenetic physiological process which is                        established in vivo and is maintained in vitro.                                Using cultured Ames dwarf fibroblasts has revealed                        that the 2Trx-ASK1 complex to dissociation in                        response to ROT treatment is consistent with our proposed mechanism that ROS                        from ETC dysfunction activates the p38 MAPK pathway via the dissociation of                        this complex.  We propose, therefore, that this mechanism is a component of the                        physiological interactions that define the overall mechanism of resistance to                        oxidative stress.                                Our proposed mechanism is supported by the response of                        the young ROT treated dwarf fibroblasts to treatment with the antioxidant,                        NAC.  For example, attenuation of the ROT mediated activation of p38 MAPK by                        NAC and resistance of the (SH)e.g., the acute phase response                        genes; (b) the transcription factors that target these genes in aged tissues                        [2Trx-ASK1 ratio in                        response to the chronic increase in ROS production 2Trx-ASK1.                         Thus, the levels of Trx in the oxidized or reduced state are dependent on the                        effects of aging on the activity of thioredoxin reductase.  Based on our                        results we propose that (a) the ratio of ASK1 : (SH)2Trx-ASK1 shifts                        toward the dissociation of the complex, which elevates the pool of free ASK1 in                        aged tissue;   (b) this is the mechanism that causes an increased basal level                        of activity of the p38 MAPK and SAPK/JNK pathways in aged tissues, and (c) the                        aged tissues become less resistant to oxidative stress .  These                        physiological processes would explain the progressive loss of resistance to ROT                        we observed in both wild-type and dwarf cells.                                Our past studies have shown that aging                        tissues develop a state of chronic stress and that this occurs in the absence                        of extrinsic challenges ,7,8.  Th         ,7; and  to oxidative stress.  This is consistent with                        the observation that ASK1 is selectively required for sustained activation of                        the p38 MAPK and SAPK/JNK pathways induced by oxidative stress [(-/-) embryonic fibroblasts in which the H2O2                        and TNF-mediated sustained activation of p38 MAPK and SAPK/JNK is lost.  These                        cells also exhibit elevated levels of the (SH)2Trx-ASK1 complex                        [(-/-) embryonic fibroblasts.  Furthermore,                        the elevated (SH)2Trx-ASK1 levels and attenuated p38 MAPK activity                        in livers of Snell dwarf mice [Interestingly, since p38 MAPK is a master regulator of                        the activity of many genes, our proposed mechanism implies that the (SH)e stress .  This w         .  We proarf mice  suggests: reduced                        Trx which determines the activity of both ASK1 and the downstream components of                        the p38 MAPK pathway suggests a lower level of endogenous oxidative stress in                        the dwarf mouse.  This proposal is further supported by the higher levels of                        reduced thioredoxin.                                Resistance to oxidative stress is an important                        physiological factor in the development of Snell and Ames dwarf longevity ,37.  Our2Trx-ASK1 levels due to the redox                        status of Trx may be a part of the mechanism of resistance to oxidative                        stress.  For example, activation of p38 MAPK in ASK-1(-/-) embryonic                        fibroblasts by H2O2 and TNF is abolished in these cells                        which are resistance to H2O2-and TNF-induced apoptosis                        [in vivo) and of the Ames dwarf                        fibroblasts (in vitro), as well as the elevated levels of reduced                        thioredoxin and (SH)2Trx-ASK1 complex may contribute to the resistance                        to oxidative stress in these long-lived mice.  Our hypothesis is supported by                        the observation that Snell dwarf fibroblasts are resistant to oxidative stress                        generated by UV light, heavy metal (Cd), H2O2, paraquat                        and heat [Our studies raise the question of whether the                        regulation or elevation of (SH)         .  ASK-1 and heat ,42.    2Trx-ASK1                        complex may represent the mechanism of an integrated simultaneous regulation of                        multiple genes whose combined levels of activity define the physiological                        status that confers resistance to oxidative stress and longevity.                                The physiological resistance to oxidative                        stress is a complex and likely integrated biological process.  The Ingenuity                        analysis of p38 MAPK protein-protein interactions shows an extensive number of                        genes whose activities are potentially important for the response and resistance                        to oxidative stress.  Both the up- and down-regulation of the p38 MAPK pathway                        activity by the ROS-mediated regulation of the level of (SH)2O2 , or indirectly by binding to signaling                        components and modulating their functions.  The complexing of Trx with the                        N-terminus of ASK-1 determines the ROS mediated regulation of activity of the                        p38 MAPK and SAPK/JNK stress response pathways [Trx can exert its protective functions either directly                        as an antioxidant by reducing the ability of oxidized Trx peroxidase to scavenge                        ROS, such as Hpathways .   Sincepathways  than in Animals and tissues                                .                  The                        initial breeding pairs of wild-type and Ames dwarf mice were obtained from The                        Jackson Laboratory.  The mice were bred at the University of Texas Medical                        Branch and maintained in accordance with the institutional guidelines and were                        treated in accordance with UTMB-IACUC-approved protocols.  The genotypes of the                        mice have been determined and described [                escribed .  Mouse Tail fibroblast isolation.                  The procedures described by Salmon et al. [o C incubator with 5%                        CO2 in air.  The cells were split six-fold 7-10 days after                        initiation of cultures to generate 1st passage cells.  The 1st                        passage cells were cultured for another 7-10 days; some of them which were                        split and seeded in 12-well culture dishes were used to generate growth                        curves.  The remaining cells were further expanded to 2nd and 3rd                        passages of cells for studies with ROT, 3-NPA, AA and H2O2.                                                n et al.  were   Cell cultures and treatments.                  The tail fibroblasts were cultured in DMEM (Gibco or                        CellGro) containing 15% fetal bovine serum (Hyclone) and supplemented with                        antibiotic plus antimycotic solution (Gibco).  For ROT, 3-NPA, and AA (Sigma)                        treatment, the cells, at passage 3 or 4, were seeded in 100 mm2                        culture dishes and cultured for 4-5 days until they reached approximately 70%                        confluence.  A day prior to inhibitor treatment, the medium was replaced with                        DMEM containing 0.5% FBS and antibiotics.  The cells were treated with 5 μM or 20 μM ROT, or with a series of ROT concentrations ranging                        from 20 μM to100 μM for 30 min; with 5                        mM or 20 mM 3-NPA, or 50 μM AA for 30-, 60-, and 120-min.; with 0.2-1.0 mM, or                        0.5-4.0 mM of H2O2 for 30 min.  The cells were pretreated                        with 20 mM of N-acetyl cysteine,  for 30 min prior to the addition                        of ROT.  Protease inhibitors and phosphatase inhibitors were added as described                        [                         .       Growth curves of fibroblasts.                  Fibroblasts (3 x 104) from 3 different                        wild-type and Ames dwarf mice of young (3-6 mos), middle aged (10-12 mos) and                        old (21-24 mos) mice at passage 3 were plated in each well of a 12-well plate.                         The cells were harvested  at passage 3 or 4 and counted every day for 8 days.                         For the growth curves of fibroblasts in the continuous presence of rotenone,                        the same number of young wild-type and dwarf fibroblasts (40 x 104/dish),                        at passage 4 were plated in a 100 mm cell culture dish and 20 μM of ROT was added to the treated groups of cells.  The cells were                        harvested and counted daily until the untreated wild-type cells become                        confluent in the dishes at the 4th day.                                                Preparation of cellular extracts                                .                  Fibroblasts                        were washed 3x with ice-cold TBS , and scrapped from the                        dish by using a rubber policeman (Fisher).  The cells were centrifuged in a                        Beckman J6B at 400 g for 5 min at 4oC.  The extracts were prepared                        following the protocols described [                escribed .  ProteiWestern blot analyses and                                immunoprecipitation assays.                                         Fibroblast cytosolic protein (30 μg) was resolved on a                        precast 4-20% gradient SDS-PAGE (Lonza) or a 4-20% Criterion SDS-PAGE (Bio-Rad)                        and transferred onto an Immobilon PVDF membrane .                         After primary and secondary antibody incubations, the proteins were detected according                        to the procedures recommended by the manufacturers using the Supersignal kits                        from Pierce  or Immobilon Western kit (Millipore).  In the cases                        where phosphorylation levels were measured, the membranes were probed with the                        antiphospho antibody first and the same membranes were washed and stripped,                        and probed with the corresponding antibodies to detect the levels of total                        proteins.  All the primary antibodies were purchased from Santa Cruz                        Biotechnology  except for   P-p38 MAPK (#9211), p38 MAPK kinase                        assay kit .  The bands detected                        by either antibody or antiphospho-antibody were imaged in a MultiImage Light                        Cabinet (Alpha Innotech) and the intensity of the image was measured.                                                oC overnight. Protein                        A-conjugated agarose beads (30 μL) was added and incubated for                        additional 2-4 hr.  The beads were washed 4x with PBS and the eluted protein                        was precipitated by boiling for 5 min.  The precipitated protein was collected                        from the supernatant after boiling for 5 min.  The supernatant was resolved on                        a precast SDS-gel and transferred, and the membranes were processed as a                        regular Western blot.  The negative control tubes which did not contain                        proteins were processed in the same manner.                                Immunoprecipitation assays                        used to measure the pool levels of Trx bound to ASK1 were performed following                        the procedure provided by the manufacturer .  Briefly,                        200 μg of pooled protein from 3 samples was precleared with                        proteon A conjugated agarose beads without antibody for 2 hr and then incubated                        with 4 μl of antibody at 4Determination of levels                                of reduced thioredoxin in fibroblasts.                                         To detect the endogenous level of reduced thioredoxin in young wild-type and                        dwarf fibroblasts we added the thiol-interacting reagent,                        4-acetamido-4'-maleimidyl stilbene-2,2'-disulfonic acid                         to 100 μg of cell extracts at a final concentration of 15 mM                        in 20 mM Tris (pH 8.0) and incubated for 2 hrs at room temperature.  The                        reaction was terminated by adding SDS running buffer and the mixture was boiled                        for 5 min. Aliquots (15 μl) of samples were loaded onto a 15% SDS-PAGE, and the                        reduced form of thioredoxin was detected using thioredoxin antibody in a                        regular Western blot analysis.                                                In Vitro                     kinase assay.                The in vitro p38 MAPK kinase assay                        was performed by modification of the procedures described by the manufacturer                        of the p38 MAPK assay kit .  Fibroblast                        extract  was mixed with 20 μl of immobilized phosphor-p38 MAPK slurry in 1x lysis buffer, and                        incubated with constant rotation overnight at 4oC.  The                        immunoprecipitated beads were washed 2x with lysis buffer and 2x with kinase                        buffer, and then resuspended in 50 μl of kinase buffer                        containing 1 μg of ATF-2 fusion protein and 4 μM ATP.  The kinase reaction was carried out at 30oC for 25                        min, and terminated by addition of 25 μl 3x SDS sample                        loading buffer.  The reaction mix was boiled for 5 min and the supernatant was                        collected and loaded on a precast 4-20% SDS-PAGE.  The gel was blotted onto a                        PVDF membrane and processed as described for Western blots using Supersignal                        West Pico detection kit.  The level of ATF-2 phosphorylated by p38 MAPK was                        determined using an antiphospho-ATF-2 antibody. The intensity of the                        phosphorylated ATF-2 band represents the relative p38 MAPK kinase activity                        precipitated by antiphospho-p38 MAPK antibody.                                Statistical analysis                                .                                         Statistical analyses were performed for age-matched comparisons with the single                        dependent variable being the Ames dwarf mutants.  The normalized values of                        protein and phosphoprotein bands or the average numbers of cells were analyzed                        using the two-tailed t-test to test the difference in means between                        age-matched groups at a significance level of 0.05.  The symbols (*) indicate                        statistical significance for the values represented by the bar or time point.


The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA2 activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA2 phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA2 protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA2 activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis.Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A Alcohol abuse is a well-recognized cause of damage to the liver, brain and gastrointestinal tract, and its excessive consumption is associated with an increased risk of cancer of the liver, pharynx, larynx, esophagus, and the oral cavity –3. MoreoAlthough the maintenance of mucosal integrity along the alimentary tract relays on multiple molecular processes, the two of the most prominent are the production of NO by the nitric oxide synthase (NOS) system and the formation of prostaglandins generated from AA by the action of cyclooxygenase (COX) enzymes –14. Furt2 (cPLA2) enzyme arachidonic acid for 4 h [3H]arachidonic acid by scintillation spectrometry. The measurement of cPLA2 activity in the acinar cells following various experimental conditions was carried out using cPLA2 assay kit (Cayman) with thioarachidonoylphosphatidylcholine as substrate [To assess the release of AA from the acinar cells of salivary gland into the incubation medium, aliquots of the cell suspension (1 ml) were labeled in DMEM with 20  for 4 h , and resubstrate . 2 S-nitrosylation was carried out utilizing a biotin switch procedure for protein S-nitrosylation [μg/ml) or L-NAME (400 μM) + ghrelin or PD98059 (30 μM) + ghrelin and incubated for 2 h in the presence of 3% ethanol. Following centrifugation, the recovered cells were lysed in HEN lysis buffer and the unnitrosylated thiol groups were blocked with S-methyl methanethiosulfonate reagent [μl of elution buffer  containing 1% 2-mercaptoethanol [Detection of cPLAsylation , 38. Theoethanol . The obtμg protein/lane [2 protein at 4°C for 16 h. After 1 h incubation with the horseradish peroxidase-conjugated secondary antibody, the phosphorylated proteins were revealed using an enhanced chemiluminescence detection kit (Pierce). Membranes were stripped by incubation in 1 M Tris-HCl (pH 6.8), 10% SDS, and 10 mM dithiotreitol for 30 min at 55°C, and reprobed with antibody against total cPLA2. Immunoblotting was performed using specific antibodies directed against cPLA2 and phospho-cPLA2 (Ser505) .The acinar cells from the control and experimental treatments were collected by centrifugation, washed with phosphate-buffered saline and resuspended in ice-cold lysis buffer . Followiein/lane . The sepP < .05.All experiments were carried out using duplicate sampling and the results are expressed as means ± SD. Analysis of variance (ANOVA) followed by nonparametric Kruskal-Wallis test was used to determine significance and the significance level was set at μg/ml of ghrelin  for the suppression of the cytotoxic effect of ethanol, resulted in a 28.3% stimulation in arachidonic acid release. This effect of ghrelin on was subject to inhibition by the cNOS inhibitor, L-NAME, while the iNOS inhibitor, 1400W had no effect. Moreover, the ghrelin-induced up-regulation in the acinar cell arachidonic acid release was inhibited by Src kinase inhibitor, PP2 and MAPK/ERK1/2 inhibitor, PD98059 (We next sought additional leads into the involvement of cNOS in ghrelin-induced signaling leading to up-regulation in salivary gland cell PGE2 generation. As the initial and rate limiting step in prostaglandin production is the liberation of arachidonic acid from membrane phospholipids by highly selective cPLA2 –24, we e PD98059  The stim PD98059 . 2 for rapid release of arachidonic acid involves Src kinase-dependent MAPK/ERK activation of the enzyme through phosphorylation [2 enzymatic activity. We found that preincubation with ghrelin countered the detrimental effect of ethanol on arachidonic acid release and evoked a 71.8% increase in the cPLA2 activity (2 activity, furthermore, was subject to suppression by Src inhibitor, PP2 and ERK1/2 inhibitor, PD98059 as well as to the inhibitor of cNOS, L-NAME (2 activation for the increase in PGE2 generation.As the activation of cPLArylation , 25, 27,activity . The ghr, L-NAME , whereas, L-NAME . These r, L-NAME , point t2 enzymes [2 (2 activation by ghrelin, the acinar cells prior to ghrelin incubation, were pretreated with cNOS inhibitor, L-NAME or ERK1/2 inhibitor, PD98059, and the lysates subjected to biotin switch procedure were examined with antibodies directed against phospho-cPLA2 and total cPLA2. We observed that ghrelin prevention of the ethanol-induced cytotoxicity was reflected in the increase in cPLA2 protein phosphorylation as well as S-nitrosylation (2 phosphorylation, whereas ERK1/2 inhibitor, PD98059, caused the blockage in cPLA2 protein phosphorylation as well as S-nitrosylation. These data demonstrate that the activation of cPLA2 in sublingual salivary gland acinar cells by ghrelin involves both the phosphorylation and S-nitrosylation events, and that the enzyme protein phosphorylation is a prerequisite for its S-nitrosylation.Recent literature data indicate that up-regulation in NO production exerts the modulatory effect on PGE2 synthesis through protein cysteine S-nitrosylation of cyclooxygenase and cPLA enzymes , 20, 28. enzymes , 37, 38,zymes [2 . To assesylation . PreincuInvestigations into the nature of factors involved in the maintenance of mucosal integrity along the alimentary tract, including that of oral cavity, have brought to the forefront the role of ghrelin in processes of mucosal defense and repair –33. This2 activity and AA release. Moreover, a significant loss in the countering effect of ghrelin on the ethanol-induced toxicity was attained with cNOS inhibitor, L-NAME as well as indomethacin and a specific COX-1 inhibitor, SC-560, while specific COX-2 inhibitor, NS-398, and a selective iNOS inhibitor, 1400W had no effect. These results, are thus in keeping with the literature data demonstrating that the detrimental effects of ethanol on gastric mucosal integrity are associated with the impairment in NO synthesis and PGE2 generation controlled by cNOS and COX-1 enzyme systems [As the diminished secretion of saliva and oral mucosal inflammatory changes are well-recognized consequences of alcohol abuse on the health of oral cavity –4, in th systems , 33. Mor2 [2 activation. Employing the acinar cell labeled with AA, we demonstrated that ethanol cytotoxicity was manifested in a diminished AA release, while preincubation with ghrelin led to cPLA2 activation as evidenced by the increase in AA release. This stimulatory effect of ghrelin was subject to suppression by cNOS inhibitor, L-NAME but not the iNOS inhibitor, 1400W or the inhibitors of COX-1 and COX-2 enzymes. Moreover, we found that up-regulation in AA release and cPLA2 activity evoked by ghrelin was also suppressed by the inhibitor of ERK1/2, PD98059. Hence, the ghrelin-induced acinar cell cPLA2 activation for the increase in PGE2 production to counter ethanol cytotoxicity requires cNOS and MAPK/ERK participation. This interpretation of our results is supported by the literature data indicating that activation of cPLA2 for rapid increase in AA release and eicosanoid generation occurs through posttranslational MAPK/ERK-dependent enzyme protein phosphorylation on the critical Ser505 residue that plays a crucial role in Ca2+-dependent translocation of cPLA2 from cytosol to membrane to gain access to phospholipid substrates [Since the initial and rate limiting event in prostaglandin production is the release of AA from membrane phospholipids by highly selective cPLA2 –24, we fbstrates –27. 526 residue has been linked to the NO-induced enhancement in catalytic activity of COX-2 [2 activation through S-nitrosylation at cysteine152 was reported to be responsible for up-regulation in AA release in human epithelial cells [2 activation for the protection of salivary gland acinar cells against cytotoxic effect of ethanol, we examined the cPLA2 activity, and its protein S-nitrosylation and phosphorylation. We found that, In keeping with well known susceptibility of S-nitrosylated proteins to reduction by ascorbic acid [2 activity and AA release was liable to suppression by ascorbate. Preincubation with cNOS inhibitor, L-NAME led to the blockage in ghrelin-induced cPLA2 protein S-nitrosylation but had no effect on its phosphorylation, whereas ERK1/2 inhibitor, PD98059 caused the blockage in both the cPLA2 protein phosphorylation and S-nitrosylation. Thus, the activation of cPLA2 in the acinar cells by ghrelin for the increase in AA release and PGE2 synthesis involves the enzyme processing through the events of phosphorylation and S-nitrosylation. It is also apparent that the cPLA2 phosphorylation is a prerequisite for its S-nitrosylation.Interestingly, recent evidence indicates that in addition to posttranslational activation by phosphorylation, the increase in prostaglandin formation may also result from NO-induced enzyme protein S-nitrosylation , 20, 28.of COX-2 , and cPLbic acid , 37, 38,While our results on the mechanism of ghrelin protection of salivary gland acinar cells against ethanol cytotoxicity are obtained employing the in vitro system and pharmacological concentrations of the peptide, it should be noted that protective effects of the peripherally administered ghrelin against acute gastric mucosal injury induced in rat stomach by ethanol also required considerably higher concentrations of the peptide than that of central ghrelin , 32, 34.2 activation through S-nitrosylation for the increase in PGE2 generation. In summary, our findings demonstrate that ghrelin protection of salivary gland acinar cells against ethanol cytotoxicity involves cNOS-derived NO induction of cPLA


We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated  The corresponding trans-acting factor for the ARE is the nuclear factor erythroid 2-related factor 2 (Nrf2), a protein belonging to the bZIP (basic-leucine zipper) transcription factor family Disruption of redox homeostasis is associated with the toxic effects of many environmental insults and pathogenesis of aging-related diseases such as cancer and neurodegenerative disorders The Nrf2-ARE system is responsible for both basal and inducible expression of many genes involved in antioxidant responses, such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), glutathione S-transferase A1 , glutamate-cysteine ligase  modifier subunit (GCLM) and catalytic subunit (GCLC) Nrf2 is activated by a plethora of stress inducers, phyto-antioxidants, and pathological conditions. The current model of the molecular mechanism for Nrf2 activation is that oxidative stress modifies cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for degradation, resulting in compromised E3 activity and enhanced Nrf2 protein stability. The subsequent elevation in Nrf2 protein levels leads to Nrf2 nuclear accumulation, increased ARE-Nrf2 binding, and transactivation of its downstream genes c-jun N-terminal kinases (JNKs), the p38 kinases and other relatively less characterized kinases, all of which catalyze phosphorylation on either serine or threonine residues adjacent to a proline residue. MAPKs are activated in a cascade fashion, by MAP2Ks  that are in turn activated by MAP3Ks  in vivo at all. Another question that remains unanswered is whether MAPKs affect Keap1-mediated control of Nrf2 protein stability in vivo. Therefore, the exact role of MAPKs in Nrf2 activation, as well as the underlying molecular mechanism, remains poorly understood.The mitogen-activated protein kinases (MAPKs) are a family of highly related kinases consisting of the extracellular signal-regulated protein kinases (ERKs), the in vivo. Combined alanine substitution of those residues leads to only a very moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, which is primarily controlled by Keap1, is not altered in vivo by Nrf2 phosphorylation. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating the Nrf2 signaling pathway. We propose that MAPKs regulate Nrf2 mainly through indirect mechanisms.Here we report the first evidence indicating that Nrf2 is phosphorylated at several serine and threonine residues by MAPKs Plasmids expressing JNK1/2, ERK2, p38, MEKK3 and MEKK4 were generous gifts from Dr. Richard R. Vaillancourt at University of Arizona. Plasmids for HA-Nrf2 and Keap1-CBD proteins have been previously described HEK293T, MDA-MB-231 and NIH3T3 cells were purchased from ATCC. Cells were maintained in either Dulbecco's modified Eagle's medium (DMEM) or Eagle's minimal essential medium (MEM) in the presence of 10% fetal bovine serum (FBS). Transfections were performed with Lipofectamine Plus (Gibco BRL) according to the manufacturer's instructions.Antibodies against the HA epitope (Santa Cruz), chitin-binding domain (CBD) (New England Biolab), α-tubulin (Santa Cruz), and phosphorylated serine/threonine followed by a proline (pS/TP) (Abcam) were purchased from commercial sources. For detection of protein expression in total cell lysates, cells were lysed in sample buffer  48 hours following transfection. For immunoprecipitation assays, cells were lysed in RIPA buffer  containing 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Sigma). Cell lysates were pre-cleared with protein A beads and incubated with HA antibody-conjugated beads (Santa Cruz) or chitin beads (New England Biolab) overnight at 4°C. After washing three times with RIPA buffer, immunoprecipitated complexes were eluted in sample buffer by boiling for 5 minutes, electrophoresed through SDS-polyacrylamide gels, and subjected to immunoblot analysis. Indirect immunofluorescence analysis was performed as described before using anti-HA primary antibodies in vivo by immunoblot, transfected cells were lysed by boiling in a buffer containing 2% SDS, 150 mM NaCl, 10 mM Tris-HCl and 1 mM DTT. This rapid lysis procedure inactivated cellular phosphatases and therefore preserved phosphorylated-Nrf2 conjugates present in cells prior to lysis. These lysates were then diluted five-fold in buffer lacking SDS, followed by immunoprecipitation with HA antibody-conjugated beads.To detect phosphorylated Nrf2 3 pieces. Gel pieces were then subjected to a modified in-gel trypsin digestion procedure Mass spectrometry analysis was performed by the Harvard Taplin mass spectrometry core facility. Briefly, immunoprecipitated proteins were visualized by Coomassie staining and recoverd from the gel. Excised gel bands were cut into approximately 1 mmtert-butylhydroquinone (tBHQ), sulforaphane (SF), sodium arsenite (As), and cadmium chloride (Cd) were purchased from Sigma . Hydrogen peroxide (H2O2) was purchased from Mallinckrodt . Oridonin  was purchased from LKT Laboratories . 15-Deoxy-Δ-12, 14-prostaglandin J2 (PGJ2) was a kind gift from Dr. John W. Regan at University of Arizona.A plasmid encoding renilla luciferase was included in all samples to control for transfection efficiency. Reporter assays were performed using the Dual-Luciferase reporter gene assay system (Promega). Nrf2 inducers The real-time RT-PCR analysis and primer/probe sequence information was previously described t- test was used to determine the significant difference between two samples from three assays in reporter gene analysis and qPCR analysis.Student's in vitrobona fide substrate of MAPKs in vivo. To address this question, HEK293T cells were cotransfected with expression vectors for HA-tagged Nrf2 and the indicated MAPKs  were identified . Three oGenetic approaches were taken to explore the function of phosphorylation on Nrf2. Single or combined alanine substitutions on the phosphorylation sites were constructed, and the transcriptional activity of the resultant Nrf2 mutants were tested in the ARE-dependent reporter gene assays using the ARE sequence taken from either the NQO1 gene promoter  or the Gin vivo phosphorylation assay  failed to significantly change the GCLM ARE-dependent reporter gene activities in the absence or presence of pyrrolidine dithiocarbamate (PDTC), an Nrf2 inducer 2O2 , making  mutants . AnotherOne possible indirect mechanism is translational control on Nrf2 protein synthesis by MAPKs. MAPKs have been shown to modulate mTOR signaling pathways in controlling eukaryotic initiation factor 4 (eIF4) complex assembly, a critical step in Cap-dependent translational initiation, through inhibiting tuberous sclerosis complex 2 (TSC2) and activating 90-kD ribosomal protein S6 kinases (RSKs) Figure S1HEK293T cells were co-transfected with expression vectors for HA-Nrf2 wild-type or mutants and different kinases. Cell lysates were collected in denaturing conditions, and phosphorylation analysis was performed as described in (0.81 MB TIF)Click here for additional data file.Figure S2HEK293T cells were co-transfected with expression vectors for NF-κB p65, along upstream kinases IKKα and IKKβ in HEK293T cells. Phosphorylation of p65 at S536 by IKKs was confirmed by an antibody that recognizes this site-specific modification . The antibody for phospho-S/TP did not pick up any signal above background, indicating the specificity of this antibody.(0.83 MB TIF)Click here for additional data file.


We describe a case of multiple myeloma (MM) and secondary plasma cell leukemia (PCL) secreting IgE-kappa immunoglobulin. To our knowledge, only 2 cases of IgE-producing secondary PCL have been reported in the medical literature. In our patient, the only tumor marker available for monitoring the therapeutic response to chemotherapy and allogeneic stem cell transplantation was the quantitative M component at serum protein electrophoresis (SPEP), because serum free light chains were in the normal range, Bence-Jones proteinuria was absent, and quantitative serum IgE levels provided inaccurate and erratic results, due to the prozone effect. This is a laboratory phenomenon that occurs when antigen excess interferes with antibody-based methods requiring immune complex formation for detection. It is important to recognize the presence of a prozone effect, because it can produce falsely normal results, and therefore it could lead clinicians to incorrect assessment of the response to therapy. IgE myeloma is a very rare subtype of MM, and it represents < 0.01% of all plasma cell dyscrasias , approxiWe describe a case of IgE-kappa MM and secondary PCL with falsely normal serum levels of IgE due to the prozone effect.® (Flanders NJ), using the Total IgE method. All serum IgE dilutions were performed manually, using the manufacturer's diluent.A 53 year-old Caucasian man with unremarkable past medical history was diagnosed with MM in November of 2006. He presented with back pain, and MRI of the spine revealed multiple compression fractures. Skeletal survey was negative for lytic lesions. Bone marrow aspirate revealed 75% kappa-restricted atypical plasma cells, establishing the diagnosis of MM. Cytogenetic analysis was normal, and the translocation t11;14) was the only abnormality detected by the MM FISH panel. IFE was positive for monoclonal IgE-kappa proteins, IgE level was 5,300,000 IU/mL, serum free kappa was normal, and Bence-Jones proteinuria was absent. Patient received treatment with multiple regimens, which included dexamethasone, thalidomide, bortezomib, and lenalidomide. However, 28 months after the diagnosis, MM became refractory to those agents, and patient was referred to our Institution for autologous stem cell transplantation. Our review of the peripheral smear showed circulating atypical plasma cells, representing 52% of the WBC , and we made the diagnosis of secondary PCL. Bone marrow aspirate contained 80% plasma cells, harboring the original cytogenetic features. At flow cytometry, these cells were positive for CD38, CD138, and negative for CD56 and CD20. Initially, serum level of IgE was reported as normal, but a distinct M peak was present on SPEP. The result of the IgE level was found to be falsely normal due to the "prozone effect". Our laboratory observed the paradoxical increase of the IgE levels with progressively increasing dilutions of the serum sample , patienPCL is distinguished in "primary PCL", which occurs as a de novo presentation of the leukemia, and "secondary PCL", which is the leukemic transformation of a previously diagnosed MM. Our patient had the secondary form, because it developed 28 months after the initial diagnosis of MM. To our knowledge, 8 other cases of IgE-producing PCL have been reported in the medical literature, and only 2 of them were secondary PCL ,13. The An important aspect of our case was the unreliability of quantitative IgE levels in the assessment of disease response, due to the prozone effect. Response to therapy in our case was best monitored with the quantification of the M component at SPEP. The recent introduction of the quantitative serum free light chains (FLC) assay has offered another useful tumor marker for monitoring response to therapy in MM . Due to The prozone effect is a laboratory phenomenon that occurs when antigen excess interferes with antibody-based methods requiring immune complex formation for detection. For immunometric immunoassays, detection of the analyte  requires that each molecule binds to two separate reagent antibodies in an antibody-analyte-antibody complex: one antibody that "captures" the antigen and the second that provides a detection signal. With excessive amounts of analyte, each reagent antibody binds to separate analyte molecules, not forming the complex essential for detection. The presence of this "high-dose hook" effect should be suspected when the result on a diluted sample is higher than in the undiluted sample. The prozone effect is a well know phenomenon that may complicate the interpretation of various quantitative assays, including those for IgG and IgA ,20, and The authors declare that they have no competing interests.GT was responsible of the patient's treatment and conceived the study. WC carried out acquisition of data, laboratory analyses, and their critical interpretations. ND coordinated the study and helped to draft the manuscript. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Signaling through MEK→ERK1/2 and PI3 kinases is implicated in many aspects of cell physiology, including the survival of oxidant exposure. Oxidants play a role in numerous physiological and pathophysiological processes, many of which rely on transport in and out of the nucleus. However, how oxidative stress impacts nuclear trafficking is not well defined.To better understand the effect of stress on nucleocytoplasmic trafficking, we exposed cells to the oxidant diethyl maleate. This treatment activated MEK→ERK1/2 as well as PI3 kinase→Akt cascades and triggered the inhibition of classical nuclear import. To define the molecular mechanisms that regulate nuclear transport, we examined whether MEK and PI3 kinase signaling affected the localization of key transport factors. Using recently developed tools for image acquisition and analysis, the subcellular distributions of importin-α, CAS, and nucleoporins Nup153 and Nup88 were quantified in different cellular compartments. These studies identified specific profiles for the localization of transport factors in the nucleus and cytoplasm, and at the nuclear envelope. Our results demonstrate that MEK and PI3 kinase signaling as well as oxidative stress control nuclear trafficking and the localization of transport components. Furthermore, stress not only induced changes in transport factor distribution, but also upregulated post-translational modification of transport factors. Our results are consistent with the idea that the phosphorylation of importin-α, CAS, Nup153, and Nup88, and the O-GlcNAc modification of Nup153 increase when cells are exposed to oxidant.Our studies defined the complex regulation of classical nuclear import and identified key transport factors that are targeted by stress, MEK, and PI3 kinase signaling. Elevated levels of reactive oxygen species play a major role in human disease by contributing to type 2 diabetes, ischemia/reperfusion damage, cardiovascular diseases, stroke, Alzheimer's disease as well as numerous neurodegenerative disorders and syndromes Macromolecular trafficking across the nuclear envelope is mediated by nuclear pore complexes (NPCs), and for most cargos it relies on a specific transport apparatus. In particular, members of the importin-α and β families are crucial to move proteins in and out of the nucleus In addition to carriers and adaptors like importin-α, nucleoporins, also called nups, are essential to move cargoes across the nuclear envelope. Nucleoporins contribute to different aspects of nuclear trafficking; for instance, nucleoporins with FG repeats provide docking sites for import complexes during their translocation across the NPC. Some nucleoporins are stably bound to NPCs, whereas others are mobile and play a more dynamic role in trafficking Publications from several groups have demonstrated that classical nuclear import is sensitive to various forms of stress For the studies described here we have exposed HeLa cells to mild stress in order to limit the irreversible damage of cellular functions. We have previously shown that under these conditions nuclear envelopes remain intact; moreover, most of the cells remain viable and will recover from this treatment Many of the endogenous cargos for classical nuclear import interact with other cellular components, either in the nucleus, the cytoplasm or both. Such interactions may anchor the cargo in different cellular locations, thereby affecting its nucleocytoplasmic distribution. To avoid this complication, we generated the reporter NLS-mCherry, a small fluorescent protein that contains SV40 nuclear localization sequence (NLS) fused to the monomeric protein mCherry. The small size of NLS-mCherry allows diffusion across the NPC, whereas nuclear accumulation of the reporter protein relies on active nuclear import. Thus, the nuclear/cytoplasmic (nuc/cyt) distribution of NLS-mCherry serves as an indicator of classical import activity. Using tools recently developed for image acquisition and quantification of fluorescence in different cellular compartments MEK and PI3 kinase signaling cascades are essential components that regulate cell survival; this includes signaling events triggered by various physiological or pathological changes. Since both MEK and PI3 kinase signaling pathways are activated in response to DEM treatment Incubation of non-stressed or DEM-treated cells with these pharmacological kinase inhibitors led to several significant changes in classical import. First, MEK inhibition stimulated classical import under non-stress conditions. Second, classical import was diminished by DEM; however, this stress-induced inhibition decreased significantly when MEK signaling was abolished . TogetheFor most macromolecules nuclear trafficking is mediated by a specialized transport apparatus that consists of soluble factors and nucleoporins. Many nuclear transport factors reside in multiple cellular compartments, and changes in their steady-state distribution may impinge on nuclear trafficking. Thus, we determined how stress and pharmacological kinase inhibitors altered the localization of transport components. In The analyses of four transport components under eight different experimental conditions (s. 2–5) demonstrated that depending on the cellular compartment, stress and kinase inhibitors may have distinct effects. Moreover, individual factors displayed unique profiles of sensitivity towards stress and pharmacological kinase inhibitors. Therefore, in the following sections Classical nuclear import is mediated by the dimeric carrier importin-α/β1, which requires importin-α as an adaptor. As previous studies did not reveal drastic stress-induced changes in the localization of importin-β1 upon DEM treatment The effect of PI3 kinase and MEK inhibitors on importin-α distribution was dependent on the presence of DEM. Without oxidative stress, PI3 kinase and PI3 kinase/MEK inhibition, but not MEK inhibition alone, led to an increase of importin-α in the nucleus. By contrast, in stressed cells PI3 kinase and MEK inhibitors in combination elevated substantially the concentration of importin-α in nuclei . At the Taken together, importin-α pools in the nucleus or at the NE responded differently to changes in cell signaling. Without DEM, PI3 kinase inhibition elevated the concentrations of importin-α in nuclei, but not at the NE. In DEM-treated cells, prevention of PI3 and/or MEK kinase activation led to an increase of importin-α both in nuclei and at the NE.Oxidant treatment induced significant changes to CAS concentrations in the nucleus, and it elevated the nuc/cyt ratio of the exporter . PharmacCollectively, these data indicate drastic changes for CAS distribution in cells that were treated with oxidant. These changes were even more pronounced when PI3 kinase and/or MEK signaling was abolished.The mobile nucleoporin Nup153 shuttles between nuclei and the cytoplasm, at steady-state the protein is concentrated at the nuclear envelope . IncubatTaken together, results for Nup153 suggest that under stress conditions both PI3 and MEK kinase cascades contribute to the distribution of Nup153, whereas in unstressed cells PI3 kinase signaling is a key player.Like the nuclear transport factors described above, Nup88 displayed a unique profile for the changes in localization that were caused by stress or kinase inhibitors . The amode novo synthesis of the nucleoporin. In summary, data obtained with pharmacological compounds suggest that the combined inhibition of PI3 kinase and MEK signaling had the opposite effect when compared to the inhibition of either pathway alone.In the absence of pharmacological drugs, stress diminished the concentration of Nup88 in the cytoplasm and at the NE, leading to an increase in the nuc/cyt ratio. Prevention of PI3 or MEK kinase signaling, either alone or in combination, had similar consequences in control or stressed cells. Specifically, inhibition of the PI3 or MEK signaling pathway reduced the amount of Nup88 in nuclei, the cytoplasm and at the NE, whereas simultaneous interference with both kinase cascades increased drastically the amount of Nup88 in all three compartments. Possible explanations for this rise of Nup88 concentrations in the nucleus, cytoplasm and at the NE under these conditions are changes in the turnover and/or Oxidant exposure may trigger the post-translational modification of proteins, and nuclear transport factors are potential candidates for such modifications. We tested this hypothesis by Western blot analysis of extracts generated from control and stressed cells . Side-bySeveral repeat-containing nucleoporins are modified by O-linked glycosylation, and the level of O-GlcNAc modification may increase upon stress 32P] under normal and stress conditions. To this end, HeLa cells were incubated in the presence of radioactive phosphate, and Nup153 was immunoprecipitated under denaturing conditions. Aliquots of the immunopurified material were separated by SDS-PAGE, and radiolabeling of the immunoprecipitated proteins was measured. In parallel, we determined the amount of immunopurified Nup153 by quantitative Western blotting. From these data, the relative phosphorylation was calculated for Nup153  equal amounts of protein were separated in SDS-PA gels. Proteins were blotted to nitrocellulose and blots processed as described HeLa cells were grown on dishes to 50 to 70% confluency. Cells were stressed as described above, washed with PBS and stored at −70°C until use. Crude extracts were prepared by solubilizing proteins in gel sample buffer, pH 8.0, containing protease inhibitors , 20 mM β-glycerophosphate, 1 mM NaN3, 2.5 mM NaF and a cocktail of protease inhibitors . DNA was sheared by vortexing with glass beads during the incubation period and insoluble material was removed by centrifugation . Supernatants were diluted 1∶2 in water and precipitated with 10% TCA for 1 h on ice. Sediments were resuspended in 100 mM Tris HCl pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.025% Triton X-100 and incubated with 200 U/ml calf intestinal phosphatase  for 1 h at 37°C. Controls were incubated under identical conditions without adding CIP.HeLa cells were incubated with ethanol or 2 mM DEM for 4 hours, extracted with digitonin to remove cytoplasmic proteins and boiled in 40 mM Tris HCl pH 8.0, 1% SDS, 50 mM DTT containing 1 mM NaN2, 2 mM DTT, and twofold concentrated protease inhibitors. Samples were incubated for 20 h with 100 U/ml hexosaminidase and 200 U/ml CIP and subjected to Western blotting. Hexosaminidase was sufficiently active under these conditions to remove GlcNAc moieties from proteins . Samples were pre-adsorbed with Protein G-Plus agarose (Santa Cruz Biotechnology) for 30 min at 4°C and beads were removed by centrifugation. After 1 hour incubation with antibodies at 4°C, Protein G-Plus agarose was added and samples were kept overnight at 4°C. Beads were collected by centrifugation and washed three times with PBS/1 mM NaN3. Bound material was eluted by incubating in twofold concentrated gel sample buffer for 10 min at 95°C. Resin was removed by centrifugation and supernatants were subjected to SDS-PAGE and Western blotting.HeLa cells were labeled with [HeLa cells treated with ethanol or DEM were rinsed with PBS and frozen at −70°C. Samples were resuspended in 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 30 mM Tris, 20 mM DTT, 1% Bio-Lyte 3–10 (Bio-Rad), vortexed with beads to shear DNA and centrifuged for 5 min at 13,000 rpm (microfuge). Supernatants were applied to 7 cm IPG strips 4–7 or 3–10 NL (non linear gradient), all purchased from Bio-Rad. After 14 h of rehydration, focusing was carried out with a Protean IEF cell (Bio-Rad) as recommended by the manufacturer. Strips were either immediately processed for the second dimension or frozen at −70°C. SDS-PAGE and Western blot analysis followed the procedures described above.Figure S1Effect of pharmacological kinase inhibitors and stress on importin-α, dually phosphorylated ERK1/2 (p-ERK1/2), and Akt phosphorylated on Ser473 (p-Akt473) in nuclei. Changes in the nuclear concentration of importin-α shown in  are comp(8.82 MB TIF)Click here for additional data file.Figure S2Hexosaminidase is active under conditions used for hexosaminidase and CIP double digests. Samples were probed with antibodies against O-GlcNAc to monitor the activity of hexosaminidase under the conditions used for (3.67 MB TIF)Click here for additional data file.