We cultured (5 days) rat alveolar epithelial cells to investigate the role of mitogen-activated protein kinase (MAPk) signaling in ventilator induced epithelial barrier dysfunction. Cells were stretched to a magnitude of 12% or 37% change in surface area at a rate of 0.25 Hz with and without pretreatment with either the JNK inhibitor SP600125 or the ERK inhibitor U0126. Following stretch , MAPk phosphorylation was examined, monolayer permeability to the uncharged tracer carboxyfluorescein measured , and occludin expression determined (0 and 60 min of stretch). Stretch to 12%, previously shown not to increase monolayer permeability, did not alter phosphorylation of any MAPk or occludin expression at any time point. Following stretch to 37%, phosphorylation of JNK, ERK, and p38 was significantly higher by 10 minutes than in unstretched monolayers. Phosphorylation of JNK and p38 subsided as stretch continued, and by 30 minutes returned to unstretched levels. Phosphorylation of ERK remained significantly elevated compared to unstretched levels at all stretch durations. Epithelial permeability increased significantly by 10 minutes of stretch compared to unstretched controls, with further significant increases by 60 minutes. Inhibition with U0126 and SP600125 prevented stretch-induced phosphorylation increases of ERK and JNK, respectively, however neither prevented increases in permeability following 10 minutes. Separately, inhibition of JNK or ERK prevented subsequent additional permeability increases as stretch continued to 60 minute time points. Inhibition of JNK, not ERK, prevented loss of occludin, and minimized loss of cell-cell contact following 60 minutes of stretch. These data suggest that stretch-induced JNK signaling modulates epithelial permeability through regulation tight junction protein expression, and is a potential target for clinical treatments during mechanical ventilation. Ventilator induced lung injury (VILI) is a significant complication to mechanically ventilated patients, as 5–15% of these patients will develop VILI, and of those approximately 35–60% of these cases are fatal Recent work in our lab has shown that 1 hour of cyclic (0.25 Hz) stretch of cultured alveolar epithelial monolayers to a peak magnitude corresponding to 100% TLC increases trancellular permeability The MAPk family of serine-threonine protein kinases is a highly conserved family consisting of the three primary members; extracellular signal-related kinases (ERK), c-Jun amino-terminal kinases (JNK), and p38. MAPks play an active signaling role following multiple stimuli, some of which include irradiation, osmotic stress, inflammation, growth factors, and mechanical loading In the lung, MAPk signaling pathway activation has been observed following mechanical ventilation. MAPk activation was also shown to increase with increased magnitude of ventilation and to correlate with increases in mortality, circulatory failure, and pulmonary edema In this communication, we examined phosphorylation of ERK, JNK, and p38 following the application of cyclic stretch to a magnitude previously shown to produce barrier dysfunction. Using inhibitors to block specific pathways, we investigated functional relationships of these pathways in transducing stretch to barrier permeability. We concluded that the JNK and ERK signaling pathways negatively regulate barrier function following stretch, however JNK, not ERK, is associated with observed stretch induced reductions in occludin expression.et. al.6 cells/cm2) on either an elastic substrate or permeable co-polyester membranes , both of which were mounted in custom-built polysulfone wells. The permeable membrane was previously corona discharge-treated, and coated with poly-L-lysine  and fibronectin  to improve cell adhesion. All wells were incubated for a period of 5 days, at which point we have demonstrated they lose alveolar epithelial type II phenotypic markers and take on type I phenotypic markers Primary rat alveolar epithelial type II cells were isolated from pathogen-free male Sprague-Dawley rats (250–350 g) via an elastase digestion technique adopted from Dobbs Following 5 days in culture, wells were washed with Delbecco's Modified Eagle's Medium (DMEM) and serum deprived for 2 hours, then treated for 1 hour with 1.5 mls of DMEM containing either SP600125 (20 or 35 µM) or U0126 (10 or 20 µM) to inhibit JNK or ERK phosphorylation respectively . DMSO and U0124 (20 µM) were used as a negative control in viability and dose response studies for the inhibitors SP60125 and U0126 respectively. DMSO was used as a vehicle control for both inhibitors in all permeability studies.in vivo at an inflation from functional residual capacity to 70% or 100% total lung capacity (TLC), or tidal volume 12ml/kg or 30 ml/kg in the rat with no end-expiratory pressure (PEEP). Three sets of stretch experiments were conducted as follows. First, the appropriate dose of each inhibitor and vehicle control was determined by stretching a group of 8 total wells from 2 rat isolations for 10 minutes, as this was the stretch duration found to produce peak MAPk phosphorylation in pilot studies. The dose which best inhibited stretch induced activation of the targeted MAPk while not being toxic to the cells was chosen. Dose response for SP600125 was evaluated at concentrations of 20 and 35 µM, and for U0126 at 10 and 20 µM. Second, stretch-induced MAPk activation was determined by stretching a group of 16 wells (with DMEM) from 4 separate isolations for four stretch durations . Finally, the effect of MAPk inhibition on epithelial paracellular permeability was determined by stretching groups of 4 wells (with DMEM and the inhibitor) from 3 isolations for a total of 12 wells per inhibitor at three stretch durations . The temperature of the stretch device was maintained at 37°C.Monolayers were stretched in the presence of DMEM, DMEM and a MAPk inhibitor, or DMEM and vehicle control (DMSO). Wells were mounted in a custom-made stretch device capable of applying a uniform and equibiaxial cyclic stretch to the cells at a magnitude of 12% or 37% ΔSA and frequency of 0.25 Hz A subset of wells was used to determine cellular toxicity of the inhibitors SP600125 and U0126 at the maximum concentration used in the dose response study, 35 µM and 20 µM respectively. We used 2 wells from each of the following unstretched groups: DMSO vehicle, SP600125, U0126, and each of the following stretched  groups: DMSO vehicle, SP600125, U0124, and U0126. Each well was treated, immediately prior to stretch, with Ethidium Homodimer-1 (7.5 µM), which stains the nuclei of dead cells, and Calcein AM (16 µM) to label live cells . Following the 60 minute stretch period, wells were imaged in three locations (20× objective) using a fluorescent microscope , the number of dead and live adherent cells were determined, and toxicity was calculated as the ratio of dead cells divided by the total number of cells (dead plus live cells). Average toxicity was determined from the mean of the 6 images (3 regions from each of 2 wells) for each treatment and stretch condition. Comparisons were made across treatments, and between stretched and unstretched fields.After a 10 or 60 minute stretch period, wells (N = 12 per group) were removed from the stretch device and mounted into Ussing chambers within approximately 2 minutes. The apical to basal paracellular permeability of the monolayers was assessed using methods similar to those previously described A0C = initial apical tracer concentration, B0C = initial basal tracer concentration, B(t)C = basal concentration at time t, P = paracellular permeability of the monolayer to a specific tracer (units = length/time), S = surface area over which permeability occurs. Since all variables but P are known, we were able to solve for permeability directly. Previously, we assessed permeability change in 15 minute intervals up to 2 hours post stretch, and reported no change in monolayer permeability over this time period We assumed diffusive transport of the tracer, with no sources or sinks, and used well-established equations governing conservation of mass Rat alveolar epithelial cells (3 isolations of 4 wells per group) in primary culture were scraped from the silastic membrane in the presence of chilled radio-immunoprecipitation assay (RIPA) buffer containing 4.3 mM ethylenediaminetetraacetic acid (EDTA) and a cocktail of protease and phosphotase inhibitors, and placed on ice to minimize cellular activity. Equal protein lysates were run on SDS-polyacrylamide (10%) reducing gels, transferred onto polyvinylidene fluoride membranes (PVDF) and non-specific binding was blocked in tris buffered saline (TBS) containing 5% non-fat powdered milk and 0.05% Tween-20 at 4°C. The PVDF membranes were initially probed for phosphorylated MAP kinases or occludin then stripped and reprobed for total MAP kinases or actin . MAPk activity and occludin and actin concentration was calculated through densitometric analysis using Kodak 1D Image Analysis Software . Band intensities for phospho-MAPks were normalized by respective total protein levels, and occludin was normalized to actin concentration. Stretched data was then normalized to the unstretched control lane on each blot for comparisons across blots.Following the stretch period (0 or 60 minutes), cells (2 isolations of 2 wells per group) were washed with phosphate-buffered saline (PBS), fixed for 15 min with 1.5% paraformaldehyde, washed again in PBS, and treated for 5 min with 0.1% Triton X-100 in PBS. Cells were blocked for 1 hour in 10% normal goat serum, then incubated overnight with either 2.0 µg/ml occludin or 1.0 µg/ml ZO-1 antibodies (Invitrogen). After washing in PBS, the cells were incubated for 2 hours in secondary antibody (Jackson Laboratories), mounted, and imaged .An initial analysis was used to determine effects of different doses of MAPk inhibitors SP600125 and U0126 on JNK and ERK phosphorylation compared to their vehicle controls DMSO and U0124, following a stretch duration of 10 min. Student's t-tests were conducted between inhibitor and vehicle control data for each MAPk to determine the effectiveness of the inhibitor. Significance was defined as p≤0.05.To determine the effect of stretch duration on MAPk phosphorylation in cultured monolayers, separate analyses was performed for each kinase JNK, ERK, and p38, comparing across stretch durations  using a one-way ANOVA. Subsequently, post hoc Tukey tests were used to determine significant difference in kinase phosphorylation at individual time points To determine the effect of MAPk inhibition on epithelial permeability, separate analyses for each inhibitor and vehicle control combination were performed, comparing across stretch duration using a two-way ANOVA. Post hoc Tukey tests were used to analyze individual comparisons between time points for the same treatment or treatments at the same time point. Statistical significance was defined as p≤0.05.To determine the effect of stretch and MAPk signaling on occludin concentration, occludin expression was compared across stretch (0 and 60 minutes) and treatment  combinations using a two-way ANOVA. Post hoc Tukey tests were used for individual comparisons.All animal use was done in accordance with, and with the approval of, the IACUC in the Office of Regulatory Affairs of the University of Pennsylvania.Cell lysate from untreated, stretched (12% and 37% ΔSA) monolayers was examined for phospohorylated and total JNK, ERK, and p38. No alteration in MAPk phosphorylation from unstretched levels was observed at any time point in monolayers stretched to 12% ΔSA . FollowiPretreatment with 20 and 35 µM of the JNK inhibitor SP600125 significantly reduced the stretch-induced phosphorylation of JNK at 10 minutes compared to pretreatment with vehicle (DMSO) . There wWe also examined the effect of the ERK inhibitor U0126 (10 and 20 µM) on stretch-induced MAPk phosphorylation. Treatment with either concentration of the inhibitor reduced phosphorylation levels of ERK following stretch to 37% ΔSA for 10 minutes compared to negative control (U0124) treatments . HoweverTo ensure that use of the inhibitors (at the highest concentration used in the dose response studies) did not increase mortality rates in unstretched and stretched conditions, we treated separate wells with 35 µM SP600125, DMSO vehicle, 20 µM U0126 or 20 µM U0124, and either stretched  the cells or left them unstretched. In unstretched wells, the percent cell death was 0.51±0.27% for DMSO, 0.43±0.42 for SP600125 and 0.57±0.74 for U0126 treated wells. Toxicity of U0124, an inactive form of the inhibitor U0126, was not determined in unstretched wells. Following stretch, percent cell death of DMSO and U0124 treated wells (vehicle and negative control) was 9.04±3.71% and 11.08±4.37% respectively, while wells treated with SP600125 and U0126 had 9.41±5.59% and 10.19±5.20% cell death respectively. These data are consistent with our previously reported values of 0.3±0.5% in unstretched naïve controls and 9.2±4.4% in similarly stretched wells , and show that neither treatment nor vehicle control altered cell viability under our experimental conditions As we reported previously, cyclic (0.25 Hz) mechanical stretch to 37% ΔSA for 60 minutes in our control groups resulted in significant increases in untreated monolayer permeability Pre-treatment and stretch in the presence of either SP600125  stretched for 10, 30, or 60 minutes to a magnitude of 12% or 37% ΔSA, which corresponds to 70% or 100% TLC. We have shown, using this model of the alveolar epithelium, that 60 minutes of stretch to 37% ΔSA and this rate produces significant increases in epithelial permeability. We analyzed phosphorylation levels of JNK, ERK and p38 MAP kinases in either unstretched monolayers, or those stretched to each magnitude, and found significant phosphorylation  above unstretched only in the high magnitude group. Phosphorylation was increased by 10 minutes of stretch and subsided with increased stretch duration. By 60 minutes only ERK phosphorylation was significantly elevated compared with unstretched controls.Pretreatment of the monolayers with specific inhibitors of JNK (SP600125) or ERK (U0126) prevented stretched induced phosphorylation of each MAPk respectively. We did not inhibit p38, and therefore cannot comment on any potential role it plays in barrier dysfunction, as increases in p38 phosphorylation observed at 10 minutes were significantly less than that of JNK and ERK, rapidly subsided, and were not significantly greater than phosphorylation levels at 30 or 60 minutes which were no different than unstretched levels. We assessed barrier function of monolayers stretched in the presence of the inhibitors, and found that neither inhibitor had any effect on permeability following 10 minutes of stretch, but after 60 minutes of stretch, both JNK and ERK inhibition significantly attenuated further permeability increases.It is possible that the beneficial effects of JNK inhibition are due to an upregulation of the ERK signaling pathway, as ERK has been linked to cell differentiation and enhanced cell survival following a stress event Initially, we hypothesized that the beneficial effect of inhibition JNK phosphorylation, not ERK phosphorylation, could be due to inhibition of apoptosis. Historically, activation of the JNK and ERK signaling pathways have been shown to produce pro-apoptotic and anti-apoptotic cellular reactions, respectively et. al. demonstrates that blocking TNFα activation of JNK inhibited ZO-1 removal from the tight junction complex and preserved barrier function in endothelial cells Activation of JNK signaling has been associated with phosphorylation of tight junction proteins ZO-1 and occludin We had further hypothesized that inhibition of an alternative MAPk signaling pathway, ERK, would similarly prevent permeability increases following stretch, also by reducing the extent of occludin degradation. This hypothesis was based on previous studies that demonstrate ERK phosphorylation regulates tight junction disassembly following cigarette smoke damage of human cell lines and TGFβ induced epithelial mesenchymal transition in murine epithelial cells In summary, we stretched cultured alveolar epithelial cells to a magnitude shown to produce significant increases in permeability, and explored MAPk signaling as a possible mechanism of these increases. We found that JNK, ERK, and p38 MAP kinases were significantly phosphorylated by stretch. Furthermore, large magnitude stretch for 60 minutes increased monolayer permeability, produced large gaps in the monolayer, and reduced expression of the tight junction protein occludin. Inhibition of either JNK or ERK reduced the stretch-induced monolayer permeability, but inhibition of JNK also reduced alterations in occludin concentration and cellular localization. We conclude that MAPk signaling has the potential to modulate stretch-induced alveolar epithelial injury, and propose further studies should be conducted to identify opportunities for reducing tight junction disruption.


Histone chaperones are at the hub of a diverse interaction networks integrating a plethora of chromatin modifying activities. Histone H3/H4 chaperone ASF1 is a target for cell-cycle regulated Tousled-like kinases (TLKs) and both proteins cooperate during chromatin replication. However, the precise role of post-translational modification of ASF1 remained unclear. Here, we identify the TLK phosphorylation sites for both Drosophila and human ASF1 proteins. Loss of TLK-mediated phosphorylation triggers hASF1a and dASF1 degradation by proteasome-dependent and independent mechanisms respectively. Consistent with this notion, introduction of phosphorylation-mimicking mutants inhibits hASF1a and dASF1 degradation. Human hASF1b is also targeted for proteasome-dependent degradation, but its stability is not affected by phosphorylation indicating that other mechanisms are likely to be involved in control of hASF1b levels. Together, these results suggest that ASF1 cellular levels are tightly controlled by distinct pathways and provide a molecular mechanism for post-translational regulation of dASF1 and hASF1a by TLK kinases. Transmission, maintenance and expression of the eukaryotic genomes are mediated by a repertoire of chromatin transactions. A wide range of modifying activities acts on nucleosome, a central unit of chromatin, including histone chaperones, ATP-dependent chromatin remodeling factors (remodelers), and histone modifying enzymes. The accumulated evidence on chromatin modifications reveals extensive role for histone chaperones going beyond just facilitating chromatin assembly in vivo with the BRM chromatin remodeling pathway Recent studies have emphasized a critical role for histone H3/H4 chaperone ASF1 in orchestrating distinct chromatin modifying activities. ASF1 hands off histones to CAF1 and HIRA facilitating replication-coupled and replication-independent chromatin assembly respectively Given a broad spectrum of ASF1 functions in chromatin regulation, control of ASF1 levels is critical for cell viability, replication and execution of developmental programs. Depletion or overexpression of ASF1 results in severe developmental defects, altered cell cycle progression and cell death In order to understand the importance of TLK-mediated phosphorylation of ASF1, we decided to map the TLK phosphorylation sites in the Drosophila ASF1 protein. For this, bacterially produced dASF1 was phosphorylated in vitro, using dTLK expressed and purified from baculovirus-infected cells . Mass spNext, we performed a similar mapping of phosphorylation sites on human hASF1a and hASF1b with baculovirus expressed hTLK2 . We founin vivo, we performed RNAi-mediated depletion of dTLK in Drosophila S2 cells and siRNA-mediated depletion of both hTLK1 and hTLK2 genes in HeLa cells. Reduced TLK levels resulted in significant reductions in dASF1 and hASF1a protein levels -N-S-(L/M) consensus motif. Phosphorylation of ASF1 by TLKs regulates positively dASF1 and hASF1a protein levels and hampers proteasome-dependent degradation of hASF1a. In Drosophila, dASF1 degradation is only partially controlled by proteasome-dependent proteolysis and other degradation pathways could be involved, but nonetheless phosphorylation by dTLK results in significant stabilization of dASF1. Finally, hASF1b stability does not appear to depend on phosphorylation by TLKs, but recently it has been shown that hASF1b transcription is controlled by the cell-cycle regulated E2F transcription factors ASF1 and TLK activities are critical for chromatin replication and progression through S phase in vivoMajor TLK phosphorylation sites are located within non-conserved C-terminal part of Drosophila and human ASF1 proteins. However, it has been shown that in yeast constitutive expression of conserved N-terminal part is sufficient to recapitulate most of the ASF1 functions The constructs for recombinant protein purification from bacteria or baculovirus system were based on pGEX-4T and pFastBacHT plasmid series (Invitrogen) respectively. The constructs used for protein expression in Drosophila and human cells were based on pRMH3-BLPR containing BirA signal 2 column GST-ASF1 protein fusions were expressed and purified as described PLUS chemiluminescent substrate (Roche) and detected with Syngene GeneGnome CCD camera setup.Immunoblotting were performed using standard procedures with the following antibodies: anti-dASF1, anti-dTLK 2 incubator as described S2 cells were cultured in Schneider's medium (Invitrogen 21720–024) supplied with 10% FBS and Penicillin/Streptomycin mixture at 25°C FuGene6 transfectin reagent (Promega) was used for the transient transfection of cell cultures. Expressed proteins were assayed 48 h post-induction for metallothioneine promoter constructs or 48 h post-transfection for other constructs by chemiluminescent western-blotting and quantified using Syngene GeneSnap software. To inhibit proteasome-dependent degradation cells were incubated for 16 h in the presence of 5µM Lactacystine and 10 mM MG-132 (Sigma), and protein synthesis was blocked using 50µM of cycloheximide


For example, Gq/phospholipase (PL)Cβ signaling by TPα isdirectly inhibited by the vasodilators prostacyclin and nitric oxide (NO)whereas that signaling by TPβ is unaffected. Herein, we investigated whether TPαand/or TPβ regulate G12/Rho activation and whether thatsignaling might be differentially regulated by prostacyclin and/or NO. Both TPαand TPβ independently regulated RhoA activation and signaling in clonal cellsover-expressing TPα or TPβ and in primary human aortic smooth muscle cells (1°AoSMCs). While RhoA-signaling by TPα was directly impaired by prostacyclin andNO through protein kinase (PK)A- and PKG-dependent phosphorylation,respectively, signaling by TPβ was not directly affected by either agent.Collectively, while TPα and TPβ contribute to RhoA activation, our findingssupport the hypothesis that TPα is involved in the dynamic regulation ofhaemostasis and vascular tone, such as in response to prostacyclin and NO.Conversely, the role of TPβ in such processes remains unsolved. Data hereinprovide essential new insights into the physiologic roles of TPα and TPβ and,through studies in AoSMCs, reveal an additional mode of regulation of VSMcontractile responses by TXA2.In humans, thromboxane (TX) A Hence, the aim of the current study was to investigatewhether TPα and/or TPβ independently regulate G12/Rhoactivation and signaling and to establish whether that signaling isdifferentially regulated by the inhibitory prostacyclin/cAMP/PKA and NO/cGMP/PKGsystems. These studies provide essential new insights into the physiologic rolesof TPα and TPβ and, through studies in primary human aortic smooth muscle cells(1° AoSMCs), reveal an additional mode of regulation of VSM contractileresponses by the potent autocoid TXA2.While both TPα and TPβ are predominantly coupled to Gq/PLCβ activation22.1anti-HA, 3F10anti-HA-horseradish peroxidase (HRP)-conjugatedantibody and chemiluminescence detection kit from Roche;anti-RhoA 26C4 (Sc-418),anti-phospho-RhoASer188(Sc-32954-R), anti-Gα12/13 H-300(Sc-28588), anti-Gαq C15 (SC-392), HRP-conjugatedgoat anti-mouse (Sc-2005), HRP-conjugated mouseanti-goat (Sc-2354) and HRP-conjugated goatanti-rabbit (Sc-2004) antibodies from Santa Cruz;Glutathione-Sepharose 4B  and FITC conjugated goatanti-rabbit antibody from Sigma;anti-cofilin (# 3312) andanti-phospho-cofilin were from Cell Signaling; AlexaFluor® 488 phalloidin  from Molecular Probes;anti-HDJ2 antibody from Neomarkers; Opti-MEM® andOligofectamine® were from Invitrogen. All oligonucleotides were synthesisedby Genosys Biotechnologies; small interfering (si) RNAs by Qiagen. Cicaprostwas a gift from Schering AG .pcDNA3.1(+):hGαqQ209l,D277N,pCis:Gα12G228A andpCis:Gα13G225A were from the UMR cDNAResource Center (Gαq) or from Dr S. Offermanns, University of Heidelberg,Germany.U46619, SQ29,548, BW245C, FK409, FURA2/AM were purchased from CaymanChemical Company; SIN-1 and Y-27632 from Calbiochem; 3F102.2S329A HEK.TPαS331A andHEK.TPαS329,331A cell lines stably over-expressinghemagglutinin (HA) -tagged forms of TPα, TPβ,TPαS329A,TPαS331A andTPαS329,331A respectively, have been previouslydescribed 6 cells/10 cm dish in 8 ml media and co-transfectedwith 10 μg of pADVA and 25 μg of pCMV-based mammalian expression vectorusing the calcium phosphate/DNA co-precipitation procedure Human embryonic kidney (HEK) 293 cells were grown in minimal essentialmedium (MEM), 10% foetal bovine serum (FBS). HEK.TPα, HEK.TPβ,HEK.TPαPrimary (1°) human aortic smooth muscle cells (1° h.AoSMCs) werepurchased from Cascade Biologics (C-007-5C) and routinely grown in SmoothMuscle Cell Growth Medium 2  supplemented with0.5 ng/ml epidermal growth factor, 2 ng/ml basic Fibroblast growth factor,5 μg/ml insulin, 5% FBS.2.32+]i)mobilization were carried out in FURA2/AM preloaded HEK 293 cell linestransiently co-transfected with pCMV:Gαq and pADVA some 48 h previously, asdescribed 2+ mobilized(Δ[Ca2+]i(nM)) as a function of time  following ligandstimulation.Measurements of intracellular calcium in growth media containing 0.1% FBS beforestimulation for 0–30 min with 0–10 μM U46619, as indicated in specificfigure legends. To assess the effect of prostacyclin, nitric oxide (NO) orPGD2 on TP-mediated Rho signaling, cells werepre-incubated for 10 min with either 0.01–10 μM Cicaprost; 0.05–50 μM SIN-1;10 μM FK409 or 1 μM BW245C before stimulation with U46619 . As controls, cells were incubated with an equivalent volume ofthe drug vehicle, agonist or inhibitor in 0.01% ethanol in HBS  for equivalent incubation times.Activated cellular Rho was determined by interaction with a purifiedglutathione-S-transferase: rhotekin Rho-binding domain (GST-RBD) fusionprotein immobilized on Glutathione-Sepharose 4B resin 2, and 10 μg/ml each of leupeptin and aprotinin;anti-RhoA monoclonal antibody(Sc-418), followed by chemiluminescence detection anti-RhoA antibody and/or with the anti-HDJ2antibody. Similarly, to assess U46619-mediated cofilin phosphorylation andactivation, aliquots of whole cells lysates  were first immunoblotted withanti-phosphoSer3 cofilinantibody; thereafter, phospho-cofilin blots were stripped and rescreenedversus anti-cofilin antibody to normalise for totalcofilin protein expression and/or with the anti-HDJ2 antibody to confirmuniform protein loading in each of the assays. All images of RhoAexpression/pulldown or cofilin phosphorylation and/or expression werecaptured using Adobe Photoshop (V6), where band width and intensity wasquantified and represented as fold increases relative to basal levels. Toaccount for biological variations in basal activation levels, experimentswere normalised to within a comparable range based on measurements from morethen 20 individual experiments for each cell type.Thereafter, cells were lysed in 800 μl Lysis Buffer . To assess the role of prostacyclin or NO, cellswere pre-incubated for 10 min with either 0.01–10 μM Cicaprost or 0.05–50 μMSIN-1 before stimulation with U46619 . F-actinpolymerization was stained by the addition of Alexa Fluor® 488 phalloidinessentially as described by the supplier (Molecular Probes) and slides wereimaged using an Axioplan 2 Imaging Universal Microscope.2.6interference(RNAi) experiments, HEK 293cell lines  or 1° h.AoSMCswere plated at ~ 2.5 × 105 cells /35-mm plate and were allowed to attach for24 h, achieving ~ 30 % confluency. Thereafter, cells werewashed twice with serum-free Opti-MEM® and transfected for 4 h at 37 °C with0.2 μM TPα siRNA  or 0.2 μM TPβ siRNA or0.2 μM Lamin A/C siRNA (5'-CUGGACUUCCAGAAGAACAtt) using Oligofectamine®(3 μl/well) in Opti-MEM® (1 ml /well). Thereafter, 1 ml pre-warmed completemedia supplemented with 30% FCS was added per well and cells were harvestedfollowing incubation at 0, 24, 48, and 72 h. As additional controls, HEK.TPαcells were treated according to the latter conditions but using TPβ siRNAsand vice versa.For RNAanti-HA(3F10) antibody and, following stripping, were rescreened versusanti-HDJ antibody to confirm uniform proteinloading. Similarly, 1° h.AoSMCs were screened, under permabilisingconditions, by indirect immunofluorescence microscopy for TPα and TPβexpression using affinity purified isoform specific rabbitanti-TPα (3 μg/ml) andanti-TPβ (3 μg/ml) antibodies To confirm the efficacy of the siRNAs to disrupt TPα or TPβ expression,HEK 293 cell lines were harvested and subject to SDS-PAGE (25–50 μg/lane on12.5% gels) followed by electroblotting onto PVDF membranes (Roche).Membranes were initially screened versus the i disruption of TPα orTPβ expression in respective HEK 293 lines and in 1° h.AoSMCs, experimentswas scaled up 8.2-fold (2 × 106 cells on 10-cm dishes) and functional disruptionwas assessed through Rho pulldown assays or cofilin phosphorylation, aspreviously outlined herein.Thereafter, having optimised the conditions for effectiveRNA2.7d andBmax values. Statisticalanalyses were carried out using the unpaired Student'st test using the Statworks Analysis Package.p-values of less than or equal to 0.05 wereconsidered to indicate a statistically significant difference. Throughoutthe figures, ⁎ < 0.05, ⁎⁎< 0.01,⁎⁎⁎< 0.001.Radioligand binding data was analyzed using GraphPad Prism V3.0 todetermine the K33.12 receptor (TP)expressed in human tissues, to our knowledge, no such study has investigatedthe propensities or relative abilities of the individual TPα or TPβ isoformsto activate and/or regulate Rho-mediated signaling. Hence, herein weinvestigated TPα and TPβ-mediated Rho signaling in response to theTXA2 mimetic U46619 in clonal HEK 293 cell lines thatstably over-express either TPα (HEK.TPα cells) or TPβ (HEK.TPβ cells).Throughout these studies, TPα/TPβ-mediated Gq/PLCβ-dependent[Ca2+]imobilization was monitored as a comparative reference.Whilst a range of studies have investigated Gq/PLCβ-mediated signalingby both the TPα and TPβ isoforms of the TXA2+]i inresponse to U46619 stimulation, with maximal responses generated using 1 μMU46619 (12 (Gα12/Gα13)members but may also occur through Gq coupling, in certain settings at least12(Gα12G228A), but not of Gαq, significantly impairedU46619-mediated RhoA activation through both TPα(p = 0.0011and p = 0.8011respectively) and TPβ , while the dominantnegative form of Gαq  had no significanteffect , it significantly impairedU46619-induced RhoA activation by TPα expressed in HEK.TPα cells in aconcentration-dependent manner and SIN-1 (500 nM) impaired F-actin polymerization in HEK.TPα cells butneither agent affected such responses in HEK.TPβ cells  orthe NO-donor SIN-1 (5 μM) on U46619-mediatedimobilization, Rho activation, F-actin polymerization and cofilinphosphorylation by TPα and its specific site directed variantsTPαS329A, TPαS331A,TPαS329,331A defective in the Cicaprost-sensitive PKA(at Ser329), NO-sensitive PKG (at Ser331)or both  phosphorylation sites, aspreviously described by us 2+]imobilization in HEK.TPα and HEK.TPαS329A cells while havingno affect on such signaling in HEK.TPαS331A andHEK.TPαS329,331A cells  agonist BW245C also impaired RhoA activation by TPα  agonist BW245C alsosignificantly impaired RhoA activation .We next investigated TP-mediated Rho activation and cytoskeletalsignaling in a physiologically relevant, vaso-responsive model byinvestigating U46619-induced signaling in 1° h.AoSMCs, cells that expressboth TPα and TPβ h.AoSMCs A. Consistivation C and cofh.AoSMCs B and D.2 signaling through theprostacyclin (IP) and PGD2 (DP) receptors, respectively,can impair U46619-mediated Rho activation and cytoskeletal signaling in 1°h.AoSMCs. Moreover, our data generated in the HEK.TPα and HEK.TPβ cell linesover-expressing the individual TPα and TPβ isoforms, respectively, suggestthat such inhibitory responses of prostacyclin and NO are mediated, at leastin part, at the interface of the stimulatory GPCR (i.e. the TP). Morespecifically, by directly targeting TPα, prostacyclin and NO may impair itsRhoA-signaling both at the level of TPα itself in addition to at the levelof the well documented targets of cAMP/PKA and cGMP/PKG Hence, taken together, both NO-donors and prostanoid vasodilatoryagents, such as prostacyclin and PGDTPα and siRNATPβ, respectively-targeting agents to selectively disrupt or knock-down expression ofTPα and TPβ in 1° h.AoSMCs. To begin with, the siRNA agents were validatedby examining their ability to affect TPα and TPβ expression and RhoAsignaling in HEK.TPα and HEK.TPβ cells. Under optimized experimentalconditions, we observed effective isoform-specific knock-down of both TPαand TPβ expression following 72 h incubation of HEK.TPα and HEK.TPβ cellswith siRNAectivelyA, with ~Pα cells A therebyell line A. MoreovPβ cells B. ConverPαcells B.RNAi dtivation B. ConsisTPα impaired expression of TPα but not of TPβwhile siRNATPβ reduced expression of TPβ but not of TPαcells; mitogenic and hypertrophic growth of VSM cells; inhibition ofangiogenesis/vascularization 2,its synthase or its receptor have been implicated in various cardiovasculardisorders including thrombosis, myocardial infarction, unstable angina,atherosclerosis, systemic- and pregnancy-induced hypertension and ischemic heartdisease, processes in which RhoA dysfunction is widely implicated 2 signalsthrough 2 distinct isoforms referred to as TPα and TPβ 2 in humans isunknown there is substantial evidence that they may have distinctphysiologic/pathophysiologic roles The prostanoid TXA2+-independent pathways to both normal anddisease-processes within the vasculature Bearing this in mind and the growing appreciation of the critical role ofthe RhoA-mediated Ca2 mimetic U46619.Moreover, we also investigated the ability of TPα and TPβ to regulate eventsdownstream of RhoA: effector coupling by monitoring U46619-induced F-actinpolymerization and cofilin phosphorylation. The ubiquitously expressedactin-depolymerising factor cofilin readily undergoes Rho/Rho kinase-dependentphosphorylation at Ser3 either by the LIM kinase 1/212, mainly Gα13,-dependent mechanism but in certain settings, particularly at higher agonistconcentrations, may also occur through a Gq-mechanism through the specificinvolvement of the LARG (Leukemia-associated Rho guanine-nucleotide exchangefactor), but not the p115- or PDZ-, member of the RGS-containing RhoGEF family12 andGq on TP-mediated RhoA signaling and found that dominant negative forms ofGα12 (Gα12G228A), not ofGαq , significantly impaired U46619-mediatedRhoA activation and cofilin phosphorylation. Collectively, these data confirmedthat both TPα and TPβ can independently couple to Gq/PLCβ activation andGα12/Gα13/RhoGEF-RhoA activation and arein agreement with a host of studies in mouse platelets whereby the single TP inthat species couples to Gq/PLCβ and to G12/RhoA activation toindependently regulate platelet activation (aggregation and secretion) andplatelet shape change responses, respectively To this end, we investigated the ability of TPα and TPβ to mediate RhoAsignaling in established clonal HEK 293 cell lines that over-express theindividual TP isoforms 2+]imobilization by TPα, but not by TPβ, was desensitized in response to bothCicaprost and NO stimulation. In keeping with this, TPα-mediated RhoAactivation, F-actin polymerization and cofilin phosphorylation was alsospecifically impaired by Cicaprost and SIN-1 while neither agent affectedRho-mediated signaling by TPβ. As stated, while both prostacyclin and NOdesensitize TPα-mediated Gq/PLCβ signaling, they do so by entirely independentmechanisms involving direct PKA- and PKG- mediated phosphorylation of TPα atSer329 and Ser331, respectively, withinits unique C-tail domain S329A,TPαS331A, TPαS329,331A, variants of TPαdefective in the prostacyclin-sensitive PKA (at Ser329),NO-sensitive PKG (at Ser331) or both  phospho-target sites 2+]imobilization by TPα and TPαS329A, they had no affect onsignaling by TPαS331A and TPαS329,331A.Conversely, both Cicaprost and the PGD2 receptor agonist BW245Cimpaired RhoA activation, cofilin phosphorylation, F-actin polymerization and[Ca2+]imobilization by TPα and TPαS331A but had no affect on signalingby TPαS329A and TPαS329,331A. Collectively,these data suggest that both Gq/PLCβ-mediated[Ca2+]imobilization and G12/RhoGEF -dependent RhoA activation of itseffector rhotekin and cofilin phosphorylation by TPα, but not by TPβ, arespecifically impaired by the potent vasodilators SIN-1 and Cicaprost in thiscellular context, at least. Of course the inhibitory effects of bothprostacyclin and NO, and other agents that signal through cAMP and cGMP, on RhoAsignaling are widely documented and form an essential component of thehomeostatic regulatory mechanism that determines the balance between activationand inhibition, particularly within the vasculature S331A and TPαS329,331A isunaffected by SIN-1 while that signaling by TPαS329A andTPαS329,331A is unaffected by Cicaprost clearly suggestthat the observed effects of SIN-1 and Cicaprost on TPα, in the HEK 293over-expression system at least, are due to direct effects on TPα itself, namelythrough site specific prostacyclin-induced PKA (at Ser329) andNO-induced PKG (at Ser331) phosphorylation rather than at someother intermediary in the RhoA signaling cascade. Moreover, in keeping with thathypothesis, the finding that agonist-induced G12/RhoA signalingby TPβ is not affected by either prostacyclin or NO again suggests that theeffects of both vasodilators are due to direct effects on TPα itself and isentirely consistent with previous findings involving both prostacyclin- andNO-mediated desensitization of TPα and TPβ signaling through the Gq/PLCβeffector system Thereafter, we investigated the effect of the selective prostacyclinanalogue Cicaprost and the NO donor SIN-1 on TP-mediated RhoA activation andsignaling. Consistent with our previous reports 2 readily desensitize TP-mediated Rho activation andcytoskeletal signaling in 1° h.AoSMCs, findings entirely predicted from and inkeeping with outcomes from other systems TPα andsiRNATPβ reagents were validated whereby we observedeffective isoform-specific knock-down of both TPα and TPβ expression andRhoA-mediated signaling in their respective HEK 293 cell lines. Moreover, theeffective delivery and utility of the latter siRNAs in 1° h.AoSMCs was confirmedwhereby the siRNATPα reduced expression of TPα but not of TPβ,while siRNATPβ reduced expression of TPβ but not of TPα. It wasnotable that the level of siRNA-mediated impairment of TPα and TPβ expression inthe 1° h.AoSMCs was significantly higher than observed in HEK.TPα or HEK.TPβcells. The reason for this apparent discrepancy is simply owing to the fact thatthe stably transfected HEK 293 cell lines express TPα and TPβ in abundance(~ 2 pmol/mg protein) relative to that expressed in 1°h.AoSMCs (20–50 fmol/mg protein) and hence, the inability of the siRNA tocompletely konckdown TPα or TPβ expression in HEK.TPα or HEK.TPβ cells was notsurprising. Consistent with their reduced expression in the 1° h.AoSMCs, therewas a significant reduction in U46619-mediated Rho activation and cofilinphosphorylation in the presence of RNAidirected to either TPα or TPβ but not to Lamin A/C confirming that both TPα andTPβ contribute to the RhoA activation in h.AoSMCs. While SIN-1 and Cicaprostsignificantly impaired U46619-mediated RhoA activation in the presence of thesiRNA directed to Lamin A/C to levels similar to that in vehicle-treated cells,the inhibitory action of both agents on RhoA activation and cofilinphosphorylation in 1° h.AoSMCs exposed to the siRNATPα wassubstantially impaired. On the other hand, in the presence ofsiRNATPβ both SIN-1 and Cicaprost reduced U46619-mediatedRhoA, F-actin polymerization (data not shown) and cofilin phosphorylation tolevels not significantly different to those observed in vehicle-treated cells.Hence, both the NO and prostacyclin impair TP-mediated cytoskeletal changesinvolving RhoA activation, F-actin polymerization and cofilin phosphorylation in1° h.AoSMCs and they do so, at least in part, by specifically and directlytargeting TPα impairing its downstream signaling. On the other hand, neithervasodilatory agent directly target TPβ.Therefore, we extended our studies by investigating TP-mediated Rhoactivation and cytoskeletal signaling in the more physiologically relevantprimary human aortic smooth muscle cells. As expected, stimulation of cultured1° h.AoSMCs with U46619 led to a concentration-dependent RhoA activation,F-actin polymerization and cofilin phosphorylation. Moreover, Cicaprost (IPagonist) and BW245C (DP agonist) and the NO donors SIN-1 and FK409 eachsignificantly impaired such U46619-induced RhoA activation, F-actinpolymerization and cofilin phosphorylation in 1° h.AoSMCs. Hence, collectively,both NO-donors and the vasodilatory prostanoids prostacyclin andPGD12/Rho-mediated shape change while cGMP/PKG signaling inhibitsthe former Gq/Ca2+ dependent mechanism but does not affect thelatter RhoA/Ca2+ independent mechanism 188 within its hypervariable region188 does not apparently alter its association witheither RhoGEFs or RhoGAPs (GTPase activating proteins), it significantlyincreases its interaction with RhoGDI (GDP dissociation inhibitor) therebyreducing the level of membrane bound RhoA and impairing its ability to activateits key effectors including Rho Kinases in vitro and in vivo evidenceto suggest that Ser188 phosphorylation of RhoA impairsactivation of Rho kinase (ROCK 1/2), but does not affect its ability to activateother Rho effectors including rhotekin, mDia-1 and PKN 188 phosphorylation of RhoA may act as a ‘secondarymolecular switch’ capable of overriding GTP-elicited activation of certain RhoAeffectors, such as ROCK, but directing it to signal with (an)other subset of Rhoeffectors, perhaps in a cell specific manner. Returning to studies herein onTPα- and TPβ-mediated RhoA signalling, both NO and prostacyclin directly targetRhoA phosphorylation at Ser188 through their regulation of PKGand PKA signaling, respectively (data not shown). Hence, RhoA-mediated signalingby TPα is subject to regulation by both direct prostacyclin/PKA andNO/PKG-inhibition mediated through their respective phosphorylation ofSer329 and Ser331 within the unique C-taildomain of TPα in addition to the more general type of regulation throughSer188 phosphorylation of RhoA. On the other hand, TPβ isnot a direct target for either PKA or PKG phosphorylation or inhibition, but itsRhoA-mediated signaling would be sensitive to RhoA phosphorylation by eithersecond messenger kinase. Whilst it has not as yet been established whether the“molecular switch mechanism” resulting from RhoAS188phosphorylation proposed by Nusser et al. As stated, it is widely held that agents that signal through either cAMP-or cGMP-second messenger systems play a critical counter-balancing/inhibitoryaffect on RhoA-mediated signaling cascades Ser188 switch mechanism’exists within TXA2-responsive VSM or indeed in platelets, RhoAsignaling through TPβ may be directed away from one effector system, such asROCK signaling, in the direction of another effector(s), such as rhotekin,mDia-1 and PKN (Hence, as presented in our model , we prop and PKN  as propo2+-dependent andG12/RhoA/Ca2+-independent signaling byprostacyclin and NO within the vasculature. On the other hand, as TPβ remainsunaffected by either agent, at the interface of the receptor at least, thefunctional role of TPβ remains to be further clearly defined. The data hereinhighlight further critical differences between the TPα and TPβ receptor isoformsin terms of their regulation of Rho signaling that are likely to bephysiologically relevant in human tissues such as SM and suggest that selectivetargeting and impairment of TPα-mediated signaling may offer a usefultherapeutic approach in the treatment of certain vascular diseases such assystemic- and pulmonary-hypertension in which both TXA2 andRhoA dysfunction has been implicated Hence, in summary TPα- and TPβ-mediated RhoA signaling functionally divergeat the point at which prostacyclin and NO affect the RhoA signaling cascade.These data further support the hypothesis that TPα is the major regulatory TPisoform involved in vascular hemostasis being a direct target for inhibition ofboth its Gq/PLCβ/Ca


It also couples to tetratricopeptide repeat 1 (TPR1) which is linked to Ras activation. It is unknown whether PLCβ and TPR1 interact with the same regions on Gα16. Previous studies on Gαq have defined two minimal clusters of amino acids that are essential for the coupling to PLCβ. Cognate residues in Gα16 might also be essential for interacting with PLCβ, and possibly contribute to TPR1 interaction and other signaling events.Gα16. Regulations of PLCβ and STAT3 were partially weakened by each cluster mutant. A mutant harboring mutations at both clusters generally produced stronger suppressions. Activation of Jun N-terminal kinase (JNK) by Gα16 was completely abolished by mutating either clusters. Contrastingly, phosphorylations of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) were not significantly affected by these mutations. The interactions between the mutants and PLCβ2 and TPR1 were also reduced in co-immunoprecipitation assays. Coupling between G16 and different categories of receptors was impaired by the mutations, with the effect of switch III mutations being more pronounced than those in the α3 helix. Mutations of both clusters almost completely abolished the receptor coupling and prevent receptor-induced Gβγ release.Alanine mutations were introduced to the two amino acid clusters (246–248 and 259–260) in the switch III region and α3 helix of Gα16 is critical for the activation of PLCβ, STAT3, and JNK but not ERK or NF-κB. Binding of Gα16 to PLCβ2 or TPR1 was reduced by the mutations of either cluster. The same region could also differentially affect the effectiveness of receptor coupling to G16. The studied region was shown to bear multiple functionally important roles of G16.The integrity of the switch III region and α3 helix of Gα PLCβ activity can in turn regulate many downstream kinases and transcription factors, thereby modulating cellular processes such as growth and differentiation. The interactions between PLCβ and Gαq subfamily members have been examined by mutagenesis studies. Alanine scanning mutagenesis of Gαq has identified a stretch of amino acids (Ile217-Lys276) that may be responsible for PLCβ interaction. Within this region, two groups of amino acids  employ a variety of signal transduction pathways to regulate cellular functions. One of the primary signaling routes initiated upon activation of GPCRs is through the stimulation of PLCβ by members of the Gαcids Ile27-Lys276 16 is a member of Gq subfamily which can activate PLCβ  (DSP) were from Pierce Biotechnology . Pertussis toxin was purchased from List Biological Laboratories. All other chemicals were obtained from Sigma.Human TPR1 cDNA was kindly provided by Richard D. Ye . Human embryonic kidney 293 (HEK293) and monkey kidney fibroblast COS-7 cells were purchased from American Type Culture Collection (ATCC CRL-1573 and 1651). Restriction endonucleases were from Roche Applied Sciences. DNA purification columns were from Qiagen. DNA modification enzymes, custom primers and cell culture and transfection reagents were from Invitrogen. -inositol was added to each well of transfected COS-7 cells and incubated for 18–24 hr. The labeling media were subsequently replaced by 1 ml of assay medium  for 10 min and then 1 ml of assay medium containing the appropriate agonist was added to the cells for another 1 h at 37°C. Reaction was stopped by adding 750 μl of ice-cold 20 mM formic acid after aspiration and the plates were stored at 4°C for 30 min. [3H]-IP were separated from labeled inositol by ion-exchange chromatography as described previously [750 μl of inositol-free DMEM containing 5% FBS and 2.5 μCi/ml eviously .3H]-adenine (1 μCi/ml) in MEM containing 1% FBS (vol/vol) one day after transfection. After 18–24 h the labeling media were replaced by 1 ml of 20 mM Hepes-buffered MEM containing 1 mM isobutylmethylxanthine (IBMX) and the appropriate drugs and incubated at 37°C for 30 min. The treatment was terminated with 1 ml ice-cold 5% trichloroacetic acid (wt/vol) with 1 mM ATP after aspiration and stored at 4°C for 30 min. [3H]cAMP was extracted from the pool of labeled nucleotides by sequential ion-exchange chromatography as described [HEK293 cell were labeled with [escribed .3H]cAMP fractions to those of the total labeled nucleotide fractions and expressed as cAMP/. Similarly, IP levels were expressed as IP/. Data shown in the figures were the mean ± S.D. of triplicates within one single experiment. At least three individual experiments yielded similar results. One-way ANOVA with Tukey-Kramer's test were performed using GraphPad Prism 3.03 to verify the significance between different treatment groups within the experiments.For cAMP and IP accumulation assay, absolute values for cAMP or IP accumulations varied between experiments, but variability within a given experiment was in general <10%. The cAMP levels were interpreted as the ratios of the counts per minute of -ATP per sample). After 30 min incubation at 30°C with occasional shaking, the reactions were terminated by adding sample buffer, and the samples were resolved by 12% SDS-PAGE. The gel was fixed for 30 min and the radioactivity incorporated to GST-c-Jun was detected and quantified by PhosphorImager (Molecular Dynamics 445 SI).Details of the assays were basically identical as described in our previous study . Briefly1R, A2AR, A2BR: types 1, 2A and 2B adenosine receptor; AC: adenylyl cyclase; CHA: N6-cyclohexyladenosine; D1R: type 1 dopamine receptor; ERK: extracellular signal-regulated kinases 1 and 2; fMLP: N-formyl-methioinyl-leucyl-phenylalanine; GPCR: G protein coupled receptor; GTPase: GTP hydrolase; IBMX: isobutylmethylxanthine; IKK: inhibitor of κB kinase; IP: inositol phosphates; JNK: c-jun N-terminal kinase; MAPK: mitogen-activating protein kinase; MEK: MAPK/ERK kinase; MEM: Eagle's minimal essential medium; MKK: MAPK kinase; NF-κB: nuclear factor κB; PKC: protein kinase C; PLCβ: phospholipase Cβ isoform; PTX: pertussis toxin; STAT3: type 3 signal transducers and activator of transcription protein; TPR1: tetratricopeptide repeat protein 1.AThe authors declare that they have no competing interests.MY carried out most of the experiments, except the co-immunoprecipitation, which was performed by AMFL. MKCH helped to design the mutant constructs, performed molecular model constructions, analyzed and interpreted the results, and drafted the manuscript. YHW conceived of the study and participated in its design and coordination.


We have recently shown that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, inhibits angiogenesis. However, the molecular mechanisms by which EGCG inhibits angiogenesis have never been investigated. In this study, we examined the interaction of PI3K/AKT and MEK/ERK pathways on the regulation of FOXO transcription factors, which ultimately control the antiangiogenic effects of EGCG.Inhibition of PI3K/AKT and MEK/ERK pathways interact synergistically to inhibit migration and capillary tube formation of HUVEC cells and further enhanced the antiangiogenic effects of EGCG. Inhibition of AKT and MEK kinases synergistically induced FOXO transcriptional activity, which was further enhanced in the presence of EGCG. Phosphorylation deficient mutants of FOXO induced FOXO transcriptional activity, inhibited HUVEC cell migration and capillary tube formation. Inhibition of FOXO phosphorylation also enhanced antiangiogenic effects of EGCG through transcriptional activation of FOXO.Inhibition of PI3K/AKT and MEK/ERK pathways act synergistically to regulate antiangiogenic effects of EGCG through activation of FOXO transcription factors. The activation of FOXO transcription factors through inhibition of these two pathways may have physiological significance in management of diabetic retinopathy, rheumatoid arthritis, psoriasis, cardiovascular diseases, and cancer. FOXO isoforms results in different phenotypes. For example, mice homozygous for a FOXO1-/- allele, but not FOXO3a-/- or FOXO4-/- mice, die during embryogenesis from defects in vascular development [angiopoietin 1 (Ang1) modulates endothelial function [Angiogenesis is a complex event which requires endothelial cell sprouting, lumen formation, tubulogenesis and is regulated by the coordinated action of different transcription factors ,2. Theirelopment ,10. Alth/KIP1 [FOXO subfamily of forkhead transcription factors include FOXO1a/FKHR, FOXO3a/FKHRL1, and FOXO4/AFX -14. The /KIP1 ,20,22,23/KIP1 ,25, Fas /KIP1 , and Bcl/KIP1 . Consequ/KIP1 -33, indiFOXO transcription factors play a crucial role in the regulation of tissue homeostasis in organs such as the pancreas and the ovaries and complex diseases such as diabetes and cancer -8. FOXO In vitro kinase assay showed that FOXO1 was phosphorylated by ERK and p38 but not by JNK [in vivo.The Ras proteins are small (21 kDa) GTP-binding, membrane-associated proteins . They art by JNK . In NIH3Green tea is a popular beverage consumed in some parts of the world and is a rich source of polyphenols ,53. GreeThe purpose of this study was to examine whether the inhibition of PI3K/AKT and MEK/ERK pathways enhance the antiangiogenic effects of EGCG through activation of FOXO transcription factors. The data demonstrate that inhibition of PI3K/AKT and MEK/ERK pathways act synergistically to induce FOXO transcription activity, capillary tube formation and migration. Furthermore, antiangiogenic effects of EGCG are regulated through activation of FOXO transcription factors.We first measured the involvement of PI3K/AKT and MEK/ERK pathways on EGCG-induced apoptosis in HUVEC cells. Treatment of HUVEC cells with AKT inhibitor and MEK1/2 inhibitor (PD98059) alone induced apoptosis  Fig. . HUVEC cTo the best of our knowledge, this is the first study to demonstrate that inhibition of PI3K/AKT and MEK/ERK pathways acted synergistically to induce activation of FOXO transcription factors and enhanced the antiangiogenic effects of EGCG in HUVEC cells. Specifically, cell migration and capillary tube formation was inhibited by AKT inhibitor and MEK inhibitor. We have recently demonstrated that EGCG can inhibit pancreatic cancer development and angiogenesis in nude mice . EGCG inin vitro and neovascularization in vivo [Angiogenesis, the sprouting of new capillaries from the preexistent blood vessels, is of central importance in many biological processes, including embryonic vascular development and differentiation, wound healing, and organ regeneration ,59. In a in vivo ,63. We h in vivo . EGCG alThe Ras signal transduction pathway is complex with multiple intersections and bifurcations . We havein vivo [FOXO1 (FKHR) contains 15 consensus phosphorylation sites for the mitogen-activated protein kinase (MAPK) family. The phosphorylation of FOXO1 was demonstrated by ERK and p38 MAPK but not by JNK . Epidermin vivo . These d/Kip1 that trigger apoptosis [Activation of AKT through PI3K leads to phosphorylation and nuclear exclusion of the transcription factor FOXO. Accordingly, FOXO1 dephosphorylation leads to translocation to the nucleus and induces the transcription of target genes such as Fas, BIM, p27poptosis . With repoptosis . Albeit poptosis . Ang-1 ipoptosis . On the We have demonstrated that inhibition of PI3K/AKT and Ras/MEK/ERK pathways interact synergistically to activate FOXO transcription factors which, in turn, inhibit angiogenesis. Inhibition of both of these pathways further enhances the antiangiogenic effects of EGCG. In a recent study, we have demonstrated that EGCG can modulate the expression of genes known to play a role in the cancer progression, invasion, metastasis and angiogenesis . We haveEGCG was purchased from LKT Laboratories, Inc. . MEK inhibitor (PD98059), AKT inhibitor-IV and Terminal Deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay kit were purchased from EMD Biosciences . AKT inhibitor-IV is a cell-permeable benzimidazole compound that inhibits AKT phosphorylation/activation by targeting the ATP binding site of a kinase upstream of AKT, but downstream of PI3K . It has Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics  and maintained in endothelial cell growth factor medium-2  supplemented with 5% FBS. Stock solutions of the EGCG were prepared in DMSO and diluted with complete medium, and an equal volume of DMSO  was added to the controls.in vitro angiogenesis, subconfluent HUVECs were resuspended in complete medium and added to Matrigel containing wells (1 × 104 cells/well), and exposed to EGCG or DMSO (control). The plates were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Capillary tube formation was assessed after 24 h by counting the total number of capillary like tubular structures from three randomly chosen fields using an inverted microscope.Cell migration assay was performed as we described earlier . In brie4 cells in 0.2 ml) cells in complete medium was mixed with desired concentration of EGCG or DMSO (control), and the cell suspension was added to the upper chamber. The lower chamber contained 0.6 ml of complete medium with the same concentration of EGCG or DMSO. Migration through the membrane was determined after 24 h of incubation at 37°C. Cells remaining on the topside of the transwell membrane were removed using a cotton swab. The membrane was washed with ice-cold PBS. Cells that had migrated to the underside were fixed with 90% methanol and stained with giemsa. Cell migration was quantified by counting the number of cells per field in five random fields.Cell migration assay was performed as we described earlier . In brieHUVECs cells were pretreated with AKT inhibitor-IV (1 μM) or MEK1/2 inhibitor PD98059 (10 μM) in the presence or absence of EGCG (40 μM). Cells were harvested and TUNEL assay was performed as per manufacturer's instructions (Promega).HUVEC cells were transfected with empty vector, FOXO1-TM, FOXO3a-TM or FOXO4-TM along with reporter plasmids, p6xDBE-luc and pRL-TK. The FOXO expression vectors (wild type and phosphorylation deficient mutants) and FOXO-luciferase constructs have been described elsewhere ,69. AfteThe mean and SD were calculated for each experimental group. Differences between groups were analyzed by one or two way ANOVA using PRISM statistical analysis software . Significant differences among groups were calculated at P < 0.05.The author(s) declare that they have no competing interests.


Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses. Unlike ions of other transition metals such as iron, copper, and vanadium, ZnTo characterize the role of oxidative stress in zinc-induced toxicity.2O2)-specific fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells.We used an integrated imaging approach that employs the hydrogen peroxide (H2+ ionophore pyrithione of A431 skin carcinoma cells preloaded with the H2O2-specific indicator PG1 resulted in a significant increase in H2O2 production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H2O2 formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts.Zinc treatment in the presence of the Zn2O2 formation, and a shift toward positive redox potential in cells exposed to zinc. These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative stress in toxicological responses.Taken together, these findings show a disruption of mitochondrial integrity, H Oxidative stress is a common feature of the mechanism of injury induced by a broad range of environmental agents . Such en2DCF-DA)  . Unfortu2DCF-DA) .Mitochondria are a known source of partially reduced oxygen species generated as a by-product of oxidative metabolism in the cell . Dysregu2O2) levels, and redox status in living cells (4) was used as a soluble form of the zinc ion (Zn2+) that is also environmentally relevant, because sulfate salts are known to predominate among soluble metal salts released by combustion processes. In addition, we used zinc pyrithione, which is used as a mildewcide in outdoor paints, making skin cells a relevant cell type to study with this agent.In the present study, we used an integrated imaging approach to the investigation of environmental oxidative stress resulting from mitochondrial dysfunction. By applying established and recently introduced indicators, this integrated approach allows real-time measurement of mitochondrial membrane potential, hydrogen peroxide  in six-well culture plates at 150,000–250,000 cells per well. Cultures were deprived of growth factors overnight prior to study. The A431 cells were preloaded with Peroxy Green 1 (PG1) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethy-lbenzimidazolylcarbocyanine iodide [JC-1 ], and the cover slip cultures were fitted into a custom-made stainless-steel chamber filled with 500 μL phosphate-buffered saline (PBS), which was supplemented with 1 g/L glucose and kept at 37°C with a stage heater. Conventional and spectral confocal microscopy analyses were conducted using a Nikon Eclipse C1Si confocal microscope  that was equipped with TE 2000 microscope. Light was delivered to the sample with a 60 × Plan Apo 1.4 numerical aperture (NA) objective; the system also uses diode lasers of 404 nm, 488 nm, 561 nm, and 633 nm. Prior to each experiment, the confocal microscope was tested for field illumination alignment, optical efficiency, colocalization, and axial resolution  at concentrations between 10 μM and 100 μM with or without 4 μM of the Zn2+-specific ionophore pyrithione, given at 5 min. The inhibitors apocynin (100 μM) (2+. H2O2 (1 mM), CCCP (10 μM), or dithiothreitol  was added at the end of experiments as positive controls.A431 human skin carcinoma cells  were used for live cell imaging experiments. The cells were cultured in Dulbecco’s modified Eagle’s medium  and supplemented with 10% fetal bovine serum (FBS) and 5 μg/mL gentamicin at 37°C, 5% COsolution ; and the(100 μM) , wortman(100 μM) , carbony(100 μM) , and Ly2(100 μM)  and EHT (100 μM)  were app2O2 production was monitored using the fluorescein-like Peroxy Green 1 (PG1) dye (2O2. A431 cells grown on glass cover slips were labeled in 5 μM PG1 for 15 min at 37°C in PBS glucose solution prior to measurement. PG1 fluorescence was excited using an argon laser (at λ = 488 nm), and the emission spectrum was monitored in a range of λ = 490 nm to 570 nm over 32 channels with 2.5 nm band pass. Signal intensity was quantified at the PG1 emission peak at 523 nm.HPG1) dye . PG1 is The mitochondrial membrane potential was monitored using the fluorescent indicator JC-1. In the presence of physiological mitochondrial membrane potentials, JC-1 forms aggregates that fluoresce with an emission peak at 588 nm. Loss of membrane potential favors the monomeric form of JC-1, which has an emission peak at 530 nm. Cells were labeled with 5 μM JC-1 in DMEM supplemented with 10% FBS and 1 μg/mL gentamicin at 37°C. After 15 min incubation, cells were washed with PBS twice and placed in the chamber with 500 μL PBS glucose. JC-1 fluorescence intensity was monitored with dual excitation at 488 nm and 561 nm and an emission scan range of 490–650 nm . Mitochondrial membrane potential was inferred from the ratio of fluorescence intensity of emission maximum at 593 nm and 538 nm, which represented the J-aggregate and monomeric forms, respectively.ad libitum access to both water and food. Animal care was given in accordance with institutional guidelines, and animals were treated humanely, with regard to alleviating suffering. The studies were conducted with approval by the EPA Institutional Animal Care and Use Committee. Mice were euthanized with an intraperitoneal injection of sodium pentobarbital (80 mg/kg body weight), and hearts were excised and weighed. Freshly isolated mitochondria were prepared from the ventricles by differential centrifugation. Briefly, heart tissues were homogenized with three strokes of a polytron homogenizer in ice-cold homogenization buffer containing 225 mM mannitol, 75 mM sucrose, 5 mM morpholinepropanesulfonic acid, and 2 mM taurine, with 0.2% bovine serum albumin . The homogenate was transferred to a glass homogenizer and homogenized for five strokes on ice. After centrifugation at 2,500 rpm for 5 min at 40°C, the supernatant was removed and centrifuged at 8,000 rpm for 5 min. The pellet was sequentially washed with homogenization buffer three times and resuspended in homogenization buffer plus 5 mM KH2PO4. The protein concentration was determined with BSA as a standard by a Bradford assay.Adult pathogen-free female CD-1 mice, which were purchased from Charles River , were used as the source of the cardiac mitochondria. Animals were housed at the U.S. Environmental Protection Agency (EPA) animal care facility  and given 2PO4 at room temperature. After adding 10 mM glutamate and 2 mM malate, the light scattering of mitochondria was measured at 540 nm for 40 min with a 96-well plate spectrophotometer . We initiated calcium- or zinc-induced mitochondrial swelling by adding 250 μM calcium or 100 μM zinc and measured for another 20 min. The absorbance was normalized to the initial absorbance.The mitochondria (50 μg) were incubated in buffer containing 120 mM KCl, 10 mM Tris HCl, and 5 mM KHThe genetically encoded mitochondria- targeted form of the genetically encoded fluorescent reporter redox-sensitive green fluorescent protein (MTroGFP1) was used for the measurement of redox potential in mitochondria . Fugene t-test; a p-value of < 0.05 was considered statistically significant.Imaging data were collected with Nikon EZ-C1 software and quantified by EZ-C1 and Nikon Elements. Figures were plotted with mean ± SE, with three repeat experiments. An average of 5–10 cells with different fluorescence intensities were collected as regions of interests in each experiment and quantified with Nikon EZ-C1 and Nikon Elements software (Nikon Instruments). Pairwise comparisons were carried out using Student’s 2+, a non-redox-active metal that is ubiquitously found associated with particulate matter in ambient air and occupational settings. Addition of noncytotoxic concentrations (2+ and pyrithione (10–100 μM ZnSO4 plus 4 μM pyrithione) to A431 cells resulted in a time-dependent elevation in intracellular concentrations of H2O2 as detected by an increase in PG1 fluorescence intensity (2O2 added as positive control resulted in a marked increase (550%) in PG1 fluorescence  induced spontaneous swelling, indicated by a 17% decrease in absorbance at 4 min, whereas the addition of zinc induced a similar effect, with a 15% decrease in absorbance  and reducing agents (DTT) within 2 min. Our findings show that Zn2+ induces a rapid decrease of mitochondrial reducing potential, consistent with the independent measurements of reduced mitochondrial membrane potential and opening of the mitochondrial permeability pore.A critical element of the approach applied in this study is monitoring redox potential using redox-sensitive variants of green fluorescent protein (roGFPs). These genetically encoded reporters were first described by 2+-induced inhibition of mitochondrial respiration by binding to the bc1 complex , it is possible to elucidate the mechanism of oxidative stress induced by exposure to environmental agents. The conclusion that oxidative stress induced by Zn2+ originates in the mitochondria is supported by the following findings in the present study: a) the partial inhibition of Zn2+-stimulated H2O2 production in the cytosol by the mitochondrial respiration uncoupler CCCP, b) the observation that Zn2+ exposure induces mitochondrial depolarization, c) the finding that Zn2+ induces mitochondrial swelling (in isolated cardiac mitochondria), and d) the decrease of reducing redox potential induced by Zn2+ exposure.As our findings with Znp-benzoquinone induce H2O2 production and a change in redox potential in both cytosolic and mitochondrial compartments .The methodologies used in this study are broadly applicable to other oxidative stressors such as redox active transition metals and organic oxidants. Using BEAS 2B cells as a model of the human airway epithelium, we have observed that the diesel exhaust organic constituents 1,2-napthoquinone and 2+ results in mitochondrial dysfunction, possibly an arrest of electron transport (2O2 detected as increased PG1 fluorescence subsequent to mitochondrial depolarization and redox changes.The timeline of events measured with the integrated approach using real-time imaging of living cells in this study seems to support a sequence in which exposure to Znransport , causing2+-induced depolarization of mitochondria reported by the color shift in JC-1 fluorescence. However, it is likely that the relative timing of the changes in fluorescence reported by the sensors and indicators used in this study is also influenced by differences between their individual response rates. Thus, it is not currently possible to precisely establish the sequence of events based on changes in the fluorescence intensity of different sensors. Future studies will focus on refining this integrated approach to factor in response rates in a dynamic system and thus more accurately reflect the temporal sequence of cellular events involving mitochondrial dysfunction and ROS generation leading to oxidative stress induced by ambient toxicants.The change in redox potential detected by the MTroGFP1 sensor appears to precede Zn


Increased superoxide levels are implicated in the pathogenesis of diabetic retinopathy. We have shown that functional activation of a small molecular weight G-protein, H-Ras, is one of the signaling steps involved in glucose-induced apoptosis of retinal capillary cells. The goal of this study was to elucidate the mechanism(s) by which oxidative stress could result in the activation of H-Ras in diabetes.2O2, or the cells in which glucose-induced superoxide accumulation was inhibited either by superoxide dismutase mimetic (MnTBAP) or by overexpressing mitochondrial superoxide dismutase (MnSOD). The in vitro experiments were complemented with in vivo experiments using the retina from mice overexpressing MnSOD.Experiments were performed in isolated retinal endothelial cells that were treated with H2O2 activated H-Ras and its downstream signaling pathway, including Raf-1 and phosphorylation of p38 (p-p38) MAP kinase. Inhibition of superoxide significantly attenuated glucose-induced activation of H-Ras, Raf-1 and p-p38 MAP kinase. Overexpression of MnSOD in mice prevented diabetes-induced activation of both H-Ras and p-p38 MAP kinase.HOur results clearly indicate that the activation of H-Ras and its downstream signaling pathway in the retina and its vasculature could be under the control of superoxide, and H-Ras activation in diabetes can be prevented by inhibiting superoxide accumulation. Diabetic retinopathy, a slow progressing complication of diabetes, is considered a multifactorial disease. Although many hyperglycemia-induced metabolic abnormalities are implicated in its pathogenesis, the exact mechanism of the development of retinopathy remains elusive. Our previous studies have suggested a role for the small molecular weight G-protein, H-Ras, in the development of retinopathy in diabetes. We have shown that functional activation of H-Ras is one of the signaling steps involved in glucose-induced capillary cell apoptosis. Inhibitors of H-Ras function reduce glucose-induced increased apoptosis of retinal capillary cells, and the therapy that inhibits apoptosis of retinal vascular cells and retinopathy in diabetes, inhibits diabetes-induced increase in Ras expression and mRNA levels in the retina -3.Small molecular weight G-proteins act as one of the key regulators of the signaling cascade triggered by oxidative stress , and Ras2O2 on the activation of H-Ras, and determined the effect of overexpression of MnSOD on glucose-induced activation of H-Ras and apoptosis. In vivo experiments were performed using the retina from the mice overexpressing MnSOD (MnSOD-Tg) to investigate the role of mitochondrial superoxide in the activation of retinal H-Ras in diabetes. The results presented demonstrate that superoxide regulate the activation of retinal H-Ras in diabetes.Oxidative stress is elevated in the retina and its capillary cells and remains elevated when the histopathology can be seen in the vasculature ,9. Oxida2 and 5% CO2 [Retinal endothelial cells, prepared from cow eyes , were cultured in Dulbecco's modified eagle medium (DMEM) containing 15% fetal calf serum (heat inactivated), 5% Nu-serum, heparin (50 μg/ml), endothelial growth supplement 25 μg/ml) and antibiotic/antimycotic in 95% O5 μg/ml ad 5% CO2 . Conflued 5% CO2 . This dud 5% CO2 ,17. Cont2O2 for 1 h. The cells were quickly washed with DMEM and incubated in 5 mM glucose and 20 mM glucose media for 96 h. At the end of the incubation period, the cells were washed with PBS, scraped, and used to determine the activation of H-Ras and phosphorylation of p38 MAP kinase, and cell apoptosis.Hydrogen peroxide levels are elevated in the retina in diabetes . In ordeRetinal endothelial cells were transfected with MnSOD expression plasmid DNA using a procedure described previously. In brief, 60-80% confluent endothelial cells from fourth passage were incubated with a transfection-complex comprising of MnSOD expression plasmid DNA and superfect transfection reagent  for 8 h. The transfected cells were then incubated in a fresh medium containing 2.5% fetal calf serum (heat inactivated), 10% Nu-serum, heparin (50 μg/ml), endothelial growth supplement (2.5 μg/ml) and antibiotic/antimycotic supplemented with 5 mM or 20 mM glucose for 96 h. As with our observations in previous studies, we found the efficiency of the transfection to be about 30% .Hemizygous MnSOD-Tg mice, generated using human β-actin-MnSOD expression construct were bred with wild-type B6C3 F1 mice to generate experimental animals. The litters were genotyped by Southern blot analysis. A group of wild-type mice (WT) and MnSOD-Tg mice  were made diabetic by intraperitoneal injection of streptozotocin (60 mg/kg/body weight) for five consecutive days. We have shown that MnSOD Tg mice can be made diabetic successfully by giving similar doses of streptozotocin as their wild-type counterparts . Body weRetinal endothelial cells incubated in either low (5 mM) or high (20 mM) glucose for 96 h were used to quantify the relative abundance of GTP-bound active H-Ras using a Raf-1 binding assay kit . This assay takes advantage of the high affinity of Ras-GTP for the Ras binding domain (RBD) of Raf-1. The cell extract was added to Raf-1RBD, and the Raf-RBD/GTP-Ras complex was pulled down by glutathione affinity beads. The beads were re-suspended in Laemmli reducing sample buffer and boiled for 5 min. The amount of activated Ras was determined by Western blot using Ras-Pan-specific antibody that was supplied in the Raf-1 binding assay kit.Protein (30-50 μg) was separated on a 12% denaturing polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked, followed by the incubation with the antibody against H-Ras or Raf-1 . After incubation with horseradish peroxidase-conjugated secondary antibody, the membranes were developed using ECL-Plus Western blotting detection kit . Kaleidoscope pre-stained molecular weight markers (Bio-Rad Laboratories) were run simultaneously on each gel. To ensure equal loading among the lanes, we also determined the expression of the housekeeping protein, β-actin. The membranes were blotted for H-Ras or Raf-1, then incubated with stripping buffer  at 50 °C for 30 min, washed, and incubated with anti-β-actin . The membranes were again washed. Next, they were incubated with horseradish peroxidase-conjugated secondary antibody, and developed using ECL-Plus Western blotting detection kit (Amersham Biosciences).Each band was quantified using Un-Scan-It Gel digitizing software , and the values in the histograms were presented as mean band density of the protein of interest  divided by the intensity of β-actin in the same sample.The activation of MAP kinase was determined by performing the Western blot analysis of the phosphorylation of p38 MAP kinase  using phospho-p38 (p-p38) antibodies from Cell Signaling Technology .PLUS kit from Roche Diagnostics . This method determines the relative amounts of mono- and oligonucleosomes generated from the apoptotic cells by using monoclonal antibodies directed against DNA and histones, respectively [Endothelial cell apoptosis was determined using Cell Death Detection ELISAectively ,15. The Each experiment was repeated three or more times, and measurements were performed in duplicates. Values are reported as mean SD. The experimental groups were compared using the nonparametric Kruskal-Wallis test followed by the Mann-Whitney test.2O2 in 5 mM glucose, and, for comparison, in 20 mM glucose medium. As shown in 2O2 in 5 mM glucose medium activated H-Ras; the activation was further increased to 30-40% when the cells were incubated with H2O2 in 20 mM glucose medium. In the same cells H2O2 significantly activated Raf-1 and increased phosphorylation of p38 MAP kinase  and apoptosis in endothelial cells incubated with HP kinase . The res2O2 in 5 mM glucose medium; however, when the cells were exposed to H2O2 in the presence of 20 mM glucose medium, cell death was increased to about 90% compared to the cells incubated in 5 mM glucose medium  compared to the non diabetic mice . Similarly, GHb values were also elevated by over 2.5 fold in diabetic mice, and the GHb values were comparable in WT and MnSOD-Tg mice: WT group had 5.3% and 13.8% GHb in non diabetic and diabetic mice, respectively, compared to 6.3 and 13.8 in the MnSOD-Tg non diabetic and diabetic mice. This clearly shows that the severity of hyperglycemia was similar in both WT-diabetic and MnSOD-Tg diabetic mice, which allowed us to investigate the effect of diabetes-induced oxidative stress, not the effect of hyperglycemia by itself, on the parameters of interest.2O2 increased activation of H-Ras and its Raf-1-mediated downstream signaling pathway in retinal endothelial cells, and overexpression of MnSOD prevented diabetes-induced activation of H-Ras and its signaling steps. Incubation of endothelial cells with H2O2 has been shown to increase vascular endothelial growth factor (VEGF) levels and cell permeability, the parameters associated with diabetic retinopathy [In diabetes H-Ras is activated, oxidative stress, including superoxide levels are elevated in the retina and its endothelial cells, and the therapies that inhibit the development of retinal histopathology in diabetes also inhibit Ras activation and superoxide accumulation ,15,16. Iinopathy .Diabetes-induced ROS act as a causal link between elevated glucose and the other metabolic abnormalities implicated in the development of complications ,13. We hOur exciting in vivo data demonstrate that in MnSOD-Tg mice the retina is protected from diabetes-induced activation of H-Ras. These results were obtained without ameliorating the severity of hyperglycemia in WT and MnSOD-Tg mice that were made diabetic with streptozotocin. This strongly supports our in vitro data obtained from the endothelial cells. Mitochondria dysfunction in the retina is associated with the pathogenesis of diabetic retinopathy; the release of cytochrome c into the cytosol and Bax into the mitochondria is increased in the retina in diabetes ,14. Bax Raf-1 is a key downstream effector protein of Ras function that in its inactive state is localized in the cytosol. We have shown that in diabetes, Raf-1 is increased in the retina. The therapies that inhibited the development of retinopathy in diabetic rats also inhibited Raf-1 activation [Ras-dependent activation of Raf-1 can activate MAP kinase, and the MAP kinase family is considered to be an effector of the Ras-Raf pathway . PhosphoIn conclusion, this is the first report showing that the activation of H-Ras and its downstream signaling pathway in the retina and its vascular cells is under the control of superoxide. Our conclusion is supported by both in vivo (retina from MnSOD-Tg mice) and in vitro  models. The results clearly demonstrate that H-Ras activation in diabetes can be prevented by overexpression of MnSOD, suggesting a complex cross-talk. The regulation of H-Ras-mediated signaling should help us identify novel pharmaco-therapeutic strategies for inhibition of retinopathy in diabetes.


The septins are a conserved family of proteins that have been proposed to carry out diverse functions. In budding yeast, the septins become localized to the site of bud emergence in G1 but have not been thought to carry out important functions at this stage of the cell cycle. We show here that the septins function in redundant mechanisms that are required for formation of the bud neck and for the normal pattern of cell growth early in the cell cycle. The Shs1 septin shows strong genetic interactions with G1 cyclins and is directly phosphorylated by G1 cyclin-dependent kinases, consistent with a role in early cell cycle events. However, Shs1 phosphorylation site mutants do not show genetic interactions with the G1 cyclins or obvious defects early in the cell cycle. Rather, they cause an increased cell size and aberrant cell morphology that are dependent upon inhibitory phosphorylation of Cdk1 at the G2/M transition. Shs1 phosphorylation mutants also show defects in interaction with the Gin4 kinase, which associates with the septins during G2/M and plays a role in regulating inhibitory phosphorylation of Cdk1. Phosphorylation of Shs1 by G1 cyclin-dependent kinases plays a role in events that influence Cdk1 inhibitory phosphorylation. G1 cyclins bind and activate cyclin-dependent kinases (CDKs) to initiate cell cycle events in G1 CLN1 and CLN2 interact genetically with a member of the septin family of proteins, which suggests that septins may function in events controlled by G1 cyclins The mechanisms used by the G1 cyclins to control G1 events are poorly understood. cln1Δ cln2Δ, and the septins become localized to the site of bud emergence in G1 Although loss of septin function causes a G2/M arrest in budding yeast, there is some evidence that the septins may also carry out functions early in the cell cycle. A unique allele of Cdc12 is synthetically lethal with In this study, we tested whether septins function in pathways controlled by G1 cyclins and identified a septin-dependent pathway that controls the pattern of cell growth early in the cell cycle. Moreover, we found that the Shs1 septin shows strong genetic interactions with G1 cyclins and is a direct target of CDKs associated with G1 cyclins. However, phosphorylation of Shs1 does not appear to play a role in septin-dependent pathways that control the pattern of cell growth during G1. Rather, Shs1 phosphorylation appears to influence events that regulate inhibitory phosphorylation of Cdk1 at the G2/M transition. Phosphorylation of Shs1 by G1 cyclin-dependent kinases could therefore play a role in mechanisms that help link entry into mitosis to successful completion of G1 events.CDC12 was previously found to show synthetic lethal interactions with cln1Δ cln2Δ, we tested whether this is also true for shs1Δcln1Δ cln2Δ shs1Δ haploids, which suggested that shs1Δ is synthetically lethal with cln1Δ cln2Δ. To further confirm that shs1Δ is synthetically lethal with cln1Δ cln2Δ, we generated a shs1Δ cln1Δ GAL1-CLN2 strain, in which the expression of CLN2 could be repressed by dextrose. This strain grew normally on galactose, but failed to grow on dextrose . The cdc12-6 cln1Δ cln2Δ cells were unable to form buds with normal morphology and largely failed to direct growth to the daughter bud . The results are summarized in The Shs1-3XHA isolated from yeast cells to map in vivo phosphorylation sites was phosphorylated on a total of nineteen sites, including all five CDK consensus sites. Four of the eight non-consensus sites that were phosphorylated by Pho85-Pcl1 in vitro were also phosphorylated in vivo. The in vivo mapping experiments may have missed some sites because they were carried out with total Shs1-3XHA isolated from asynchronous cells, rather than with specific isoforms, to ensure that there was no bias towards specific phosphorylation sites. As a result, the Shs1-3XHA used for in vivo mapping was not quantitatively phosphorylated, whereas the Shs1 phosphorylated in vitro was quantitatively phosphorylated, which may have led to better detection of some phosphorylation sites on the in vitro phosphorylated form of Shs1.shs1-ps1) shs1-ps2). We also made two mutant versions of Shs1 in which we mutated non-consensus sites in addition to the five consensus sites. In shs1-ps3, we mutated only the non-consensus sites that were phosphorylated in vitro and in vivo. In shs1-ps4, we mutated all non-consensus sites that were phosphorylated in vitro. In the mapping experiments we sometimes identified phosphorylation sites that occurred at two or more adjacent serines or threonines, and it was impossible to unambiguously identify which serine or threonine was phosphorylated. In these cases, it seemed likely that the adjacent serines or threonines could be phosphorylated in vivo. We therefore mutated each of the adjacent serines or threonines at the site to ensure that all phosphorylation would be eliminated. All four shs1-ps mutants were integrated at the endogenous SHS1 locus.To assess the significance of Pho85-Pcl1 dependent phosphorylation of Shs1, we generated four phosphorylation site mutants of Shs1 . In one shs1-ps1, shs1-ps2, shs1-ps3, and shs1-ps4 cells (shs1-ps1 and shs1-ps2) showed a decrease in the most hyperphosphorylated upper form of Shs1, whereas the mutant versions that also included the non-consensus site mutations (shs1-ps3 and shs1-ps4) showed a more dramatic loss of phosphorylation. These results demonstrate that both CDK consensus and non-consensus sites are phosphorylated in vivo. Note that the shs1-ps4 mutant was less phosphorylated than the shs1-ps3 mutant. This suggests that the four non-consensus sites that were mapped in vitro, but not in vivo, are genuine phosphorylation sites in vivo. We also tested whether the upper form of Shs1 that remained in shs1-ps1 and shs1-ps2 became hyperphosphorylated during the cell cycle . The additional features of the strains used in this study are listed in GAL1 promoter and 3XHA was integrated upstream of CLN2 using standard procedures (oligos: ACTCTATAGCTGCCAATTCATTCGCTTAC-CACATCATAATGAATTCGAGCTCGTTTAAAC and TGATGACGAGTCCCAT-ACGGGGTCTTGGTTCAGCACTAGCGCACTGAGCAGCGTAATCTG) SHS1 using standard procedures . DK573 was made by using PCR to amplify pcl1Δ::natMX6 from BY1404 (gift from Brenda Andrews) and pcl2Δ::kanMX6 from the yeast haploid deletion collection (Open Biosystems) followed by transformation of the PCR products into DK186 using standard procedures. Similarly, DK1051 was made by using PCR to amplify swe1Δ::URA from SH24 followed by transformation of the PCR product into DK1031. To create DK1068, a plasmid (pCLN2-HA) that contained Cln2 fused to three repeats of the HA epitope was linearized with PvuII to target integration at the CLN2 gene and then transformed into DK573. To create the shs1-ps mutants a plasmid that included the wild type SHS1 gene was made by using PCR to amplify the SHS1 open reading frame and flanking control regions (oligos: GCGGGATCCGCGACTTGA-ACCATTCAGTC and GCGGCATGCGAAGTTACGGGAAATCATGATAG). This fragment was then cloned into the BamH1 and Sph1 sites of the YCPlac111 vector to create pTE2. pTE2 was mutagenized by site-directed mutagenesis to convert T6 to an alanine to create pTE15. To create shs1-ps1, pTE15 was further mutagenized to convert S447 to an alanine (pTE17). To create shs1-ps2, pTE15 was cut with restriction enzymes Msc1 and BspEII to drop out a 328 bp fragment. This fragment was replaced with a synthesized fragment that contained mutations of the four consensus sites at T386, S416, S441, and S447 to alanines (pTE13). The shs1-ps3 and shs1-ps4 alleles were constructed by using PCR to amplify a 1308 bp fragment (oligos: CCGCGATAAAATTGCTCAA-TTGGCACCA and ATTAGGACCAATTTCTTCAGCGTTGGCC) of pTE15 and a 787 bp fragment (oligos: GGCCAACG-CTGAAGAAATTGGTCCTAAT and TATGTGTGTTCCCTTCTTGAAGGCTGTG) from plasmids pTE18 and pTE19. pTE18 contained a synthesized fragment that included mutations of the twelve sites at T386, S416, S441, S447, S460, T462, S519, S520, S521, S522, S525, and S545 to alanines and was used to make shs1-ps3. pTE19 contained a synthesized fragment that included mutations of the nineteen sites at S350, T351, T386, S416, S441, S447, T454, S460, T462, S519, S520, S521, S522, S525, S529, T530, T539, T541, and S545 to alanines and was used to make shs1-ps4. These PCR fragments were gel purified and pieced together by PCR amplification (oligos: CCGCGATAAAATTGCTCAATTG-GCACCA and TATGTG-TGTTCCCTTCTTGAAGGCTGTG) to create full length shs1-ps3 and shs1-ps4. The shs1-ps mutants were integrated into the genome at the SHS1 locus as previously described shs1-ps mutants was verified by sequencing. All gene synthesis was carried out by DNA 2.0, Menlo Park, California.Standard yeast media were used. YPD and YPGal media were supplemented with 40 mg/liter of adenine. All strains are isogenic to W303  and the column was washed in the same buffer without PMSF. The elution buffer was the same as the lysis buffer except that it contained 0.5 mg/ml HA dipeptide, 0.01% Tween-20, and no PMSF. To purify Shs1-3XHA treated with lambda phosphatase, cells were lysed in buffer containing 50 mM HEPES-KOH, pH 7.6, 1 M KCl, 1 mM MgCl2, 1 mM EGTA, 0.45% Tween-20, 5% glycerol, 1 mM PMSF. After the extracts were incubated with the immunoaffinity beads, the beads were washed twice batchwise with 15 ml of lysis buffer and then twice with 15 ml of phosphatase buffer . The beads were transferred to a 1.6 ml tube and then washed 5 times with 1 ml of phosphatase buffer. The beads were pelleted, the supernatant was removed, and the beads were incubated with 20 µL of lambda phosphatase (New England Biolabs) for 45 min at 30°C, with gentle mixing every 5 min. The beads were transferred to a 1.5-ml Biospin column (Bio-Rad) and washed with 5 ml of wash buffer  by pipeting 1-ml aliquots of buffer on top of the column and allowing the buffer to flow through by gravity. The column was eluted as described for the Shs1-3XHA that was not treated with phosphatase.Immunoaffinity purification of Shs1-3XHA for in vitro phosphorylation by Pho85-Pcl1 was carried out in the presence of 1 M KCl using the same protocol used previously to purify Gin4, with the following changes PCL1 was amplified by PCR and cloned into a Gateway entry vector (pDONR221) to create pTE4 (oligos for first round of PCR: AACCTGTACTTCCAGTCCATGTGTGAATACAGCAAGGC and GTACAAGAAA-GCTGGGTCCTAAAACCCATGTTGACTCATG and oligos for second round of PCR: GGGGACAAGTTTGTACAAAAAAGCAGGCTCCGAAAACCTGTACTTCC-AG and GGGGACCACTTTGTACAAGAAAGCTGGGTCGGACTGGAAGTACAG-GTT). The DNA was verified by sequencing and then recombined into a Gateway destination vector (pDEST15) to fuse glutathione-S-transferase (GST) to the N-terminus of Pcl1 (pTE6). Full length PHO85 from a cDNA library was amplified by PCR and cloned into Gateway entry vector PCR8/TOPOA to create pTE7 (oligos: CGCATGTCTTCTTCTTCACAATTTAAGC and CGCTTATGAAGCGT-GGTGGTAGTAC). The DNA was verified by sequencing and then recombined into a Gateway destination vector  to fuse six histidines at the N-terminus of Pho85 (pTE9). pTE6 and pTE9 were co-transformed into BL21 Arabinose cells and plated on LB plates containing 50 µg/ml kanamicin and 100 µg/ml ampicillin. Cells carrying these plasmids were grown to an optical density of 1.0 at 37°C, chilled on ice until they reached 25°C, and then induced with 0.2% L-Arabinose for 4 hours at room temperature. Cells were pelleted, frozen in liquid nitrogen, and stored at −80°C. Purification of the 6XHIS-Pho85/GST-Pcl1 complex was carried out by successive glutathione and nickel affinity chromatography as previously described for Gin4, with the following changes 2 and frozen in liquid nitrogen. 3XHA-Cln2/Cdk1 was purified by immunoaffinity chromatography and used in kinase assays as previously described Full length To demonstrate that Shs1 is directly phosphorylated by Pho85-Pcl1, increasing amounts of purified 6XHIS-Pho85/GST-Pcl1 were added to the purified dephosphorylated Shs1-3XHA in the presence of kinase assay buffer . The reactions were incubated at 30°C for 1 hour and terminated by the addition of 2.5 µl of 4X sample buffer. The samples were incubated at 100°C for 5 minutes and loaded onto a 10% SDS polyacrylamide gel, which was transferred to nitrocellulose and probed with α-HA antibody. Reactions with purified 3XHA-Cln2/Cdk1 were carried out in a similar manner.To map in vitro phosphorylation sites on Shs1, we scaled up the reactions in Coomassie blue-stained bands corresponding to Shs1 protein were reduced, carboxiamidomethylated, and digested independently with trypsin and chymotrypsin for increased sequence coverage. Peptide mixtures were separated by microcapillary (125 µM×18 cm) reverse-phase (MagicC18AQ) chromatography and online analyzed on a hybrid mass spectrometer , in a data-dependent fashion. Precursor masses were collected at high resolution; MS/MS spectra were triggered for the ten most abundant ions and acquired in the linear ion trap. MS/MS spectra were searched using the Sequest algorithm with serine, threonine, and tyrosine phosphorylation and methionine oxidation as dynamic modifications. Peptide matches obtained were deemed correct after applying several filtering criteria, including mass error <10 ppm, tryptic ends for trypsin digested samples. Redundant identifications of phosphorylation sites derived from different digestions added confidence to our results. Further, all spectra corresponding to phosphopeptides were manually inspected for correct sequence identification validation and site assignment based on the presence of site determining fragment ions. Ambiguity is denoted when applicable see  legend.600 0.7 were resuspended in YPD containing 30 µg/ml benomyl, followed by growth at room temperature for 3 hours. Cells were pelleted, resuspended in 3 ml of 50 mM HEPES-KOH, pH 7.6, and aliquoted into two 1.6-ml screw-top tubes, pelleted again, and frozen in liquid nitrogen after removing the supernatant. Extracts and immunoprecipitation were carried out as previously described Immunoaffinity beads for the precipitation of Gin4 were made as previously described Figure S1Shs1 phosphorylation site mutants localize normally. shs1-ps2-3XHA, shs1-ps3-3XHA, and shs1-ps4-3XHA cells were grown to log phase. Shs1 localization was determined with an anti-HA antibody.(4.56 MB TIF)Click here for additional data file.