These results indicate that bistability of myosin phosphatase activity is responsible for the bistability of MLC phosphorylation, and the sustained phosphorylation of MLC is attributed to this feature of bistability.Myosin light chain (MLC) phosphorylation plays important roles in various cellular functions such as cellular morphogenesis, motility, and smooth muscle contraction. MLC phosphorylation is determined by the balance between activities of Rho-associated kinase (Rho-kinase) and myosin phosphatase. An impaired balance between Rho-kinase and myosin phosphatase activities induces the abnormal sustained phosphorylation of MLC, which contributes to the pathogenesis of certain vascular diseases, such as vasospasm and hypertension. However, the dynamic principle of the system underlying the regulation of MLC phosphorylation remains to be clarified. Here, to elucidate this dynamic principle whereby Rho-kinase regulates MLC phosphorylation, we developed a mathematical model based on the behavior of thrombin-dependent MLC phosphorylation, which is regulated by the Rho-kinase signaling network. Through analyzing our mathematical model, we predict that MLC phosphorylation and myosin phosphatase activity exhibit bistability, and that a novel signaling pathway leading to the auto-activation of myosin phosphatase is required for the regulatory system of MLC phosphorylation. In addition, on the basis of experimental data, we propose that the auto-activation pathway of myosin phosphatase occurs  Mathematical analysis has proven to be an efficient tool for the elucidation of these regulatory system mechanisms.Signaling networks regulate cellular functions through an interconnected set of proteins that transmit signaling information via the network. An impaired balance of signaling molecule activities induces several diseases. Accordingly, understanding the principle of system regulation of the balance of signaling molecules’ activities in cells is essential to establish new strategies for the prevention and treatment of diseases. However, it is difficult to monitor the balance of highly interconnected signaling molecule activities Small Rho GTPase and Rho-associated kinase (Rho-kinase) play an important role in the regulation of MLC phosphorylation Abnormal smooth muscle contraction causes diseases such as hypertension, vasospasm, and asthma in vivo. However, such quantitative experimental data have not been sufficient for the analysis of dynamics. In endothelial cells, Maeda and colleagues quantitatively measured thrombin-induced Rho activation and the phosphorylation of MLC and MYPT1 in human umbilical vein endothelial cells (HUVEC) Dynamic analysis requires quantitative experimental data, for example, time dependent changes in activities and concentrations of signaling molecules in vitro in the following experiment: in an in vitro phosphatase assay using purified myosin phosphatase, Rho-kinase, and MLC, they found that the purified myosin phosphatase holoenzyme directly dephosphorylates its regulatory subunit, MYPT1, which has been phosphorylated by Rho-kinase We reconsidered the signaling pathway regulating MLC phosphorylation through Rho/Rho-kinase. In common with MLC, other substrates of Rho-kinase  are regulated by Rho-kinase and myosin phosphatase in vivo. Moreover, we showed that the activity of myosin phosphatase and MLC phosphorylation indicate bistability in our model. The bistable system can adopt either of two alternative steady states in response to small changes in stimulus In the present study, to examine the characteristics of the system regulating MLC phosphorylation through Rho/Rho-kinase, we developed a mathematical model including a hypothetical pathway by which myosin phosphatase regulates MYPT1 phosphorylation by Rho-kinase. Our model reproduced the sustained phosphorylation of MLC and MYPT1 induced by transient Rho-kinase activation. Thus, myosin phosphatase auto-dephosphorylation, as an additional pathway, is considered to be a necessary component of the system regulating MLC phosphorylation by Rho/Rho-kinase. We also demonstrated experimentally that myosin phosphatase undergoes auto-dephosphorylation All calculations were performed using Mathematica 7 (Wolfram). All reactions are represented by enzymatic reactions total) is conserved throughout the reaction.  is the concentration of the inactive form of myosin phosphatase containing phosphorylated MYPT1. kcat1 and kcat2 are the turnover numbers of myosin phosphatase and Rho-kinase, respectively. Km1 and Km2 are Michaelis constants. k1 is the rate constant for the reaction. k1 assumes the activation of myosin phosphatase by other signaling pathways.In our model, the activation of myosin phosphatase is described by the following combined equation composed of enzymatic reactions and a first order reaction:At steady state, the concentration of activated myosin phosphatase becomes constant. Therefore, by setting equation (1) equal to zero, we obtained equation (2).total) is conserved throughout the reaction.  denotes the concentration of non-phosphorylated MLC. kcat3 and kcat4 are the turnover numbers of Rho-kinase and myosin phosphatase, respectively. Km3 and Km4 are Michaelis constants of Rho-kinase and myosin phosphatase. k2 denotes the rate constant for the reaction. k2 assumes the phosphorylation of MLC by other signaling pathways.Phosphorylation of MLC is also described by the equation consisting of the enzymatic reactions and the first order reaction:Inhibitor and antibodies were obtained from commercial sources. Calyculin A was obtained from Wako Co. . cDNA-encoding human CPI-17 was purchased from Invitrogen Co. . pCAGGS-myc-kk1 vector was provided by Dr. K. Kaibuchi .cDNA fragment encoding CPI-17 full length was amplified by polymerase chain reaction (PCR) using the obtained cloned as a template, containing the entire open reading frame of human CPI-17. The CPI-17 mutants, T38D and T38A, were generated with a site-directed mutagenesis kit  by replacing Thr-38 with Asp-38 or Ala-38. The cDNA fragments were subcloned into pCAGGS-myc-kk1 vector.2 atmosphere at constant humidity. Transfection of plasmid into cells was carried out using Hillymax reagent  according to the manufacturer’s protocol. For the treatment of Calyculin A, cells were seeded onto 6 well plates coated with poly-D-lysine (PDL) and cultured overnight.HEK293 cells were grown in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C in an air-5% COAfter treatment with 0.1 µM Calyculin A for 10 min and transfection, cells were harvested with 10% (w/v) trichloroacetic acid. The resulting precipitates were subjected to immunoblotting with anti-phospho-MYPT1 (Thr-853) antibody , anti-MYPT1 antibody , and anti-myc antibody .. Rho-kinase phosphorylates MYPT1, a regulatory subunit of myosin phosphatase. The activity of myosin phosphatase is suppressed through MYPT1 phosphorylation by Rho-kinase. Myosin phosphatase incorporating non-phosphorylated MYPT1 can be regarded as an active form of myosin phosphatase. In our model, the concentration of non-phosphorylated MYPT1 is considered to represent that of activated myosin phosphatase. Some substrates of Rho-kinase are regulated by myosin phosphatase, and MYPT1 is a substrate of Rho-kinase. Thus, we assumed that myosin phosphatase dephosphorylates MYPT1, as a hypothetical pathway (myosin phosphatase auto-dephosphorylation pathway) (Dashed line in ). Mathematical details of the model are provided in the (Table I).To examine the dynamics whereby Rho/Rho-kinase regulates MLC phosphorylation, we constructed the mathematical model based on previous experimental results showing thrombin-dependent MLC phosphorylation in endothelial cells . Rho-kinin vivo, whereas stimulation with a low concentration leads only to transient phosphorylation of MLC and MYPT1 in vivo. With the high peak activation signal, the concentration of activated Rho-kinase was transiently raised from 0.030 µM to 0.047 µM for only 100 seconds (100–200 sec) (Red line in). On the other hand, with the low peak activation signal, the concentration of activated Rho-kinase was increased from 0.030 µM to 0.037 µM over the same 100 second interval (100–200 sec) (Blue line in). Each signal returned to the basal level of Rho-kinase activity (0.030 µM) after transient activation.To test the validity of our mathematical model, the simulation results were compared with the already-known experimental data (phosphorylation profiles of MLC and MYPT1). It has been reported that stimulation with a high concentration of thrombin gives rise to sustained phosphorylation of MLC and MYPT1 . We used 0.1 µM phosphorylated MLC as the initial condition. The low peak activation of Rho-kinase transiently increased phosphorylation of MLC, which then returned to the basal level of phosphorylation (Blue line in). In contrast, the high peak activation of Rho-kinase also increased the phosphorylation of MLC, which was maintained (Red line in). Correspondingly, we analyzed the dynamic behavior of MYPT1 phosphorylation. With high and low transient activation of Rho-kinase, the phosphorylation of MYPT1 against time t was simulated using equation (1) . We used 0.86 µM activated myosin phosphatase as an initial condition. The high peak activation of Rho-kinase induced the sustained phosphorylation of MYPT1, whereas low peak activation of Rho-kinase transiently increased the phosphorylation of MYPT1 (Blue and Red lines in). These results are consistent with the experimental dynamics of thrombin-induced phosphorylation of MLC and MYPT1 We analyzed the dynamic behavior of MLC phosphorylation. We simulated the time course of phosphorylation of MLC according to equations (1) and (3) in . We used. We used. We used. We usedin vivo2 in 2 values  of Rho-kinase, using equation (1) in . Correspondingly, the phosphorylation of MLC against time t was calculated using the various kcat2 values  for phosphorylation of MYPT1 by Rho-kinase, using equations (1) and (3) in . In these simulations, the transient high peak of Rho-kinase activity was used (Red line in ). We showed that the phosphorylation of MYPT1 and MLC was altered from sustained to transient by the stepwise reduction in Rho-kinase turnover number (kcat2) towards MYPT1 . Furthermore, we demonstrated that a slight increase in turnover number produced sustained phosphorylation of MYPT1 and MLC (Orange and green lines in ). These results suggest that there is a threshold, above which there is a sustained MLC phosphorylation response, in this reaction. We also assessed the effect of suppression of the turnover number  for direct phosphorylation of MLC by Rho-kinase, and using equations (1) and (3) in . Despite the suppression of direct MLC phosphorylation by Rho-kinase, MLC phosphorylation was maintained, even though phosphorylation level in the sustained phase was low . As compared with the results of the suppression of kcat2 and kcat3, the suppression of kcat2 has an influence on the sustained MLC phosphorylation. On the regulation of MLC phosphorylation by Rho-kinase, our simulation results suggest that the major effect of Rho-kinase is suppression of myosin phosphatase activity rather than direct MLC phosphorylation. These results may agree with the previous observation that myosin phosphatase plays a pivotal role in controlling phosphorylation of MLC in response to physiological stimuli. We reproduced the sustained MLC phosphorylation induced by transient activation of Rho-kinase; this is a signal upstream of MLC, in our simulation, incorporating the hypothesis that myosin phosphatase dephosphorylates its regulatory subunit, MYPT1. On the basis of our simulation results, it is reasonable to assume that MYPT1 is dephosphorylated by myosin phosphatase in our mathematical model.It has been reported that the sustained phosphorylation of MLC and that of MYPT1 are repressed by the Rho-kinase specific inhibitor Y-27632 kcat3 in  value of. These results indicate that MYPT1 is dephosphorylated by PP1 or PP2A, in agreement with previous reports . These results indicate that myosin phosphatase regulates the phosphorylation level of MYPT1. Therefore, it is appropriate to assume that myosin phosphatase directly dephosphorylates MYPT1 in our model.We determined experimentally whether MYPT1 is dephosphorylated by myosin phosphatase, using a phosphatase inhibitor and myosin phosphatase inhibitory protein. Calyculin A is an inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A), and myosin phosphatase is a PP1. We found that Calyculin A induced an increase in MYPT1 phosphorylation at Thr-853, which is a phosphorylation site of Rho-kinase . These r. At the steady states, the concentration of activated myosin phosphatase, [MP], is constant. In other words, the reaction velocity is zero, which means that the differentiation of activated myosin phosphatase concentration against time, d[MP]/dt, is zero. The plots showed the presence of three intersections with the horizontal axis , each of which corresponds to a steady state, because the rate of change at these points is zero . d[MP]/dt exhibits the rate of change of [MP], for example, d[MP]/dt >0 indicates an increase in concentration of [MP], whereas d[MP]/dt <0 indicates a decline in [MP]. The plots indicate that [MP] converges to S1 or S3, when the system is given a slight perturbation in the vicinity of S1 and S3. However, the perturbation around S2 induces convergence to S1 or S3, but not S2. Thus, the system acquired two stable steady states, S1 and S3, separated by the unstable steady state S2 . These results suggest that the activity of myosin phosphatase is bistable in our model under these conditions.To investigate the dynamics whereby Rho/Rho-kinase regulates the phosphorylation of MLC, we analyzed the steady states of myosin phosphatase activity using the mathematical model. Since MYPT1 phosphorylated by Rho-kinase decreases its phosphatase activity, myosin phosphatase consisting of non-phosphorylated MYPT1 is assumed to represent the active form. We plotted the concentration of myosin phosphatase active form, denoted [MP], against its derivative with respect to time, which is denoted by d[MP]/dt using equation (1) in . At the . At the . At the in vivo2 values  of Rho-kinase against MYPT1 . The number of intersections with the horizontal axis was altered from three to one by the stepwise change of kcat2 value . To characterize the steady states of myosin phosphatase activity against Rho-kinase, d[MP]/dt was plotted against [MP] using the various Michaelis constant values  for phosphorylation of MYPT1 by Rho-kinase, and using equation (1) in . The stepwise change of Km2 value also altered the number of intersections with the horizontal axis from three to one . These results suggest that Rho-kinase inhibition altered the system from bistable to monostable, thereby inhibiting sustained MLC phosphorylation.A bistable system toggles between two discrete alternative stable steady states, in contrast to a monostable system, which slides along a continuum of steady states 1 values  of myosin phosphatase (for dephosphorylation of MYPT1), and using equation (1) in . Likewise, to examine the steady states of myosin phosphatase activity against Rho-kinase activity, d[MP]/dt was plotted against [MP] using the various Km1 values  for dephosphorylation of MYPT1 by myosin phosphatase, and also using equation (1) in . The number of intersections with the horizontal axis was shifted from three to one by the stepwise change of kcat1 and Km1 value  in . Likewis. The bifurcation diagram revealed the steady states of activated myosin phosphatase ([MP]) against Rho-kinase activities ([ROCK]) . The hysteretic behavior of the system can be deduced from the results shown in 1). This plot suggests that a wide range of Rho-kinase activities induce myosin phosphatase activity .We first examined the relationship between Rho-kinase activity and the steady states of myosin phosphatase activity in our mathematical model. We obtained a bifurcation diagram from equation (2) in . The bif. The bif. The bifurcation diagram indicated the bistability of MLC phosphorylation against Rho-kinase activity. Therefore, the bistability of myosin phosphatase activity leads to the bistable phosphorylation of MLC, and the property of bistability is responsible for the sustained phosphorylation. These results suggest that the steady state of MLC phosphorylation converges from low to high in excess of the threshold, due to the transient Rho-kinase activity. Consequently, transient Rho-kinase activation induces sustained MLC phosphorylation; the pharmacological action of a Rho-kinase inhibitor induces the transition from high to low steady state of MLC phosphorylation.Secondly, to investigate the correlation between the steady states of MLC phosphorylation and Rho-kinase activity, we obtained the value of [MP] against [ROCK] from equation (2) in In this study, to elucidate the system dynamics, we developed a mathematical model in which Rho/Rho-kinase regulates MLC phosphorylation. We hypothesized that myosin phosphatase dephosphorylates MYPT1 phosphorylated by Rho-kinase. We demonstrated that our mathematical model incorporating the myosin phosphatase auto-dephosphorylation pathway reproduces the sustained phosphorylation of MLC and MYPT1 induced by transient activation of Rho-kinase. These results support our hypothesis that myosin phosphatase dephosphorylates the regulatory subunit of MYPT1. We showed that the activity of myosin phosphatase and MLC phosphorylation are bistable. Furthermore, the transition of myosin phosphatase activity from monostable to bistable depends on Rho-kinase. The bistability of myosin phosphatase activity is responsible for the bistability of MLC phosphorylation, and the sustained phosphorylation of MLC is attributed to the feature of bistability.2+/calmodulin-dependent MLC kinase (MLCK) and Rho-kinase, and myosin phosphatase 2+/Calmodulin-dependent MLCK signaling, whereas the sustained phosphorylation of MLC is regulated by Rho/Rho-kinase signaling MLC phosphorylation status is determined by the balance between activities of kinases including Cain vivo. These results suggest that the phosphorylation of MYPT1 is regulated by PP1 or PP2A. Since myosin phosphatase is a PP1, these previous observations support the hypothesis that myosin phosphatase regulates the phosphorylation of MYPT1. Moreover, we found that the phosphorylation of MYPT1 was elevated by overexpression of CPI-17 phospho-mimic mutant (CPI-17 T38D), which suppresses myosin phosphatase activity . In addition to our experimental data, Kimura and colleagues have reported that the purified holoenzyme of myosin phosphatase exhibits phosphatase activity toward myosin phosphatase phosphorylated by Rho-kinase in vitroRho-kinase has been reported to phosphorylate MYPT1 at Thr-696 and Thr-853 (Table I), whereby the activity of myosin phosphatase indicates bistability. Since the phosphorylation of MLC is regulated by myosin phosphatase, the phosphorylation of MLC also exhibits bistability. ERM family proteins, adducin, Tau and MAP2 are found to be substrates of both Rho-kinase and myosin phosphatase A bistable signaling system must include a positive feedback loop, a double-negative feedback loop, or the equivalent Xenopus oocyte maturation, ERK1/2 achieves a bistable form following a single stimulation with progesterone Bistability can adopt either of two alternative steady states in response to a constant stimulus, and can toggle between alternative stable steady states in response to small changes in stimulus or transient stimulus in vivo. Therefore, the pharmacological action of Rho-kinase specific inhibitors may cause transition from a higher to a lower steady state.Rho-kinase specific inhibitor Y-27632 suppresses the sustained phosphorylation of MLC and MYPT1 in vivo. Understanding the dynamics by which Rho/Rho-kinase regulates MLC phosphorylation in living cells will be a challenge for further experimental investigations.Understanding the dynamics regulating MLC phosphorylation is essential to establish new strategies for the prevention and treatment of diseases of blood vessels and several other areas. This study sheds light on the dynamics underlying the regulation of MLC phosphorylation by Rho/Rho-kinase signaling. We predict that the positive feedback loop is required for the sustained MLC phosphorylation, and that the sustained MLC phosphorylation is regulated by bistability of myosin phosphatase activity. However, further experimental studies are required to determine whether myosin phosphatase dephosphorylates MYPT1 phosphorylated by Rho-kinase, and whether the phosphorylation of MLC and MYPT1 indicates bistability Figure S1Effects of Rho-kinase and hypothetical pathway. (A) d[MP]/dt was plotted against [MP] using the various Km2 values  for phosphorylation of MYPT1 by Rho-kinase. (B) d[MP]/dt was plotted against [MP] using the various Km1 values  for dephosphorylation of MYPT1 by myosin phosphatase.(TIF)Click here for additional data file.Figure S21.Estimation of Rho-kinase activity and rate constant k The steady states of activated myosin phosphatase against Rho-kinase activity and rate constant (k1). X, Y, and Z axes show the Rho-kinase activities, rate constant k1, and the steady states of myosin phosphatase, respectively.(TIF)Click here for additional data file.


Furthermore, these compounds that contain anα-β-unsatured carbonyl moiety and function as potent Michael reactionacceptor, induce a rapid drop in intracellular glutathione (GSH) concentrationby direct interaction with it, thereby triggeringS-glutathionylation of STAT3. Dehydrocostunolide (HCS), thereduced form of CS lacking only the α-β-unsaturated carbonyl group,fails to exert any inhibitory action. Finally, the glutathione ethylene ester(GEE), the cell permeable GSH form, reverts the inhibitory action of DCE and CSon STAT3 tyrosine phosphorylation. We conclude that these two sesquiterpenelactones are able to induce redox-dependent post-translational modification ofcysteine residues of STAT3 protein in order to regulate its function.The main purpose of the present study is to envisage the molecular mechanism ofinhibitory action ofdehydrocostuslactone (DCE) andcostunolide (CS), twonaturally occurring sesquiterpene lactones, towards the activation of signaltransducer and activator of transcription 3 (STAT3). We report that, in humanTHP-1 cell line, they inhibit IL-6-elicited tyrosine phosphorylation of STAT3and its DNA binding activity with EC The Janus kinases (JAKs) and signal transducers and activators of transcription(STATs) pathway is responsible for signal transduction of a large number ofcytokines and growth factors that regulate responses to inflammation or immunechallenge and address the future of cell decision during development and neoplasticgrowth Seven STATs proteins have been identified so far. In physiological conditions STATsactivation is transient and usually lasts from a few minutes to several hours. Thisis assured by a variety of negatively acting events thatblock further activation,decrease DNA binding or result indephosphorylation of STATs. The final outcome ofcytokine/growth factor stimulation reflects how different cell types can interpretthe complex and often contrasting signals they receive.The pleiotropic cytokine IL-6 activates predominantly STAT3 binding to Gp130 receptorcomplex and modulate the expression of genes encoding mediators crucial for theclassic physiological acute phase response and for the apoptotic pathway Although STAT3 activation normally leads to the physiological response, deregulationof this transduction cascade could lead to the tissue damage and directly orindirectly could be involved in different pathologies. A number ofinflammation-correlated diseases such as Crohn's disease, pleurisy, psoriasisetc. are characterized by hyperactivationof STAT3 The cellular redox state is a crucial mediator of multiple metabolic, signalling andtranscriptional processes in cells, and a fine balance between oxidizing andreducing conditions is essential for normal function and survival of cells Laurusnobilis L., Magnolia sieboldii L. andSassureacustus L., exhibit various biological and immunologicalactivity, including antinflammatory and antifungal one Dehydrocostuslactone (DCE) and costunolide (CS), two sesquiterpene lactones presentin a number of plants such as In the present study we hypothesized that decrease in the intracellular GSH level,induced by DCE and CS, triggers the inhibition of STAT3 tyrosine phosphorylationmediated by its glutathionylation. To evaluate this, we examined the effect of twoterpenes on the activation of STAT3 induced by IL-6 in human acute monocyticleukemiaTHP-1 cells. We present data indicating the direct involvement of GSH deficiency inthe inhibition of STAT3 pathway through the glutathionylation of STAT3.All chemicals used throughout the present study were of the highest analyticalgrade, purchased from Sigma Chemical Company, Milan, Italy, unless otherwisespecified. RPMI 1640 medium, Dulbecco's modified Eagle's medium (DMEM)and fetal bovine (FBS) serum were obtained from Lonza, Verviers, BE. DCE and CS werepur705STAT3, anti-phospho-Ser727STAT3,anti-phospho-Tyr1054/55TyK2 and anti-JAK2 were purchased fromCell Signaling Technology, Beverly, MA; rabbitanti-phospho-Tyr1022/1023JAK1 andanti-phospho-Tyr1007/1008JAK2 antibodies were obtained fromMillipore, Bedford, MA; rabbit anti-SOCS-3 was from ImmunoBiologicalLaboratories, Tokyo, Japan; rabbit anti-STAT3, anti-Tyk2 and anti-JAK1antibodies werefrom Santa Cruz Biotechnology, Inc., Santa Cruz, CA.Rabbit anti-phospho-Tyr2 atmosphere at 37°C. Human cervical carcinoma HeLacells (American Type Culture Collection) and human colorectal adenocarcinomaDLD-1 cells (American Type Culture Collection) were cultured in DMEMsupplemented with 10% FBS under the same conditions.Human monocyticleukemia THP-1 cells  were cultured in RPMI 1640 supplemented with 10% FBS, 100 UI/mlpenicillin, 100 µg/ml streptomycin and 40 µg/ml gentamycin in a5% COTyk2 was amplified by PCR with the following primer sets:GAATTCATGCCTCTGCGCCACTGG-3′5′-CG(sense)GCGGCCGCTCAGCACACGCTGAACAC-3′5′-AC(antisense).PCR primers included 5′-NotI, 5′-EcoRI restriction sites (boldface)to aid cloning. PCR products were digested completely with NotI, EcoRI,gel-purified, and insert into the identical sites of pcDNA 3.0 to giveTyk2-pcDNA 3.0. The nucleotide sequence of this expression plasmid was verifiedby DNA sequencing.5 in DMEM without antibiotics. After 18 hours,the DMEM was replaced with the serum-reduced medium OPTI-MEM  and the cells were transfected with 1 µg Tyk2-pcDNA 3.0expression vector and 10 µllipofectamine 2000 according to themanufacturer's instructions . After 24 hours, thecells were treated with IL-6 (20 ng/ml) for 5 minutes in order to achieve Tyk2activation. Effect of two terpenes on TyK2 activation was examined by treatingcells for 30 minutes before IL-6 stimulation. The cells were harvested and usedfor Western blot analysis.For a transient transfection, HeLa cells were plated into 60-mm plates at adensity of 8×106 THP-1 or DLD-1 cells, after 4 hours starvation, weretreated with IL-6 (20 ng/ml) for 15 minutes in order to achieve STAT3activation. Effect of two terpenes on STAT3 activation was examined by treatingcells for 30 minutes before IL-6 stimulation. Nuclear extracts of THP-1 cellswere prepared according to Osborn et al. 4 cpm of 32P-labeleddouble-stranded oligonucleotides, the consensus STAT3 DNA binding site from the c-fospromoter (5′-gtcgaCATTTCCCGTAAATCg-3′), in a 15 µlreaction mixture containing 20 mM Hepes, pH 7.9, 50 mM KCl, 0.5 mMdithiothreitol, 0.1 mM EDTA, 2 µg of poly(dI-dC), 1 µg of salmonsperm DNA, and 10% glycerol. Products were fractionated on anon-denaturing 5% polyacrylamidegel. The gels were dried andautoradiographed and the intensity of hybridization was quantified using thepublic domain NIH Image 1.61 program . Supershift assay wasperformed by incubating the nuclear extracts in a binding buffer for 1 hour at4°C with 1 µl of antibody before addition of labelledoligonucleotide.3×10Cells, treated as described above, were homogenized at 4°C in 20 mM HEPES, pH7.4, containing 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet-P40(NP-40), 20% glycerol, protease cocktail inhibitors  and phosphatase cocktail inhibitors. Aliquots of the celllysate  were loaded on 7.5%SDS-polyacrylamide gels. Electrophoresis was performed at 100 V with a runningbuffer containing 0.25 M TrisHCl, pH 8.3, 1.92 M glycine, and 1% SDS. Theresolved protein were electroblotted onto a PVDF membrane  and incubated overnight at 4°C with the indicatedprimary antibodies. After washing, membraneswere developed using anti-rabbit oranti-mouse IgG peroxidase-conjugated antibody  andchemiluminescent detection system . Blotted proteins were detected and quantified using theChemiDoc XRS Imaging System . After stripping, membraneswere re-hybridized with the correspondent antibodies.The intracellular oxidized and reduced glutathione weredeterminedspectrophotometrically using the GSH recycling method as previouslydescribed 5/mL were loaded with 2.5 µM of themembrane-permeable probe5-(and-6)-chloromethyl-2′7′-dichlorodihydrofluorescein diacetateacetyl ester  for 1 hour at37°C. They were then washed with HBSS and stimulated with 6, 12, 25 µMDCE or CS or 500 µM diamide (positive control) and placed back into theincubator. After 30 minutes or 1 hour they were washed and resuspended in PBS.ROS generation was evaluated in flow cytometry  by measuring the green fluorescence signal of DCF, the oxidationproduct of CM-H2DCFDA by free radicals. The emitted fluorescence wasdetected in Fl-1 using a 530/30 nm band-pass optical filter. FlowJo 8.8.2software  was used to analyze data.THP-1 cells resuspended in HBSS  at5×1010 µM DCE and CS were incubated with 5, 25 and 100 mM GSH in RPMI mediumwithout FBS, at 37°C for 30 minutes and analysed by HPLC .The reaction mixtures were charged on Mightysil RP-18 GP reverse-phase column, maintained at 35°C andeluted with 35% acetonitrile at a flow rate of 1.0 ml/min. The elutionprofiles were monitored at 215 nm by UV detector. Retention times of DCE and CSpeaks were 7.3 and 8.1 minutes, respectively.3VO4) supplemented with protease cocktail inhibitorfor 15 minutes on ice with occasional vortexing. Equal amounts of proteins fromthe clarified cell lysates were incubated overnightat 4°C with rotation inthe presence of 2 µg STAT3 antibody. The immune complexes were collectedbyaddition of protein A sepharose (Millipore Corp.), washed extensively, eluted ina non-reducing sample buffer  and separated on a 5%SDS–polyacrylamide gel. After electrophoresis, proteins were transferredto a PVDF membrane and nonspecific binding was blocked by incubation in3% BSA diluted in TBST. Membranes were then probed with primarymonoclonal antibodies against GSH  or STAT3. Afterwashing, blots were incubated with anti-rabbit IgG peroxidase-conjugatedantibody . Protein-antibody reactions were detectedwith chemiluminescent detection system . The S-glutathionylated proteins on membranes weredetected and quantified using the ChemiDoc XRS Imaging System (Bio Rad).Cells were lysed in RIPA buffer Nonidet P-40, 1 mM EDTA, 10% glycerol, 100 mM NaF, 1 mMNaData are reported as means ± SD of four independent experiments(n = 4); statistical analyses were performed usingStudent's t test. Differences were considered significantwhenp≤0.05.50 value of 10 µM. for 15 minutes. IL-6 increased predominantly the STAT3 DNA-bindingactivity, as indicated by EMSA/supershift experiment with anti-STAT3 antibody inline with previous report . The admf 10 µM..705 phosphorylation of cytosolic STAT3 without affectingthe total amount of STAT3 protein expression In order to envisage the molecular mechanism of the inhibitory action of DCE andCS on STAT3 phosphorylation, we further hypothesized that the change inintracellular GSH level may play a critical role.Spectrophotometric analysis measuring GSH and GSSG concentrations in THP-1 cellsshowed that under normal conditions the amounts of GSH (13±0.8 nmoles/mgprotein) are far higher than those of GSSG (0.09±0.02 nmoles/mg protein)and that DCE and CS induced dose- and time-dependently the drop in GSHconcentration, only slightly affecting GSSG level . The rapTo evaluate whether this event is crucial in the modulation of STAT3 pathway,THP-1 cells were pre-treated overnight with 1 mM glutathione monoethyl ester(GEE), the cell permeable GSH, and thereafter with the two sesquiterpenelactones up to 25 µM for 30 minutes before IL-6 administration. Westernblot analysis showed that GEE reverts tyrosine-phosphorylation of STAT3 and thiseffect is correlated with the administrated sesquiterpenes' concentration. Also inAll these data further suggest the critical role played by intracellular GSHlevel in regulating IL-6-elicited-STAT3 activation.2DCFDA and treated with DCE or CSexhibited a clear fluorescence increase as respect to untreated sample. Thisdata is consistent with an enhancement of intracellular ROS. THP-1 cells loaded with CM-HPrevious works reported that some sesquiterpene lactones including CS, decreasethe intracellular thiols level, without clear demonstration of the direct bondformation between them HPLC analysis of the solutions containing the sesquiterpene lactones plus GSH,prepared as described under DCE and CS, that contain an α-β-unsatured carbonyl moiety, function aspotent Michael reaction acceptor. To analyze the importance of this group in theinhibitory action of these sesquiterpene lactones, we examined the effect ofdehydrocostunolide (HCS), the reduced form of CS , on IL-6S-glutathionylation of STAT3 leading to the inhibition ofits tyrosine phosphorylation A recent report describes that the strong oxidant diamide elicitedAs shown in Receptor-associated JAK family tyrosine kinases, JAK1, JAK2 and Tyk2, plays a crucialrole in the STATs signal transduction pathway activated by a number of cytokines andgrowth factors. Following the binding of these ligands to their specific membranereceptors, JAK kinases associated to cytoplasmic portion of receptors, tyrosinephosphorylate themselves and successively STAT proteins. Although this signallingpathway is finely regulated to guarantee the physiological response,hyper-activation of STATs has often been observed in a number of pathologies,especially that of STAT3 in cancer and in some inflammatory diseases. Therefore,attention has recently been paid to the compounds able to modulate STAT3 pathway inorder to alleviate the detrimental effect of its hyper-activation.50 = 10µM) and demonstrates that DCE and CS rapidly suppress cytokine-inducedtyrosine-phosphorylation of JAK1, JAK2 and Tyk2.The present study identifies two structurally related naturally occurringsesquiterpene lactones, DCE and CS, as inhibitors of IL-6-elicited STAT3activationin human cell line THP-1  is akey factor in keeping the cysteine residues of intracellular proteins in the reducedform. The decrease in GSH content, leading to the drop in the cellular redoxpotential, is often induced by oxidative stress. In the present study we show that,in line with above-described notion, in THP-1 cells GSH content is far higher thanGSSG and that DCE and CS dose-dependently induce the consistent drop inintracellular GSH level without significantly affecting GSSG content . The droThe direct interaction between DCE or CS and GSH may be chemically mediated by theα-β-unsaturated carbonyl group present in their moiety , as descS-glutathionylation of cysteine residues ofsensitive proteins S-glutathionylated with concomitant loss of its phosphorylation inHepG2 cells treated with diamide, a strong oxidant compound, pointing out that thissignal transcription factor is susceptible to redox regulation The disturbance in the GSH/GSSG homeostasis is implicated in the induction ofreversibileS-glutathionylation of STAT3 with concomitant decrease in STAT3tyrosine phosphorylation EMSA shows that DCE and CS dose-dependently decrease DNA-binding activity ofSTAT3 activated by 20 ng/ml IL6 in DLD-1 cells. (b) Western Blot analysisshows that DCE and CS dose-dependently decrease tyrosine705phosphorylation of STAT3 induced by 20 ng/ml IL-6 in DLD-1 cells withoutchanging the total amount of STAT3. (c) DCE and CS slightly decreasetyrosine1022/1023 phosphorylation of JAK1 andtyrosine1007/1008 phosphorylation of JAK2 in DLD-1 cells. (d)Western Blot analysis shows that inhibitory action of DCE and CS on IL-6induced STAT3 Tyr705 phosphorylation is reverted by 1 mMglutathione monoethyl ester (GEE). The total amount of STAT3 is not affectedduring the experiments.(TIF)Click here for additional data file.


However, the role of TG2 phosphorylation in the modulation of NF-κB, Akt and PTEN is not explored.Transglutaminase 2 (TG2) and its phosphorylation have been consistently found to be upregulated in a number of cancer cell types. At the molecular level, TG2 has been associated with the activation of nuclear factor-kappa B (NF-κB), protein kinase B (PKB/Akt) and in the downregulation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN). However, the underlying mechanism involved is not known. We have reported that protein kinase A (PKA) induced phosphorylation of TG2 at serine-216 (Sertg2-/-) overexpressing native TG2 or mutant-TG2 (m-TG2) lacking Ser216 phosphorylation site with and without dibutyryl cyclic-AMP (db-cAMP) stimulation. Functional consequences on cell cycle and cell motility were determined by fluorescence activated cell sorting (FACS) analysis and cell migration assay respectively.In this study we have investigated the effect of TG2 phosphorylation on NF-κB, Akt and PTEN using embryonic fibroblasts derived from TG2 null mice (MEFtg2-/- cells resulted in an increased activation of NF-κB and Akt phosphorylation in comparison to empty vector transfected control cells as determined by the reporter-gene assay and immunoblot analysis respectively. These effects were not observed in MEFtg2-/- cells overexpressing m-TG2. Similarly, a significant downregulation of PTEN at both, the mRNA and protein levels were found in cells overexpressing TG2 in comparison to empty vector control and m-TG2 transfected cells. Furthermore, Akt activation correlated with the simultaneous activation of NF-κB and a decrease in PTEN suggesting that the facilitatory effect of TG2 on Akt activation occurs in a PTEN-dependent manner. Similar results were found with MCF-7 and T-47D breast cancer cells overexpressing TG2 and m-TG2 further supporting the role of TG2 phosphorylation in NF-κB activation and in the downregulation of PTEN.PKA activation in TG2 overexpressing MEF216 plays a role in TG2 mediated activation of NF-κB, Akt and in the downregulation of PTEN. Blocking TG2 phosphorylation may provide a novel strategy to attenuate NF-κB activation and downregulation of PTEN in TG2 overexpressing cancers.Collectively, these data suggest that phosphorylation of TG2 at Ser Transglutaminase 2 (TG2) is the most diverse and ubiquitous member of the TG family of enzymes that catalyze posttranslational modification of proteins by crosslinking proteins via ε-(γ-glutamyl)lysine isopeptide bonds or through incorporating primary amine at glutamine residues,2. In adTG2 has been found to be upregulated in a number of cancers including breast, ovarian, pancreatic, colon cancers and shown to confer resistance to chemotherapeutic drugs and promotes invasive potential of cancer cells-15. In chttp://www.phosphosite.org/proteinAction.do?id=4135&showAllSites=true. However, the kinase responsible for TG2 phosphorylation and functional consequence of TG2 phosphorylation in cancer cell is not known. We have reported that TG2 undergoes phosphorylation at serine-216 (Ser216) residue in response to PKA activation[216 on TG2 mediated modulation of NF-κB, PTEN and Akt in embryonic fibroblasts derived from TG2 null mice (MEFtg2-/-) and in breast cancer cells. Herein we report that phosphorylation of TG2 at Ser216 has an important role in TG2 mediated activation of NF-κB and in the downregulation of PTEN resulting into Akt activation.Posttranslational modification of proteins by phosphorylation plays a critical role in the regulation of protein functions and in protein-protein interactions. For exativation. In additivation,10. Moretivation. The cAMtivation. It is p216 residue[216 was further confirmed using MEFtg-/- cells overexpressing Myc tagged wild-type TG2 and Ser216Ala-TG2 mutant (m-TG2) lacking Ser216 phosphorylation site . Expression level of wild-type TG2 and m-TG2 was confirmed by immunoblotting using anti-Myc antibody  in TG2 is also involved in this process and is consistent with the phosphorylation level of TG2 and m-TG2 observed in response to db-cAMP in MEFtg2-/- cells . Activation occurs via phosphorylation of IκB followed by proteasome-mediated degradation that results in the release and nuclear translocation of active NF-κB . As a prelude to NF-κB activation, we examined IκBα protein levels in cells overexpressing TG2 and m-TG2. IκBα was found to be significantly downregulated in TG2 overexpressing cells in comparison to empty vector transfected and m-TG2 overexpressing cells with db-cAMP stimulation FigureB-C. No stg2-/- is not cell specific and pertinent to TG2 protein, experiments were repeated using MCF-7 and T-47D breast cancer cells. A similar downregulation of PTEN protein level in MCF-7 and T-47D cells were found as in the case of MEFtg2-/- cells overexpressing TG2 /Akt signaling pathway,32. The 216 in TG2, we studied the effect of overexpression of TG2 and m-TG2 on cell migration using a two chamber technique. Overexpression of TG2 alone led to significant increase in the migration of MEFtg2-/- cells in comparison to empty vector transfected control cells which was further enhanced after db-cAMP stimulation , we unraveled a novel role of TG2 phosphorylation at Ser216 in the activation of NF-κB, Akt and in the downregulation of PTEN. We provide evidence that phosphorylation of TG2 at Ser216 facilitates TG2 mediated NF-κB activation, which induces downregulation of PTEN, resulting in Akt activation. As TG2 induced activation of Akt was found in the absence of receptor tyrosine kinase or PI3K stimulation, this would imply that TG2 induced activation of Akt under the experimental condition used in this study mainly occurs through downregulation of PTEN. Most importantly, the loss of phosphorylation at Ser216 in TG2 completely blocked TG2-induced effect on NF-κB activation and downregulation of PTEN mRNA as determined by reporter-gene assay and real-time PCR respectively.PKA signaling plays an important role in the pathogenesis of a number of NF-κB related diseases including cancer and PKA activation confer resistance to trastuzumab in human breast cancer cells,35. In pn 14-3-3. In thisBiochemically, PTEN is a phosphatase that dephosphorylates PIP3 that is produced by PI3K and function as a ubiquitous inhibitor of PI3K-dependent signaling,34. The 216 phosphorylation site partially overlaps with a functional BH3-only motif identified in TG2[L[L and increases the accessibility of another serine residue within BH3 domain to PKA induced phosphorylation[216 in TG2 contains additional serine residue  which is also present as a PKA consensus phosphorylation site (209RDCSRRSSPVYVGRV223). It is possible that phosphorylation of TG2 at Ser216 plays a similar role in its interaction with Bcl-2 family members which in turn may attenuate interaction of TG2 with pro-apoptotic members or facilitate interaction with anti-apoptotic members of Bcl-2 family of proteins. Furthermore, we have reported that phosphorylation of TG2 at Ser216 facilitates its interaction with protein 14-3-3, a phospho-serine/threonine binding protein[The motif containing Serd in TG2. The BH3in TG2[L. In thisrylation. The mot protein. Moreove protein. It is c216 as a PKA phosphorylation site, the phosphorylation of Ser216 by other kinases may not be ruled out. This may also explain the differences observed in Akt phosphorylation and pPTEN/PTEN levels in three different cell lines used in this study. For example, TG2 has been identified as a downstream target of EGFR, and EGFR expression level is known to significantly vary in various breast cancer cell lines including MCF-7 and T-47D cells[216 with phospho-motif servers (http://networkin.info/version_2_0/search.php andhttp://www.hprd.org) predicted Ser212 and Ser216 as potential sites of phosphorylation by a number of kinases known to be involved in tumorigenic signals such as calmodulin-dependent protein kinase II, casein kinase II, MAPK, PKC, RSK and cdk5. In addition to Ser216, phosphorylation of TG2 at Tyr219 and Tyr369 has been identified in a number of cancer cells[As TG2 has been identified as a phospho-protein in multiple cancer cells/tissues, this would imply that TG2 may serve as a target protein for a number of upstream kinases and mediate the effect of tumor promoting factors. For instance, although we have identified Ser7D cells. Moreove7D cells. Analysier cells,26. It i216 and other potentially important phosphorylation sites will hopefully provide information for the development of novel strategies to reduce the enhanced cancer growth associated with TG2 upregulation. Continued research will determine if interfering with the phosphorylation of TG2 has preventive or therapeutic application in cancer.In summary, data presented here provides new insight into the underlying mechanism involved in TG2 mediated activation of NF-κB, Akt and in the downregulation of PTEN that have been implicated in the invasive and chemoresistant potential of cancers. Further studies to identify the factors that are involved in the regulation of TG2 phosphorylation at Sertg2-/- cells were obtained from TG2 null mice[MEFull mice,10. MCF-TG2 knockout C57BL/6 mice were generously provided by Dr. Nikolaos Frangogiannis  with permission from Dr. Gerry Melino . Experiments involving mice were performed as approved by the Animal Care Committee of the University of Manitoba.216 phosphorylation sites was made by a site-directed mutagenesis kit using two complementary nucleotide primers  containing desired mutation and Myc-TG2 as the template. Authenticity of all constructs was confirmed by DNA sequencing. Transfections with various constructs were performed using FuGENE HD transfection reagent  according to the manufacturer’s instructions. Cells treatments were performed 48 h post-transfection. Cell culture medium was exchanged for serum free media, and cells were serum starved for 6 h. Subsequently cells were treated with db-cAMP (100 μM) for 1 h or as otherwise stated in the presence and absence of PKA inhibitor H89 (100 nM).MEF and breast cancer cell culture and treatments were performed as described before,10. The Cell lysates were prepared using lysis buffer (Sigma-Aldrich), containing protease inhibitor cocktail as described previously. Proteintg-/- and breast cancer cells transfected with empty vector, wild type TG2 and mutant-TG2  constructs. The cDNA was synthesized from total RNA (1 μg) using reverse transcriptase and oligo-deoxythymidine primers[Total RNA was extracted using RNA easy plus mini kit  from MEF primers. The foltg-/- cells were transfected with various TG2 plasmids for 48 hours. Subsequently cells were collected by trypsinization, pelleted at 800 x g for 10 min, and fixed in 70% ethanol. DNA content was evaluated by flow cytometry with propidium iodide (PI) staining.Cell cycle analysis was performed by fluorescence activated cell sorting (FACS) as described before. In brietg2-/- cells were used for the cell motility assays. Polycarbonate filters  with cells which were transfected with various TG2 constructs were placed in wells of 6-well plates. Incubation was carried out with and without db-cAMP (100 μM) in the presence and absence of PKA inhibitor H89 (100 nM). The filters were removed and fixed in 1:10 diluted formalin for 1 h at RT. Cells on upper filter surface were removed carefully with cotton swab. The filters were stained in hematoxylin for 10 min, and cells on the lower surface of the filter were counted under a light microscope[MEFcroscope.tg2-/- and breast cancer cells were co-transfected with TG2 and pNFkB-MetLuc2-Reporter  constructs as per manufacturer’s protocol. Subsequently, cells were serum-starved for 6 hours, treated with 100 μM db cAMP for 1.5 h. Samples of supernatant media (100 μL) were collected. Subsequently 10 μL of 1 x substrate/reaction buffer was added to each sample and activity was measured using a Luminometer (Lumat LB 9507). The transfection efficiency was normalized by adding substrate buffer to cell lysates of each sample and light signals were recorded using a Luminometer[MEFinometer.Experimental results are shown as means ± SEM. One-way ANOVA with Dunnett’s test was used for multiple comparisons. P values <0.05 were considered significant difference.tg2-/-: Mice embryonic fibroblast derived from TG2 null mice; m-TG2: Mutant-transglutaminase 2; NF-κB: Nuclear factor-kappa B; PAkt: Phosphorylated Akt; PCR: Polymerase chain reaction; PI: Propidium iodide; PI3K: Phosphatidylinositol 3 kinase; PIP3: Phosphatidylinositol triphosphate; PKA: Protein kinase A; PKC: Protein kinase C; PTEN: Phosphatase and tensin homologue deleted on chromosome 10; PPTEN: Phosphorylated PTEN; RQ: Relative quantification; RSK: Ribosomal S6 kinase; SDS-PAGE: Sodium dodecyl polyacrylamide gel electrophoresis; SEM: Standard error of mean; Ser: Serine; S phase: Synthesis phase; TG2: Transglutaminase 2; Thr: Threonine; Tyr: Tyrosine.Akt: Protein kinase B/Akt; Ala: Alanine; ATP: Adenine nucleotide triphosphate; BSA: Bovine serum albumin; cdk5: Cyclin-dependent kinase 5; db cAMP: Dibutyryl cyclic adenosine monophosphate; ECL: Enhanced chemiluminescence; EGFR: Epidermal growth factor receptor; FACS: Fluorescence activated cell sorting; GTP: Guanine nucleotide triphosphate; HRP: Horse radish-peroxidase; MAPK: Mitogen activated protein kinase; MEFThe authors declare that they have no competing interests.YW and SRA performed the experiments and help in design of the experiments and data analysis, SM participated in design of the experiments, data analysis and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/277/prepub


Some 10 years ago it emerged that at sufficiently high concentrations certain monoclonal mouse IgEs exert previously unsuspected effects on mast cells. Thus they can both promote survival and induce activation of mast cells without the requirement for antigens. This was a wake up call that appears to have been missed (or dismissed) by the majority of immunologists. The structural attributes responsible for the potency of the so-called “highly cytokinergic” or HC IgEs have not yet been determined, but the events that ensue when such IgEs bind to the high-affinity receptor, FcεRI, on mast cells have been thoroughly studied, and are strikingly similar to those engendered by antigens when they form cross-linked complexes with the receptors. We review the evidence for the cytokinergic activity of IgE, and the structural features and known properties of immunoglobulins, and of IgE in particular, most likely to be implicated in the phenomenon. We suggest that IgEs with cytokinergic activity may be generated by local germinal center reactions in the target organs of allergy. We consider also the important implications that the existence of cytokinergic IgE may have for a fuller understanding of adaptive immunity and of the action of IgE in asthma and other diseases. In the classical model for allergenic activation, a multivalent antigen – the allergen – enters the body and is captured by a pre-existing complex of an IgE antibody bound to its high-affinity receptor, FcεRI, on the membrane of mast cells, and basophils. Cross-linking of the IgE-FcεRI complex triggers mast cell activation and the release of substances that cause the symptoms of allergy Figure . AllergiTissue targets are strategically located as barriers against the environment, and the sensitization of mast cells to antigens by antigen-specific IgE antibodies may be one of the many mechanisms, including those of both innate and adaptive immunity that have evolved to impede the entry of pathogens or toxins into the body and thus avert worse mayhem than that generally caused by mere allergic reactions  with certain hybridoma IgEs has the striking effect of increasing cell survival after removal of growth factors  inflammation by, for example, inducing B cells to make more IgE by a positive feedback mechanism , ablated this activity in accordance with the later demonstration of IL-3’s importance for mast cell survival in the absence of antigen  or the use of HLMC in place of CBMC would require comparison of the different IgEs in the two types of mast cells. The same group later demonstrated that the cytokinergic activity of their IgE depended on the autocrine activity of IL-6 . With H1 DNP-ε-206 and IgE-E3 by contrast, the cells failed to migrate. The kinetics of the migration of the cells toward SPE-7 IgE were similar to those of IgE-sensitized BMMC migrating toward DNP-HSA. This did not occur when BMMC from FcεRIα−/− mice were placed in the upper well. In marked contrast, BMMC from the FcεRIα−/− mice responded vigorously to SCF in control experiments. Wild type BMMC pre-sensitized with PC IgEs also migrated toward DNP-HSA. Migration of BMMC toward the HC IgE was specific to both IgE isotype and antigen. It was necessary to coat the filter between the wells with fibronectin, vitronectin, or laminin, but not by coating with collagen and bovine serum albumin (BSA). Migration was inhibited in the presence of a blocking antibody against integrin β1  and S. aureus enterotoxin B (a T cell superantigen) to stimulate a vigorous IgE response, an approach designed to provide a model of AD  required for induction of IL-3 synthesis. This is not seen with antigen-dependent stimulation of mast cells. Inhibition of ROS generation with pharmacological inhibitors, e.g., the phospholipase C inhibitor U73122, converts slow to fast kinetics, while the addition of H2O2 results in IL-3 expression and increased survival of mast cells.Later studies confirmed these observations, clarified the points at which the signaling pathways diverge and identified the resulting downstream events . How does this occur? The available evidence points to the involvement of contact sites in the N-terminal (Fv) domains of IgE when it is bound to FcεRI on the mast cells. The fact that monoclonal IgEs with different variable regions, regardless of specificity, linked to the same (ε) constant region, exhibited varying degrees of cytokinergic activity implicates this domain. One caveat is that differential glycosylation of PC and HC IgE may vary the exposure of a potential “active site” in the Fc region of IgE. A key role for the Fv domain is also supported by the observation that specific monovalent haptens inhibit the cytokinergic activity, as noted above. The hapten may simply occupy the “active site” or binding may stabilize an alternative conformation of the domain in which the site is occluded or deformed. We enlarge upon the latter possibility below and in Sections “Conformational Isomerism as a Basis of Cytokinergic Allergy?”The rank order of cytokinergic activities of different IgEs was preserved in BMMC tested in serum-free medium  or in CBMC  by assaying β-hexosaminidase release (degranulation) and/or IL-8 cytokine secretion. Independent experiments in the two laboratories led to the same conclusion. Thus, a given VL combined with one Vε was highly cytokinergic, while in combination with a different Vε it exhibited only vestigial activity. Likewise, the cytokinergic activity was not consistently associated with the presence of a particular Vε sequence. About one-third of all the recombinant IgEs exhibited a range of activities up to the level observed with mouse SPE-7. These results suggest that the conformation of the whole Fv domain may be important in determining the cytokinergic activity of an IgE; further experiments comparing the cytokinergic activities of recombinant IgEs with natural and scrambled Vε and VL pairs are in progress to resolve this question. In the following sections we review what is known about the conformational properties of antibody Fv regions and how they may impact on cytokinergic activity.To gain further insight into the structural requirements for cytokinergic activity we constructed recombinant human IgEs with different combinations of VH, and J gene segments encoding the variable regions of the Ig heavy- and light-chains. There is an accompanying contribution to diversity by non-templated nucleotide insertions and deletions at the DH-JH junction. However, in practice, the comparatively low number of the body’s circulating primary germline-repertoire B cells (ca. 106) limits this genetic diversity. The extra diversity required for recognition of a wider range of potential antigens originates, it has been suggested, comes from conformational flexibility of the complementarity-determining region (CDR) loops  has a broad and flat conformation that resembles the state in antibody-protein complexes. By contrast, the minor constituent, Ab2 (ca. 20% of SPE-7 molecules) has a deep and narrow conformation, reminiscent of antibody-hapten complexes  engender an “induced-fit” rearrangement to a third state, Ab3  is instructive in this context . Nevertheless, in early 2012 binding of recombinant mouse HRF to certain mouse monoclonal IgE+ were investigated. Their Fabs were as active in binding HRF as intact IgE molecules, and binding was inhibited by antigen, implying that HRF binds at a site in the variable regions. Interestingly, there appeared to be a relation to the presence of κ-chains derived from two particular members of the κ germline gene family (8–30 and 2–137), paired with heavy-chains from different VH families. Recombinant HRF was also split into smaller fragments, two of which inhibited HRF activities in vitro and in vivo in mice. The activity of the corresponding HRF peptides in the human system remains to be evaluated; clinical applications may be envisaged.The structural properties of IgE+-mediated BMMC or peritoneal mast cell activation  show that the two activities are not equivalent  and many types of cells express CD23 (the “low-affinity” IgE receptor). FcεRI is expressed in Langerhans cells, dendritic cells, monocytes, smooth muscle cells, and epithelial cells, while CD23 is expressed in many of these cell types and also in B cells, T cells, and follicular dendritic cells  may be critical for cytokinergic IgE activity see . In so fhorror autotoxicus; Ehrlich, H)J recombination in the bone marrow up to 75% of B cells are self-reactive . Helminths induce Th2-type immune responses and regulatory T cells that produce anti-inflammatory cytokines. This is accompanied by the synthesis of abundant polyspecific IgE, little or none of which is specific for the parasite antigens  or other proteins .After affinity maturation of binding to a specific antigen, residues from the three CDR loops on the heavy- or light-chains, and often both, directly contact the antigen, an event in which CDRH3 often plays a dominant role . The likelihood therefore is that, in the normally attainable concentration range, interaction between the molecules we are considering would be undetectably low in solution. Schweitzer-Stenner and Pecht  and slow dissociation (t1/2 ∼ 14 days in tissue) imply that IgE is effectively permanently attached to FcεRI on the cell membrane. IgE has one more pair of Ig domains in its Fc region than IgG, which causes IgE to adopt an asymmetrically bent structure , showing that there needs to be sufficient proximity of the cross-linked complexes (<70 Å apart) for significant stimulation of degranulation, although signaling upstream of the CaHuman IgEs do not bind to mouse FcεRI, but mouse IgE does bind to human FcεRI  inflammation in the target organs of allergy → somatic hypermutation, receptor revision, and class switching to IgE → IgEs with instability in Fv due to random combination of VWhat then might be the biological function of local receptor revision and the production of polyspecific cytokinergic IgE in the respiratory tract and gastrointestinal mucosa and skin? All these are barrier tissues involved in immune defense against multitudinous ever-changing external threats. It may be vital to maintain the diversity of the local antibody repertoire , and need to be extended to other cytokinergic IgEs to establish the generality of the conclusions. Nor has anything yet been undertaken to relate the highly variable level of cytokinergic activity to amino acid sequence differences. Additional questions also arise: are differences in cytokinergic IgE potency linked to degree of conformational mutability? Are the structural and dynamic characteristics of the IgEs governed to any significant extent by the VThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Decades of theory and experimental evidence underscore the critical importance of inhibitory functions to flexible, context-appropriate and goal-directed action  response inhibition can be either a control process (to override a prepotent response tendency) or it can itself be a prepotent response tendency (to be overridden); (2) response inhibition processes and response selection and execution processes are fundamentally the same; and (3) we learn to inhibit a response in fundamentally the same way that we learn to select and execute a response.The purpose of this Opinion is to highlight some alternative ideas and approaches in neuroscience research on response inhibition and inhibitory control that have emerged in the literature, in order to stimulate debate and suggest hypotheses for future research. In particular, building on recent neuroscience-based accounts of response inhibition  requires a deliberate, effortful inhibition of a prepotent, automatic response, creating the illusion that inhibitory control is always deliberate and effortful whereas the response to be inhibited is always prepotent and automatic. But one can think of conditions and situations in which this relationship is reversed, e.g., a patient with social anxiety may have to intentionally and effortfully overcome his or her prepotent tendency to refrain from public speaking, and many of us would face a similar challenge if asked to get up on the stage and sing.by default; instead, it may function as either a control process (to override a prepotent response tendency) or as a prepotent response tendency itself (to be overridden), depending on the situational demands and/or the relative strengths of the two competing goal representations. I expand on the idea of inhibitory goal representation in Proposition 2, and I discuss the notion of automatic inhibition in Proposition 3.Therefore, I argue that response inhibition is not a control process Evidence from human functional MRI (fMRI) studies using Go/NoGo, Stop-Signal, and similar tasks suggests that inhibition of a motor response engages the same network (or networks) of brain regions that are engaged during selection and execution of this motor response. In particular, response inhibition processes engage a distributed network of both cortical and subcortical regions, including the inferior frontal gyrus , insula, anterior cingulate cortex (ACC), pre-supplementary motor cortex (pre-SMA), dorsolateral prefrontal cortex (DLPFC), parietal regions, and basal ganglia [e.g.,  the activity of downstream glutamatergic neurons, leading to activation rather than inhibition at the local-circuit or larger-network level. Conversely, activation of glutamatergic neurons may be required to activate a group of GABAergic neurons and trigger inhibition. Thus, successful response inhibition likely relies on reciprocal interactions between inhibitory GABAergic neurons and excitatory glutamatergic neurons, rather than on inhibitory GABAergic transmission alone.Similarly, at the level of synaptic transmission, response inhibition processes may not be fundamentally different from response selection and execution processes. It is sometimes assumed that response inhibition must rely on inhibitory synaptic transmission to a larger degree than response selection and execution processes. But why should it be the case? Inhibitory synaptic transmission involving the neurotransmitter gamma-aminobutyric acid (GABA) is known to be as important as excitatory glutamatergic transmission both at cortical and subcortical levels  should not fundamentally differ from the neural representations of response selection and execution goals , and the underlying learning processes should also be fundamentally the same. In the influential horse-race models (Logan and Cowan, Furthermore, although counterintuitive, growing evidence suggests that response inhibition processes may be stimulus-driven to the same extent that response selection and execution processes are stimulus-driven. In fact, one and the same stimulus can activate both a goal representation to carry out a behavior and a goal representation to inhibit this behavior. For instance, food-related cues may activate both a goal to consume the food and a competing goal to stay on a diet (Fishbach et al., automatic inhibition—following training in a Go/NoGo task was also demonstrated by reduced corticospinal excitability on Go trials preceded by NoGo trials, relative to Go trials preceded by Go trials (Chiu et al., Finally, following the same logic, there is no reason why response inhibition should not become automatic—and inhibitory control habitual—with appropriate and sufficient practice. Specifically, if response inhibition can be triggered by a Stop stimulus in the same fashion that response selection and execution is triggered by a Go stimulus, then a consistent mapping between a specific Stop stimulus and the inhibition of a specific response should result in practice-related improvements and eventually automaticity (Verbruggen and Logan, In this Opinion article, drawing on recent neuroscience-based accounts of inhibitory control mechanisms in the human brain (e.g., Mostofsky and Simmonds,


Autophagy is a cell digestion process that contributes to overcoming nutrient deprivation and is initiated under stress conditions. However, whether autophagy is a cell survival or cell death mechanism remains under discussion and may depend on context. Nevertheless, autophagy is a cellular process that determines cell fate and is tightly regulated by different signaling pathways, some of which, for example MAPK, PI3K and mTOR, are tightly regulated by 14-3-3 proteins. It is therefore important to understand the role of 14-3-3 protein in modulating the autophagic process. Within this context, direct binding of 14-3-3 to mTOR regulatory proteins, such as TSC2 and PRAS40, connects 14-3-3 with autophagy regulatory processes. In addition, 14-3-3 binding to human vacuolar protein sorting 34 (hVps34), a class III phosphatidylinositol-3-kinase (PI3KC3), indicates the involvement of 14-3-3 proteins in regulating autophagosome formation. hVps34 is involved in vesicle trafficking processes such as autophagy, and its activation is needed for initiation of autophagy. Chromatography and overlay techniques suggest that hVps34 directly interacts with 14-3-3 proteins under physiological conditions, thereby maintaining hVps34 in an inactive state. In contrast, nutrient starvation promotes dissociation of the 14-3-3–hVps34 complex, thereby enhancing hVps34 lipid kinase activity. Thus, 14-3-3 proteins are regulators of autophagy through regulating key components of the autophagic machinery. This review summarizes the role of 14-3-3 protein in the control of target proteins involved in regulating the master switches of autophagy. The cellular decision to live or die is finely controlled by tightly regulated signaling pathways. 14-3-3 proteins have a key role in this decision by controlling many of the signaling pathways that mediate this process.14-3-3 proteins comprise a large family of acidic proteins that are expressed within all eukaryotic cells and function as homodimers and heterodimers . They peThe role of 14-3-3 in apoptosis has been well documented and several reports indicate that 14-3-3 protein interaction with target binding partners initiates events that support cell survival, thus mediating an essential anti-apoptotic signal . It has Macroautophagy (hereafter referred to as autophagy) is a self-digestion process that is enhanced by several cancer-related stimuli, as well as starvation, TNFα and ceramide. Autophagy may promote cell adaptation to starvation conditions produced by reduced extracellular or intracellular nutrients as a consequence of loss of growth factor signaling. Autophagy is characterized by the engulfment of the cell cytoplasm and organelles in double membrane vesicles, or autophagosomes, resulting in their degradation . AutophaThe autophagic machinery includes groups of proteins that control autophagosome formation and thus the rate of degradation of sequestered material and cellular energy levels. Autophagosomes fuse with lysosomes to form autolysosomes, in which cellular components are degraded by lysosomal hydrolases  through Under different experimental conditions, autophagy has been reported to constitute a stress adaptation to avoid cell death, a failed attempt to rescue stressed cells from death or an alternative cell death pathway ,45,46,4714-3-3 proteins form a ubiquitous eukaryotic adaptor protein family involved in numerous cell biology processes, including those related to cell survival or death decisions. This review summarizes the involvement of 14-3-3 family members in cell fate, and emphasizes their recently discovered regulatory roles in autophagy.Autophagy is tightly controlled by signaling pathways that sense the energy and nutrient status of cells. The AMP-activated protein kinases (AMPKs) are important cellular sensors of energy levels that function by sensing the AMP:ATP ratio and regulating metabolic pathways. AMPKs are activated by AMP binding to their γ-subunit, leading to phosphorylation of Thr172 within the activation loop of the catalytic α-subunit. This phosphorylation is promoted by an upstream kinase, such as the tumor suppressor liver kinase B1 (LKB1/STK11) . AMPKs rIn addition to TSC2, the 14-3-3-binding partner proline-rich Akt substrate 40 (PRAS40) is reported to be essential for mediating mTORC1 signaling and triggering autophagy . PRAS40 The mTOR binding partner Raptor has been reported to be a direct substrate of AMPK. Raptor phosphorylation by AMPK at two highly conserved serine residues, Ser722 and Ser792, promotes direct binding to 14-3-3, thus leading to inhibition of mTORC1 kinase activity . AMPK phIn addition, hypoxic stress suppresses mTOR activity and induces autophagy. REDD1 (Regulated in development and DNA damages responses 1) expression is reported to be highly induced in response to hypoxia . In mammIn addition, 14-3-3 proteins may control mTORC1 through PI3K pathway regulation in response to nutrient and growth factor depletion by direct binding to IRS-1 (insulin receptor substrate-1). 14-3-3 protein interaction with IRS-1 promotes dissociation of the IRS-1–PI3K class I complex during the insulin desensitization process .Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that respond to signals such as growth factors and stress, leading to MAPK-dependent activation of a signaling cascade comprising of kinases and transcription factors. MAPK family members include ERK, which is activated by growth signals, and p38 and JNK, which are activated in response to various stresses. Extracellular signals promote activation of Ras, which then binds to and activates Raf, leading to phosphorylation and activation of the two ERK isoforms, ERK1 (p44) and ERK2 (p42) . ERK hasOn the other hand, p38 MAPK has been implicated in cell cycle inhibition, the induction of apoptosis and terminal differentiation . HoweverIn this context, MAPK/ERK kinase kinase 3 (MEKK3) is activated by autophosphorylation of Ser526. Association between MEKK3 and 14-3-3 seems to be dependent on Ser526 phosphorylation and this interaction prevents dephosphorylation of Ser526 by PP2A . Thus, t2+ and diacylglycerol and their structural characteristics, PKC isoforms involved in distinct roles in signal transduction pathways are classified as conventional , novel  and atypical (PKCζ and ɩ) [Protein kinase C (PKC) comprises a family of phospholipid-dependent serine/threonine kinases that regulate diverse cellular functions. On the basis of their requirement for Caζ and ɩ) . The hypζ and ɩ) ,96. In cζ and ɩ) . Thus, aζ and ɩ) . ER streζ and ɩ) . Despiteζ and ɩ) . In addiζ and ɩ) . Moreoveζ and ɩ) ,103. As ζ and ɩ) ,105,106 ζ and ɩ) ,108 of P2+/calmodulin-dependent kinase kinase-beta (CaMKKβ) binds 14-3-3, which may have important consequences for the regulation of Akt, CaMKI, CaMKIV and ERK signaling pathways [It is interesting to note that under ER stress, induction of autophagy facilitates the removal of unfolded proteins . Among tpathways . Under Ein vivo and in vitro. Thus, Ser555 to Ala mutation blocks 14-3-3 binding to ULK1, and in vivo ULK1 phosphorylation is inhibited by expression of a dominant-negative AMPK mutant. Although further investigation is required, these data suggest a role for 14-3-3 in regulating a key protein during the autophagy initiation process. Moreover, mTOR has been reported to interact with the ULK1 kinase domain. In addition, formation of a complex between ULK1, mTORC1, and AMPK coincides with 14-3-3–Raptor binding and initiation of autophagy [Autophagy is a highly regulated degradation process due to the energy cost involved and its important role in determining cell fate, and many different pathways cooperate to regulate this degradation process. In addition, genetic screens in yeast have identified a number of autophagy-related genes, including the serine/threonine protein kinase Atg1 that forms an active complex with Atg13 and Atg17 to regulate the autophagy initiation process ,112,113.utophagy . Under futophagy . These r14-3-3 proteins have also been reported to interact with proteins involved in the vesicle nucleation process during autophagy, such as hVps34 (human version of yeast Vps34), the class III phosphatidylinositol-3-kinase (PI3KC3). Phosphoinositide 3-kinases are enzymes that catalyze phosphorylation of the 3ʹ hydroxyl group of the inositol ring in phosphatidylinositol (PtdIns). PI3Ks are classified into three types based on their structure, substrate specificity and functionality . Class Iin vitro phosphorylation by PKC promotes hVps34–14-3-3 binding. Additionally, experiments using deletion mutants and site-directed mutagenesis indicate that the N-terminal region of hVps34 mediates 14-3-3 binding and that phosphorylation at several sites may be required for hVps34–14-3-3 binding (Thr197 and Ser212). These data are consistent with the negative regulation of autophagy by PKC and the role of 14-3-3 in negatively regulating both hVps34 and the autophagy initiation process. Data also suggest that 14-3-3 ζ overexpression inhibits autophagy promoted by C2-ceramide or starvation in HeLa and HEK293T cells. Furthermore, depletion of 14-3-3 ζ promotes autophagy in cervix and breast cancer cell lines under normal culture conditions. A significant reduction in Beclin-1 protein expression correlating with positive regulation of autophagy has been observed in U20S cell lines stably expressing inducible or transient siRNA against 14-3-3 τ (θ) [hVps34 forms a complex with Beclin-1 that participates in autophagosome formation. Regulatory mechanisms directing Beclin-1/hVps34 specificity were initially shown to involve complex formation with proteins such as Atg14L and Rubicon. Further analysis of these proteins suggested that Atg14L enhances hVps34 lipid kinase activity and upregulates autophagy, whereas Rubicon reduces hVps34 activity and downregulates autophagy . Followi-3 τ (θ) . NeverthIt is becoming clear that autophagy is a tightly regulated process and that this cellular degradation process must only be initiated under specific cellular conditions. 14-3-3 proteins provide an additional level of regulation to this process by binding and thereby controlling the function of key regulatory proteins involved in autophagy. 14-3-3 proteins can directly interact with IRS-1 and this interaction may regulate the ability of the IRS-1 to recruit and activate PI3K, leading to consequences for mTOR regulation and autophagy initiation . FurtherMore research is required to fully define the role of 14-3-3 in autophagy. However, in addition to having an anti-apoptotic role, 14-3-3 proteins, along with other anti-apoptotic proteins such as Bcl-2  during a


Escherichia coli and Bacillus subtilis, with the general belief that division site positioning occurs as a result of the combination of two negative regulatory systems, Min and nucleoid occlusion. These systems influence division by preventing the cytokinetic Z ring from forming anywhere other than midcell. However, evidence is accumulating for the existence of additional mechanisms that are involved in controlling Z ring positioning both in these organisms and in several other bacteria. In some cases the decision of where to divide is solved by variations on a common evolutionary theme, and in others completely different proteins and mechanisms are involved. Here we review the different ways bacteria solve the problem of finding the right place to divide. It appears that a one-size-fits-all model does not apply, and that individual species have adapted a division-site positioning mechanism that best suits their lifestyle, environmental niche and mode of growth to ensure equal partitioning of DNA for survival of the next generation.Spatial regulation of cell division in bacteria has been a focus of research for decades. It has been well studied in two model rod-shaped organisms,  The location of proteins inside bacterial cells is tightly regulated, with different proteins having specific cellular addresses that can change dynamically with time . This exThe first protein to localize to the division site in bacteria is the highly conserved tubulin-like protein, FtsZ. FtsZ polymerizes at this site to form a ring, called the Z ring, which then recruits all other known division proteins, about 20 in all, to form a protein complex called the divisome . The divEscherichia coli and Bacillus subtilis, Z ring formation occurs precisely at the cell midpoint .The Min system blocks Z ring assembly at the cell poles by inhibiting the polymerization of FtsZ . In bothpolymers . MinC isein MinJ , while iB. subtilis .Nucleoid occlusion prevents Z ring assembly over the nucleoid or chromosome . This efsubtilis  and SlmA and the cell poles  occurred in the central region of the cell. FtsZ production was then switched on  is consistent with a progressive step-wise potentiation of midcell , further highlighting the species-specific nature of bacterial division site placement. Below we describe the different positioning mechanisms at play in a range of recently studied organisms.The importance of Z ring positioning mechanisms other than Min and NO is further exemplified by the fact that many bacteria lack Noc/SlmA and/or Min protein homologues . Recent Caulobacter crescentus, which lacks both Min and NO proteins, Z ring positioning is governed by a bipolar gradient of the FtsZ inhibitor MipZ . Replication and segregation of the chromosomal origins then sets up a bipolar gradient in pre-divisional cells, which dislodges FtsZ from the pole and restricts Z ring formation to midcell .Importantly, MipZ provides both spatial and temporal cues for Z ring formation. In newborn Streptomyces coelicolor, which also lacks the Min and NO systems, FtsZ is recruited and tethered to the division site directly via interaction with the membrane-associated protein SsgB  may not be uniformly localized throughout the cell wall, at least during septum formation . WTA loc. aureus  indicatiS. pneumoniae frequently form over nucleoids and that cell constriction occurs concurrently with the separation of the DNA  can serve as a marker for the cell equator and hence the site for future cell division  is a Gram-negative coccus that divides in two alternating perpendicular planes , as well as the repertoire of FtsZ-binding proteins present in the organism. A striking recent example of niche-specific division site selection comes from the study of a rod-shaped γ-proteobacterium that adheres via one of its poles to the nematode Laxus oneistus. To ensure that both daughter cells remain attached to the host, this bacterium grows by increasing in width and forms its septum parallel rather than perpendicular to the length axis , mode of peptidoglycan synthesis (elongation/division modes), lifestyle (division occurs asymmetrically in gth axis . We are The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Circulating angiotensin II (AngII) is elevated in congestive heart failure (CHF), and leads to skeletal muscle wasting, which is strongly associated with poor patient outcomes. We previously found that AngII upregulates protein phosphatase 2C-alpha (PP2Cα) and dephosphorylates AMP-activated protein kinase (AMPK), a critical regulator of cellular metabolism, in skeletal muscle.To determine the role of PP2Cα in AngII-induced wasting, gastrocnemius (Gas) muscles of FVB mice were injected with scrambled or PP2Cα siRNA and mice were infused with saline or AngII for 4 days.Knockdown of PP2Cα reduced AngII wasting, blocked AngII upregulation of PP2Cα, increased p-T172-AMPK, and inhibited AngII-mediated reductions in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), in complex IV activity, and in ATP levels. AngII impaired the rate of autophagy as determined by a 2.4-fold increase in p62/SQSTM1 (p62) accumulation. This induction was reduced by PP2Cα knockdown, which also increased beclin-1 expression and microtubule-associated protein 1 light chain 3 (LC3)-II conversion in AngII-infused Gas. AngII reduced activating S555 phosphorylation of UNC-51-like kinase 1 (ULK1), a critical regulator of autophagosome formation, and increased inhibitory S757 ULK1 phosphorylation and these effects were prevented by PP2Cα siRNA.AngII inhibited AMPK activity and reduced PGC-1α and TFAM expression  and impaired ULK1 activation and autophagy , resulting in mitochondrial dysfunction, decreased ATP, and wasting. Knockdown of PP2Cα normalized AMPK activity, PGC-1α, NRF1, and TFAM levels and blocked AngII inhibition of ULK1, leading to improved mitochondrial biogenesis/recycling/function, energy production, and inhibition of AngII-induced wasting. These results demonstrate novel effects of AngII on cellular metabolism that are likely critical in mediating the muscle wasting that is a hallmark of CHF. In conditions such as congestive heart failure and chronic kidney disease, where loss of lean body mass correlates strongly with poor prognosis -3, circuWhile it has been well established that AngII causes skeletal muscle atrophy, much less is known about the metabolic and bioenergetic effects of AngII, or how these effects relate to AngII wasting. Protein breakdown in skeletal muscle is predominantly mediated by the UPP and the autophagy-lysosome system, but the specific role of autophagy in AngII skeletal muscle wasting is not known. We originally reported that AngII impaired AMP-activated protein kinase (AMPK) activation in muscle, likely via upregulation of protein phosphatase 2C-alpha (PP2Cα) (AngII did not inhibit kinases upstream to AMPK), leading to mitochondrial dysfunction and ATP depletion, and that direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevented AngII wasting ,16. The in vivo, by injecting gastrocnemius (Gas) muscles of ketamine/xylazine anesthetized FVB mice  with 2.5 μg [in vivo.Four separate siRNAs targeted to PP2Cα mRNA  were tested for knockdown efficiency h 2.5 μg ,25 of eah 2.5 μg ,25, 20 mh 2.5 μg , and 1 sIn the experimental setting, 5 μg of PP2Cα siRNA and scrambled siRNA were electroporated into contralateral Gas muscles . Three days later, mice were implanted subcutaneously with osmotic minipumps (Alzet 1007D) continuously infusing either sterile saline (control) or 1,000 ng/kg/min AngII . This dose of AngII yields a 2.8-fold increase in plasma AngII that is within the pathophysiological ranges observed in patients with congestive heart failure (CHF) and chronic kidney disease ,14,27-30Frozen tissue was blotted dry of excess Allprotect reagent and pulverized with a mortar and pestle over liquid nitrogen. Aliquots of each pulverized muscle were prepared as previously described  for mitoSerial skeletal muscle frozen cross sections were stained for LC3 and p62 using rabbit a/LC3 pAb from MBL Co., Ltd  or mouse a/SQSTM1/p62 mAb from Abcam , respectively. Sections were fixed with 4% paraformaldehyde (15 min), treated with 0.1% Triton X-100 in PBS, and stained with primary a/b for 1 h at room temperature. Biotin-conjugated anti-mouse (or anti-rabbit) IgG  were applied for sections for 45 min followed by incubation with streptavidin-Alexa594 conjugate (1:500) plus DAPI (both from Invitrogen). Antibody specificity was verified routinely by staining of serial sections with ‘normal’ IgG . We did not observe any detectable immunopositivity on ‘normal’ IgG-stained sections (data not shown).P <0.05 was considered statistically significant.All data are presented as means +/- standard errors . GraphPad software  was used to perform the statistical analysis. Differences between groups were determined by two-way ANOVA followed by Holm-Sidak’s multiple comparisons test , or by one-way ANOVA followed by Tukey post-tests  as appropriate. A value of P <0.05), while siRNA A proved to be the most effective with a 57% reduction (P <0.001) in mRNA compared to control, and siRNA D failed to reduce expression of PP2Cα  and 26% of Gas mass (P <0.001) compared to saline-infused controls at day 4 , consistent with our previous findings [P <0.001 and P <0.05 respectively, Figure P <0.05, Figure P <0.001, Figure P <0.05, Figure Infusion of AngII caused a 46% increase in PP2Cα protein expression , and PP2Cα knockdown partially blocked the effect of AngII such that complex IV activity was reduced by only 48% , which was partially inhibited to a 49% reduction by PP2Cα knockdown  was not reduced  and PP2Cα siRNA reduced that increase to only 1.6-fold, while not changing basal caspase activation , and that increased cleavage was blunted by PP2Cα siRNA , and PP2Cα siRNA blunted that increase to 1.5-fold, while not altering basal p62 , and this effect was completely reversed by PP2Cα siRNA , and this increase was also almost completely reversed by knockdown of PP2Cα , although PP2Cα siRNA did not blunt this decrease  and knockdown of PP2Cα did not prevent this reduction  facilitate fusion of the outer and inner mitochondrial membranes, respectively. Mitochondrial fission is mediated by mitochondrial fission 1 protein (Fis1), mitochondrial fission factor (Mff), and dynamin-1-like protein (DRP1). Interestingly, Mitofusin-2, OPA1, Fis1, and DRP1 expression were all reduced by Ang II, indicating that Ang II inhibits both mitochondrial fusion and fission. AngII markedly reduced expression of mitofusin-2 , and AMPK-independent .Our results indicate that PP2Cα is a critical mediator of AngII wasting. Knockdown of PP2Cα via electroporation of siRNA into Gas partially prevented AngII wasting, which is consistent with activation of AMPK being protective against wasting caused by AngII, as we have previously demonstrated . AlthougCertainly, the UPP is very important for AngII wasting, and we have previously shown that AngII-mediated effects on Akt/FOXO phosphorylation and upregulation of atrogin-1 and MURF1 mRNA was prevented by direct AMPK activation with AICAR . It is iAs AMPK is activated, PGC-1α expression increases, in turn activating a program of mitochondrial biogenesis ,32. As sHealthy mitochondrial function is normally maintained both by generation of new mitochondria via PGC-1α-NRF1-TFAM-mediated biogenesis, and by sequestration of damaged or defective mitochondria to autophagosomes and their subsequent degradation following fusion to lysosomes in a process known as mitophagy ,44. The AngII significantly increased p62 protein without altering mRNA levels, indicating impairment of autophagy. While p62 clearance/accumulation is a reasonable index of autophagic flux in the absence of transcriptional regulation, it will be informative to measure flux directly in future studies, for instance by tracking LC3 turnover alongside p62 accumulation . The parFailure of the cell to clear damaged mitochondrial proteins results in the accumulation of dysfunctional mitochondria and a reduced ability to meet cellular energetic demands. This deficit is accentuated in our model (where AMPK activation is impaired by AngII) because AMPK activation is the normal physiologic response to decreased ATP availability. Although AMPK activation can be associated with mitochondrial dependent muscle wasting, and inhibition of AMPK has been shown to restore muscle size in myofibers with altered mitochondria , the oppIn the current study, p62 accumulation with AngII indicates impairment in the rate of autophagy. This is corroborated by the accumulation of dysfunctional complex IV in AngII-infused muscle, as demonstrated by the marked reduction in complex IV activity, even while total complex IV expression tended to increase. The accumulation of dysfunctional mitochondria is reflected in the reduction of ATP in response to AngII while mitochondrial load remained unchanged. Activation of AMPK signaling via knockdown of PP2Cα acted in opposition to AngII by facilitating autophagy as evidenced by reduced p62 accumulation, and improved mitochondrial function as indicated by increased complex IV activity, and increased ATP. Further, the degree to which PP2Cα siRNA activated mitochondrial biogenesis outweighed the degree to which mitophagy was activated since total mitochondrial content was increased by knockdown of PP2Cα. As such, the net effect of PP2Cα knockdown during infusion of AngII is a greater number of total mitochondria and a simultaneously higher proportion of fully functional, more efficient mitochondria, leading to increased energy production and protection from AngII wasting.ULK1 is a critical mediator of autophagy -39, actiParallel to its inhibitory effects on the PGC-1α-NRF1-TFAM axis , ULK1 (autophagosome formation), and p62 clearance (autophagic flux), AngII also significantly reduced expression of several markers of mitochondrial fusion  and fission , suggesting that AngII also acts to inhibit both mitochondrial fusion and mitochondrial fission. Mitofusin-2 is located on the outer mitochondrial membrane, while OPA1 is localized to the inner mitochondrial membrane and both are critical for the initiation of mitochondrial fusion ,44,48. FMARCH5 is an E3 ligase that regulates the expression of both mitofusin-2 and Fis1 via UPP-mediated degradation of these proteins . Its expOur data show that AngII-mediated skeletal muscle wasting is characterized by impairment of mitochondrial biogenesis, fission, fusion, and autophagy, thereby preventing normal mitochondrial recycling and leading to mitochondrial dysfunction, ATP depletion, and wasting (summarized in Figure 1 complex; complex IV: Cytochrome C oxidase; complex V: ATP synthase; DRP1: Dynamin-1-like protein; Fis1: Mitochondrial fission 1 protein; FOXO: Forkhead box protein class O; Gas: Gastrocnemius muscle; IGF-1: Insulin-like growth factor 1; LAMP1: Lysosomal-associated membrane protein 1; LC3: microtubule-associated protein 1 light chain 3; MARCH5: Membrane-associated RING-CH5; Mff: Mitochondrial fission factor; mtDNA: Mitochondrial deoxyribonucleic acid; mTOR: Mammalian target of rapamycin; MuRF1: Muscle RING-finger protein-1, Trim63; NRF1: nNuclear respiratory factor 1; OCT: Optimal cutting temperature; OPA1: Optic atrophy 1; p62: p62/SQSTM1; PGC-1α: Peroxisome proliferator -activated receptor-γ coactivator 1-α; PP2Cα: Protein phosphatase 2C-alpha; RT-PCR: Reverse transcriptase polymerase chain reaction; SEM: Standard error of the mean; siRNA: Small interfering ribonucleic acid; TFAM: Mitochondrial transcription factor A; UCP3: Uncoupling protein-3; ULK1: UNC-51-like kinase 1; UPP: Ubiquitin proteasome pathway; WB: Western blot.AICAR: 5-aminoimidazole-4-carboxamide ribonucleoside; Akt: Protein kinase B; AMPK: AMP-Activated Protein Kinase; AngII: Angiotensin II; ANOVA: analysis of variance; ATP: Adenosine triphosphate; atrogin-1: Atrogin-1/MAFbx, Fbxo32; CHF: Congestive heart failure; complex I: NADH dehydrogenase; complex II: succinate dehydrogenase; complex III: cytochrome bcThe authors declare that they have no competing interests.AMT carried out the animal experiments, performed the molecular assays and immunoblots, participated in the design of the study, performed the statistical analysis, and drafted the manuscript. TY performed the quantification of muscle fiber cross sectional area, participated in the design of the study, and helped to revise manuscript. SS performed the immunohistochemical analysis of p62 and LC3 positive punctae in Gas cross sections and participated in the design of the study. PD conceived of the study and participated in its design, coordination, and interpretation of results, and helped to revise the manuscript. All authors read and approved the final manuscript.