Moreover, phosphorylation of the myosin targeting subunit (MYPT1) of the RLC20 phosphatase (MLCP) by the RhoA-dependent ROCK kinase, inhibits the phosphatase activity and consequently inhibits dephosphorylation of RLC20 with concomitant increase in contractile force, at constant intracellular [Ca2+]. This pathway is referred to as Ca2+-sensitization. There is, however, emerging evidence suggesting that additional Ser/Thr kinases may contribute to the regulatory pathways in SM. Here, we report data implicating the p90 ribosomal S6 kinase (RSK) in SM contractility. During both Ca2+- and agonist (U46619) induced SM contraction, RSK inhibition by the highly selective compound BI-D1870 (which has no effect on MLCK or ROCK) resulted in significant suppression of contractile force. Furthermore, phosphorylation levels of RLC20 and MYPT1 were both significantly decreased. Experiments involving the irreversible MLCP inhibitor microcystin-LR, in the absence of Ca2+, revealed that the decrease in phosphorylation levels of RLC20 upon RSK inhibition are not due solely to the increase in the phosphatase activity, but reflect direct or indirect phosphorylation of RLC20 by RSK. Finally, we show that agonist (U46619) stimulation of SM leads to activation of extracellular signal-regulated kinases ERK1/2 and PDK1, consistent with a canonical activation cascade for RSK. Thus, we demonstrate a novel and important physiological function of the p90 ribosomal S6 kinase, which to date has been typically associated with the regulation of gene expression.In the canonical model of smooth muscle (SM) contraction, the contractile force is generated by phosphorylation of the myosin regulatory light chain (RLC The second mechanism involves agonist-dependent down-regulation of MLCP, leading to increased contractility at constant intracellular Ca2+ concentration ([Ca2+]i). The release of select agonists (e.g. thromboxane A2), which act on G-protein coupled receptors (GPCRs), up-regulates the cytosolic GTPase RhoA, which in turn activates the Rho-associated protein kinase-ROCK. The latter phosphorylates the myosin targeting subunit (MYPT1) of the heterotrimeric MLCP, made up also of the catalytic subunit (PP1C) and a 21 kDa subunit 20 phosphorylation and thus enhancing the SM contractile force. Interestingly, both Ca2+ independent ZIPK and ILK also phosphorylate MYPT1 and suppress PP1C activity Over the past two decades, it has been shown that this relatively simple Ca2+-sensitization is now recognized as a major regulatory mechanism in SM, and is targeted for such conditions as hypertension, it is also increasingly clear that additional regulatory mechanisms, both Ca2+-dependent and independent, must be in place in diverse SM tissues. Evidence for Ca2+-independent activity is seen when microcystin-LR, a phosphatase inhibitor, is added to permeabilized SM in the absence of [Ca2+]i20 increases, resulting in contraction. It has also been shown that the blood vessels isolated from MLCK-null mice embryos at E14.5 to term and permeabilized with α-toxin, develop force in response to increase in [Ca2+]i,2+-sensitization can be induced in MLCK-null vessels by addition of GTPγS and this contraction can be relaxed by the Rho-kinase inhibitor, Y-27632 2+ dependent kinase(s) other than MLCK, or/and Ca2+-independent kinase(s) which are activated by Ca2+-dependent upstream signaling.Although Ca20in vitro, although the physiological consequences have never been explored Interestingly, it has been reported some time ago that the p90 ribosomal S6 kinase (RSK2), which is typically associated with regulation of gene expression, can phosphorylate RLC20 phosphorylation by RSK2 was reported, two selective inhibitors for the RSK family of kinases were discovered: the flavonoid glycoside SL0101 ex vivo assays. We show that inhibition of RSK significantly reduces contractile response in intact SM stimulated by either high [K+] or the thromboxane A2 (TXA2) analogue U46619, which typically induces Ca2+-sensitization cascade through the activation of the TXA2 receptors and the RhoA/ROCK signaling cascade. Furthermore, RSK inhibition by BI-D1870 significantly reduces phosphorylation levels of both RLC20 and MYPT1, and suppresses agonist induced Ca2+-sensitized force, indicating that RSK functions in SM not only through phosphorylation of RLC20 but also through inhibitory phosphorylation of MYPT1. Further evidence for agonist-induced activation of RSK in SM is our finding that the TXA2 receptor synthetic agonist U46619 increases ERK1/2 and RSK2 Ser227 phosphorylation. Interestingly, our data also suggest that RSK up-regulation leads to the phosphorylation of the MYPT1 subunit not only at the canonical sites of Thr853 and Thr696, but also on Ser668.Since the original observation of RLCStaphylococcus aureus α-toxin 2+ stores, strips were treated with 10 µM Ca ionophore A23187  for 10–15 min in G1. For Ca2+-activating solutions, 10 mM EGTA was used and a calculated amount of Ca2+-methanesulfonate was added to give the desired [Ca2+]. Measurements of IC50 for RSK and PDK inhibitors were carried out in muscle strips stimulated with pCa 6.7. Once the force reached a plateau, increasing concentrations of inhibitors or of the diluent (DMSO) were added, and the magnitude of force relaxation was recorded. The magnitude of relaxation was normalized to that of the pCa 6.7-induced force, taken as 100%.All procedures and protocols were approved by the Animal Care and Use Committee at the University of Virginia . The primary branches of the rabbit pulmonary artery were dissected, freed from connective tissue, cut into small strips  and mounted between two tungsten hooks on a bubble plate for force measurements 2+] was determined by activating the strips by increasing [Ca2+] from pCa>8. Muscle strips were preincubated with 100 nM of BI-D1870 or 30 µM GSK2334470 or the corresponding concentrations of DMSO as control, for 15 min in relaxing (G1) solution and then stimulated with Ca2+-activating solutions to allow for the magnitude of the force response to be measured. Force was normalized to maximal Ca2+-induced force (pCa 4.5) taken as 100%, or expressed as the absolute value in milli-Newtons (mN).The dependence of force developed in response to varying  in the presence of 25 nM, 100 nM and 1 µM BI-D1870, or the DMSO diluent. The initial large phasic component of force was significantly suppressed at the 1 µM BI-D1870 concentration (+]-induced contraction was used as a reference (i.e. 100%) to assure that the magnitude of U46619-induced contraction was significant and to compare magnitudes of contraction among different treatments. As a result, addition of Y-27632 together with BI-D1870 prior to activation with U46619 (300 nM) inhibited the contraction to a greater extent that Y-27632 alone . In the presence of the inhibitor, we observed two distinct effects: the onset of contraction was shifted to higher [Ca2+] and the maximum force was significantly reduced (∼65%) as compared for the control samples treated only with the diluent DMSO  were both significantly decreased (2+-mediated RLC20 phosphorylation may reflect its direct or indirect phosphorylation by RSK or is secondary to the decrease in MYPT1 Thr853 phosphorylation and increased phosphatase activity.Although we expected that the observed effects of BI-D1870 may be due to suppression of direct phosphorylation of the RLCecreased . The BI-20 in Ca2+ free rigor solution (ATP free) The presence of BI-D1870 (100 nM)   or GSK232+] (20 at Ser19 to ∼60% as compared to control experiments (data not shown). These results conclusively implicate the involvement of canonical regulatory mechanisms for RSK in SM contractile regulation.The constitutively active PDK1 kinase is required for upstream activation of RSK 2+]  along wi2+), i.e. with [Ca2+] well below normal contraction threshold (pCa 7.0), which attests to the phosphorylation of RLC20 catalyzed by Ca2+-independent kinases. We asked what effect BI-D1870 had under the conditions of this experiment. The addition of the inhibitor significantly increased the lag time to the onset of force measured at the intercept of the baseline with a straight line fitted to the rising phase ; and the rate of force development  , is routinely used as a reagent that allows the study of signaling phenomena independent of MLCP. In a control experiment using a rabbit pulmonary artery permeabilized with β-escin, microcystin induces a slow contraction at pCa 9.0 (1 nM Ca p<0.02) . BI-D187rylation , measure2+ independent ZIP kinase 2+ independent microcystin-induced contraction is through inhibition of ZIP Kinase.To address the possibility that the inhibition of the microcystin-induced contraction by BI-D1870 was due to the inhibition of the Ca2+-independent manner, targeting MYPT1 and consequently MLCP. We used an assay involving the synthetic agonist U46619 for the thromboxane A2 (TXA2) receptor.We next asked if agonist stimulation might also up-regulate RSK and mediate SM contractility in a Ca2+] (pCa 7.0) was suppressed in the presence of 100 nM BI-D1870 by more than ∼50% compared with untreated control  also induced an increase in phosphorylation of RSK2 at Ser227 and its upstream regulator ERK1/2  and U46619  which includes Ser668. We found that in intact pulmonary arteries, the phosphorylation level of MYPT1 at Ser668 increased by 42±10.5%, n = 3, p<0.01; following stimulation with U46619, but no change was detected with high pyridimidine group of Src inhibitors We were intrigued by a report that the p90 ribosomal S6 protein kinase (RSK2) was also able to phosphorylate RLC2+], and shifted the onset of contraction to pCa 7.1 from pCa of 8.0. Furthermore, BI-D1870 also inhibited contractions elicited by high [K+] and U46619 in intact SM demonstrating that the observed effects in permeabilized SM are not merely due to loss of cytoplasmic components. Our findings were not expected, because RSK kinases have been associated primarily with signaling downstream of growth factors and receptor tyrosine kinases and are thought to play key roles in cell cycle progression, cell growth, survival and proliferation 2+-induced contraction, we analyzed the phosphorylation levels of both RLC20 and MYPT1 in the presence of inhibitor. Both proteins were phosphorylated on the canonical regulatory sites, Ser19 and Thr853 respectively, at a significantly higher level in the absence of inhibitors, strongly suggesting that RSK simultaneously up-regulates myosin and down-regulates MLCP, resulting in a significant contribution to contractility. We also demonstrated that the mechanism of action of the RSK inhibitor is not through inhibition of the SM actomyosin ATPase.Our data show, for the first time, that RSK kinases are indeed involved in SM contractility. We found, that in the α-toxin permeabilized rabbit pulmonary artery the RSK inhibitor BI-D1780 significantly decreased the contractile force response to  2+]-induced signaling in vascular SM is under investigation in our group.It is not clear at this point in what way increased Ca2+-sensitization effect 2+-independent mechanism is also consistent with the decreased sensitivity to Ca2+ in inhibitor treated samples (2+ concentrations are required to trigger contraction in the presence of up-regulated (or rather uninhibited) activity of MLCP. Thus, RSK may be recruited in agonist-stimulated Ca2+-sensitization, through a pathway canonically identified with ROCK, as well as acting as a Ca2+-independent MLCK.A new, exciting discovery reported in this paper is that BI-D1870 has a significant impact on the phosphorylation of MYPT1, suggesting that inhibition of MLCP may constitute an important mechanism by which RSK potentiates the contractile force. Moreover, we determined that in response to stimulation by the thromboxane A2 analogue U46619 at constant pCa, RSK phosphorylates MYPT1 at Thr696 and Thr853, the two known inhibitory sites. Both these sites are targets for the RhoA-dependent ROCK kinase and their phosphorylation accounts for the Ca samples , as highin vitro2+ desensitization and relaxation of SM One of the interesting outcomes of our study is the identification of the third potential phosphorylation site in MYPT1, i.e. Ser668. This is one of several sites in MYPT1 reported by Cell Signaling Technology using LC-MS/MS platform to be phosphorylated in a number of human cancers Perhaps the functional outcome of Ser688 phosphorylation may depend on whether other sites on MYPT1 are phosphorylated, and if the combined structural effects lead to activation or inhibition of MLCP activity on myosin. Details of this mechanism remain to be determined.2+ sensitized force, MLC20 and MYPT1 phosphorylation correlates with an increase in phosphorylated ERK1/2, RSK2 S227 and PDK1 S241 are all consistent with agonist-induced RSK activation. However, the original in vitro observations that p42MAPK suppressed the ability of RSK2 (in the absence of CTKD) to phosphorylate RLC20 but not a C-terminal peptide of human 40S ribosomal protein S6 in vitro vs in vivo protocols and if so, reflects the importance of intracellular compartmentalization and concentrations of signaling molecules. One possible further explanation of the discrepancy in the findings is that in vivo activated RSK may preferentially phosphorylate MYPT1 rather than directly phosphorylate RLC20.The activation of RSK kinases involves several sequential phosphorylation steps, initiated by the docking of extracellular signal-regulated kinases (ERK1/2) at the C-terminus with subsequent phosphorylation and activation of CTKD. Our findings that U-46619-induced Ca2+-independent protein kinase activity observed with microcystin-induced force in rat ileal SM at 1 µM microcystin 20. The residual microcystin-induced force in the presence of the RSK inhibitor reflects the activity of other Ca2+ independent kinases such as ZIPK consistent with our finding that ZIPK is not eluted from a γ-phosphate linked ATP resin by BI-D1870. Constitutively active ILK is unavailable for testing.While the activation of the ERK1/2 pathway by the thromboxane A2 receptor in SM has been reported before Altogether, our findings demonstrate that the canonical upstream RSK activation signaling pathways established in other cell types are present and activated in SM, supporting a novel function for p90 ribosomal S6 kinases in the regulation of SM contractility . It remaFigure S1(A) Western Blot analysis shows absence of cross-reactivity of anti-RSK1 and anti-RSK3 antibodies with RSK2. (B) Expression profile of RSK1 and RSK2 in rabbit and mouse smooth muscle tissues. Positive control: over-expressed truncated RSK2.  Alignment of C-terminal part of human (h), mouse(m) and rabbit(rab) RSK isoforms used for the commercial antibody production. Note the perfect homology between species for given isoforms RSK1 and RSK2. Therefore, the presence of RSK1 protein in rabbit but not mouse smooth muscle tissues is not due to differences in antigen sequence.(TIF)Click here for additional data file.Figure S2Dose-response curves for RSK inhibitors (BI-D1870 and SL0101-1), PDK inhibitor (GSK2334470) and corresponding concentrations of the diluent, DMSO carried out in a-toxin permeabilized rabbit pulmonary artery SM partially contracted with pCa 6.7.(TIF)Click here for additional data file.


Calcium levels were measured by spectrofluorimetry in Fura-2AM-loaded platelets and tyrosine phosphorylation levels of receptor-proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPARγ agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions.Washed platelets were stimulated with PPARγ ligands inhibited collagen-stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P-selectin exposure. PPARγ ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPARγ agonists, implicating PPARγ in the effects observed. Furthermore, PPARγ ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPARγ was found to associate with Syk and LAT after platelet activation. This association was prevented by PPARγ agonists, indicating a potential mechanism for PPARγ function in collagen-stimulated platelet activation. Conclusions: PPARγ agonists inhibit the activation of collagen-stimulation of platelet function through modulation of early GPVI signalling.PPAR IIbβ3, this leads to the formation of a stable platelet thrombus  for 3 min and then stimulated with collagen (1.0 μg mL−1) in a luminescence spectrophotometer . The ratio of emission values (excitation:340/380 nm) was calculated and converted to calcium concentration using FLWinLab software (Perkin Elmer).Mobilization of calcium from intracellular stores was measured in platelets pre-loaded with the fluorescent dye FURA-2AM as described previously ,33. PlatTo measure α-granule secretion, surface exposure of P-selectin was assessed in whole blood by flow cytometry as reported previously . In thes6) and perfused through collagen-coated (100 μg mL−1) micro-capillaries at a shear rate of 1000 s−1 in the presence of PPARγ agonists 15d-PGJ2, rosiglitazone or vehicle control. Thrombi were subsequently visualized using a Leica DMIRE2 inverted confocal microscopy (using N PLANL 20×/0.4 objective lens with 0–2 mm correction) and thrombus volume calculated from Z series images captured using TCS SP2 software , as previously reported  for 3, 15 or 20 min prior to stimulation with collagen (1 μg mL−1) for 90 s.To determine if the natural PPARγ agonists 15d-PGJ2  on platelet aggregation induced by a range of collagen concentrations  was also examined. Levels of inhibition became reduced significantly with increasing concentrations of collagen. Inhibition was, however, maintained at higher concentrations of collagen. Incubation with 15d-PGJ2 (10 μmol L−1) for 15 min resulted in a significant inhibition of platelet aggregation in response to high concentrations of collagen (10–25 μg mL−1) (Platelet aggregation in response to collagen was found to be inhibited in a concentration-dependent manner by each of the PPAR15d-PGJ2  and rosi15d-PGJ2 . Aggrega15d-PGJ2 . The ext15d-PGJ2  or 20 mi15d-PGJ2  enabled μg mL−1) .2. Furthermore, PPARγ agonists have been previously reported to inhibit platelet aggregation induced by ADP . Thrombus size was calculated from the mean thrombus volume of five randomly selected fields of view. γ ligands, rosiglitazone and 15d-PGJ2 inhibited the thrombus formation significantly in a concentration-dependent manner, where 1 μmol L−1 rosiglitazone or 15d-PGJ2 were able to inhibit thrombus formation by 50.4 ± 14.7 % and 66.6 ± 2.7 % compared with the vehicle control (γ ligands resulted in reduced protein concentration compared with control (2 treatments. The inclusion of the PPARγ antagonist GW9662 (3 μmol L−1) was able to reverse the inhibitory effect of the PPARγ ligand 15d-PGJ2 (3 μmol L−1) on thrombus formation  or solvent control. Thrombus volume was subsequently measured by confocal microscopy. After perfusion, a concentration of 1 μmol L−1 rosiglitazone caused no effect on thrombus stability, although an approximate reduction of 10% in thrombus volume was observed at 20 μmol L−1  or vehicle [DMSO 0.1% (v/v)] for 3 min and then stimulated with GPVI-selective ligand CRP (1 μg mL−1) for 3 min and α-granule secretion was assessed by surface exposure of P-selectin by flow cytometry  or vehicle [DMSO 0.1% (v/v)] for 3 min and then stimulated with collagen (1 μg mL−1). Rosiglitazone caused inhibition of collagen-stimulated peak calcium concentrations , apyrase (2 U mL−1) and indomethacin (10 μmol L−1) to prevent aggregation and ensure the study of primary signaling events. In collagen signaling studies, where non-aggregation conditions are necessary, collagen concentrations required to observe signaling were increased (25 μg mL−1) in order to observe tyrosine phosphorylation of components of the GPVI pathway, consistent with previous reports [γ ligand concentrations used were therefore also increased.To begin to explore the mechanism through which PPAR reports ,33. PPARγ2 was investigated. Treatment of platelets with rosiglitazone was without a marked effect on the levels of collagen-stimulated tyrosine phosphorylation of Syk  for 15 min prior to their stimulation with collagen (25 μg mL−1) and immunoblot analyses were conducted to detect PPARγ. PPARγ was found to interact with Syk and LAT when platelets were stimulated with collagen in the absence of PPARγ ligands  .As in the presence of PPAR ligands . In the mol L−1) , indicatet al. [γ ligands in vitro was dependent on PECAM-1, the effect of rosiglitazone on platelet aggregation was examined using washed platelets from PECAM-1-deficient mice.Platelet endothelial cell adhesion molecule-1 (PECAM-1), has been reported to negatively regulate platelet function and thrombus formation ,30,36. Tet al.  to suggeγ ligand rosiglitazone, when compared with the vehicle control  [While platelets are anucleate cells, recent reports have demonstrated that nuclear receptors such as the glucocorticoid receptor , RXR 1717 and PPγ  and β/δ γ  are exprγ –19. PPARitazone) ,11.γ ligands have been reported to inhibit platelet aggregation in response to ADP that is accompanied by a reduction in markers of platelet activation such as P-selectin exposure, TXA2 synthesis and sCD40L release [2 mimetic U46619 and have proposed this to be mediated through suppression of Gq signaling, resulting in inhibition of mobilization of calcium from intracellular stores [γ to interact with RXR may suggest some overlapping modes of action.PPAR release . Recentlr stores . Althougγ ligands inhibit collagen-stimulated platelet aggregation, α-granule secretion and calcium mobilization. In the presence of the PPARγ antagonist GW9662, inhibition of aggregation was reversed, suggesting that this affect is at least in part, modulated by PPARγ in platelets. Failure to completely reverse inhibition indicates potential additional, and as yet uncharacterized, PPARγ-independent modes of action of these ligands. Increased concentrations of rosiglitazone or 15d-PGJ2 were associated with more accentuated levels of shape change upon stimulation with collagen. This is likely to reflect lower levels of aggregation in this optical assay, as PPARγ agonists alone do not stimulate shape change. We cannot, however, rule out the possibility that PPARγ normally serves to inhibit shape change.In this study, we have demonstrated that PPARγ agonists were found to inhibit collagen-stimulated calcium mobilization, a range of signaling proteins upstream of calcium in the GPVI collagen activation pathway were examined. Rosiglitazone did not cause marked inhibition of collagen-stimulated tyrosine phosphorylation of the kinase Syk, suggesting that the activity of upstream Src-family kinases, such Fyn and Lyn, is not modulated by PPARγ ligands. This ligand was, however, found to reduce the levels of tyrosine phosphorylation of the transmembrane adapter protein LAT and thereby PLCγ2, which is consistent with the inhibition of calcium regulation and α-granule secretion.As PPARγ stimulation also results in suppression of PI3-K signaling. In the present study, interactions of PPARγ with Syk and LAT highlight a potential novel GPVI-dependent mechanism for PPARγ action on platelet activation. PPARγ in its inactivated state interacts with Syk and LAT . These interactions correlate with phosphorylation of Syk and LAT leading to the activation of proteins downstream within the GPVI pathway. Upon ligation of PPARγ, interactions with Syk and LAT were prevented, which coincided with diminished signaling downstream resulting in a reduction in platelet activation. The addition of the antagonist GW9662 was able to prevent the inhibitory effect of PPARγ ligands on interactions between PPARγ with Syk and LAT. Taken together, this suggests that the inhibitory actions of PPARγ ligands may be mediated within the GPVI signaling pathway at the level of LAT or the LAT signalosome and that inhibition of platelets by PPARγ ligands is not because of toxic effects. Further work is required to establish whether PPARγ is recruited to a signaling protein complex with both Syk and LAT, or whether interaction with Syk and LAT occurs independently. Furthermore, whether PPARγ interactions contribute to positive signaling through the GPVI pathway remains to be established.The tyrosine phosphorylation of LAT results in the recruitment and activation of PI3-K, leading to the generation of 3′-phosphorylated inositol phospholipid second messengers. Rosiglitazone treatment resulted in diminished collagen-stimulated phosphorylation of Akt/PKBα, suggesting that the inhibitory effect of the PPARγ ligands reduce the development of atherosclerosis and myocardial ischemia–reperfusion injury through inhibition of platelet activation and intra-arterial thrombus formation in animal models [γ ligands rosiglitazone and 15d-PGJ2, inhibit thrombus formation in human whole blood on immobilized collagen under arterial flow conditions. Furthermore, perfusion with a low concentration of rosiglitazone, which is likely achievable in plasma of patients taking rosiglitazone (1 μmol L−1) [γ ligands may offer beneficial clinical actions through inhibition of thrombus formation without embolization effects. Future studies using in vivo models of thrombosis will be required to explore this further.It has been suggested that PPARl models . In suppmol L−1) , caused γ ligands are able to inhibit the function of platelets, and they possess similar abilities to modulate calcium mobilization in these cells, we sought to determine whether the acute, non-genomic actions of rosiglitazone may be dependent on PECAM-1 expression. Examination of PECAM-1-deficient mouse platelets revealed, however, that the inhibitory effect of collagen-stimulated platelet aggregation by PPARγ ligands is unaffected by the presence or absence of PECAM-1. The possibility still exists, however, that PPARγ and PECAM-1 share similarities in their modes of modulation of GPVI-stimulated signaling in platelets.Treatment with TZDs such as rosiglitazone has been reported to reduce the activity of circulating platelets in patients with coronary artery disease  and typeγ agonists may be associated with an increased incidence of congestive heart failure, myocardial infarct and death [Clinical trials have demonstrated that the treatment of diabetic patients with TZDs exerts a cardioprotective effect as evidenced by a reduction in the risk of myocardial infarction in diabetic patients –26,40. Hnd death –44. Thesγ ligands inhibit collagen-stimulated platelet function through modulation of signaling downstream of the collagen receptor GPVI.Our findings indicate that PPAR


IgE-mediated allergy to peach represents a frequent cause of severe anaphylactic reactions in Mediterranean countries. Pru p 3, a nonspecific lipid transfer protein (LTP) from peach, was identified as an important elicitor of IgE-mediated food hypersensitivity reactions to peach. Specific immunotherapy is currently the only allergen-specific and disease-modifying treatment of allergies, but has so far not been applied to food allergic patients due to the high risk of IgE-mediated side effects. Hypoallergenic Pru p 3 derivatives have therefore been constructed in order to minimize side-effects, but these molecules were not immunogenic in animal models.Escherichia coli. The proteins were purified by affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. IgE-reactivity was tested by dot blot analysis and immunogenicity was investigated by immunization of rabbits.Two recombinant Pru p 3-derivatives were developed based on the Pru p 3 amino acid sequence, by introduction of point mutations, or rearrangements of Pru p 3 fragments and oligomerization. Genes encoding the resulting Pru p 3 derivatives were cloned into the expression vector pET27b and expressed in Escherichia coli yielded high amounts of recombinant proteins. They were purified to homogeneity and exhibited a strongly reduced IgE-reactivity when exposed to sera of peach allergic patients. Upon immunization of rabbits the Pru p 3 derivatives induced a robust IgG antibody response specific for natural Pru p 3.Expression of the two recombinant Pru p 3 derivatives in The reported recombinant hypoallergenic Pru p 3 derivatives are candidates for specific immunotherapy of peach allergic patients.B Linhart: None declared, A Gstoettner: None declared, C Gamez: None declared, I Swoboda: None declared, A Mari: None declared, N Papadopoulos: None declared, R Valenta: Grant/research support from Biomay AG, Vienna, Austria.


Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling. Receptor tyrosine kinases (RTKs) are involved in countless signaling pathways and their deregulation is involved in several diseases, in particular in cancer. Despite extensive study of these signaling cascades, until recently RTK internalization was believed to determine a down-regulation of RTK activity, ultimately leading to receptor degradation. However, increasing evidence indicates that endocytosis modulates and also sustains signaling transduction throughout the downstream targets of RTKs It has recently been reported that in lung cancer IGF1R endocytosis is triggered by ligand binding, causing IGF1R ubiquitination and internalization via clathrin-coated vesicles and/or caveolae In keeping with recent publications indicating that RTKs endocytosis regulates signaling transduction, we analyzed the role of IGF1R internalization in ES. This study was performed before and after ligand binding (IGF1) with the aim of exploring the two most common RTKs internalization mechanisms: clathrin- and caveolin-dependent endocytosis.We observed that under basal conditions (without IGF1 stimulation), IGF1R was present in the plasma membrane. However, upon ligand stimulation IGF1R was internalized by clathrin-dependent mechanisms  and by CMoreover, since ES cells have scant cytoplasm, we were interested in confirming IGF1R signaling there, distinguishing this signaling from that performed by active IGF1R present in the cell membrane. As shown in In order to confirm IGF1R internalization mediated by clathrin and CAV1, we performed co-IP studies. The results showed that after stimulation with IGF1, IGF1R co-precipitated with both clathrin and CAV1 . Again, Here we tested the impact of clathrin or CAV1 interference and inhibition on IGF1R internalization and signaling using two different approaches: 1) specific siRNAs and 2) a drug approach, using MCD and CPMZ. MCD is known to deplete cholesterol, disrupting caveolae,Regarding clathrin and CAV1 interference, siRNAs effectively reduced CAV1 and clathrin protein levels (40–65% reduction) see . RegardiSeveral groups have shown that RTKs are frequently activated in endosomes, and so we were interested in determining the impact of the inhibition of clathrin and CAV1 endocytosis on IGF1R signalling. Accordingly, we also studied the consequent effects on cell growth and survival. In order to accomplish this, we compared the effects of CAV1 and clathrin inhibition and interference in 3 ES cell lines  with different levels of sensitivity to IGF1R inhibition, the A673 cell line being less sensitive, as reported previously by our group.50 of proliferation was similar in all cell lines (between 10–15 µM for CPMZ and 4–4.5 µM for MCD)  . No chanfor MCD) . Similarfor MCD) . Additiose drugs . Regardise drugs  for CAV1Previous observations obtained in our own work indicated that IGF1R signaling can be enchanced after clathrin- and/or caveolin-dependent internalization. Accordingly we were interested in testing a new drug combination that has never been assayed before in ES. Here we tested the effect of the combination of the commonly used tyrosine kinase inhibitor, AEW541(AEW), with the previously evaluated endocytosis inhibitors CPMZ and MCD on the levels of proliferation  and the As illustrated in the isobolograms shown in In this study we detected changes in the phosphorylation of several other proteins , such asIt has been described that IGF1R upregulation promotes cell survival and proliferation in many types of cancer, including carcinomas such as prostate IGF1R is able to activate several signalling pathways through the phosphorylation of different intracellular proteins, including insulin receptor substrate-1 (IRS-1) and the Shc proteins In line with this, we decided to study the involvement of IGF1R internalization in ES signaling and its possible targeting benefits. Initially, we observed that IGF1R was being internalized in ES cell lines after ligand binding, mainly due to clathrin–dependent mechanisms. We were able to show that i) clathrin or CAV1 inhibition/interference blocks IGF1R internalization, ii) IGF1R signaling in ES largely depends on receptor internalization, and iii) the pro-survival and anti-apoptotic effects of IGF1R signaling are considerably reduced when internalization is blocked.The results obtained support the initial hypothesis to the effect that IGF1R signaling is enhanced after receptor internalization in ES. This mechanism allows access of upstream active kinases to direct downstream targets, thus permitting higher signaling specificity through the different subcellular compartments. Based on our results, we propose that in ES, after ligand binding, IGF1R is internalized under two different mechanisms: i) one dependent on clathrin and ii) the other caveolin-dependent . HoweverTaken together, all these findings lead to the hypothesis that these molecular hits could be promoting ES tumorigenicity. Our results suggest that IGF1R internalization in ES is under a highly controlled spatial and temporal regulation, combining both clathrin and CAV1 mechanisms and underscoring the role of CAV1 in RTK endocytosis.In keeping with the observations discussed previously, together with Podar's studies in multiple myeloma With the studies on the effects of AEW541 and the CPMZ/MCD combination on ES cells, here we demonstrate that IGF1R tyrosine kinase inhibition is improved when combined with CAV1 or clathrin inhibition, reducing the levels of proliferation and the induction of apoptosis in ES cell lines. The effects of the drug combination had an impact on the phosphorylation status of several proteins, namely AKT, SRC p38, TOR, MSK1/2, MEK1/2, among others, confirming once again that IGF1R signaling depends on its internalization. Also, proteins such as HSP27, AKT, β-catenin, AMPKa and STAT2/4/3 exhibited the highest reduction in phosphorylation with the triple treatment, supporting the idea of combining TK inhibitors with internalization blockers in order to decrease signaling. It should be noted that RTK-blocking antibodies would, in theory, only block receptors located in the cellular membrane, not affecting the receptors already internalized. In this case, the small-molecule tyrosine kinase inhibitors would be a better choice since they would diffuse to the cytoplasm and also inhibit some of the receptors within the endocytic vesicles.In conclusion, here we show that blockade of IGF1R internalization, either by clathrin or caveolin1, inhibits IGF1R signalling in ES cells. Accordingly, we provide new evidence regarding the role of cav1/clathrin endocytosis in IGF1R internalization. Finally, we consider the combination of TK inhibitors with endocytosis inhibitors as a major and plausible strategy to reduce ES cell proliferation through IGF1R signaling.The results shown here are focused on Ewing Sarcoma, but it is extremely likely that they may find a more general application in sarcomas, and in several subsets of other more prevalent neoplasms that depend largely upon IGF1R survival signals (i.e. subsets of lung or breast carcinoma), therefore increasing the potential impact of our findings for sarcoma therapy.Mycoplasma, as screened with the MycoAlert Mycoplasma Detection kit .The A673, TC-71, SK-ES-1, A4573 and TTC466 cell lines were obtained and maintained as previously described AEW541 (AEW) was kindly provided by Novartis Pharma AG, Basel, Switzerland. Chlorpromazine (CPMZ) and Methyl-β-cyclodextrin (MCD) were purchased from Sigma-Aldrich .Caveolin1 pre-designed siRNA, clathrin-validated siRNAs and Silencer negative control siRNA were purchased from Ambion . Cell lines were treated with 100 nM siRNAs, as previously described,4CL for 10 minutes, blocked for 30 minutes with PBS 0.2% BSA (Sigma Aldrich), and incubated overnight at 4°C with one of the following primary antibodies; rabbit polyclonal antibody against caveolin1 (Abcam), or rabbit polyclonal antibody against clathrin (Abcam), or mouse monoclonal antibody against clathrin (Affinity Bioreagents). After three washes in PBS, the slides were incubated with one of the following secondary antibodies: chicken anti-rabbit AlexaFluor594 (Invitrogen) or goat anti-rabbit Cy3  or goat anti-mouse AlexaFluor594 (Invitrogen), for 45 min, and washed three times. Then, all the slides were incubated with rabbit polyclonal antibody against IGF1R  for 1 hour, washed three times, and incubated with chicken anti-rabbit Alexa 488 (Invitrogen) for 1 hour. Following this, cells were washed three times and nuclear DNA was counterstained with DAPI (2.5 mg/mL) (Invitrogen). Cells were mounted using the Slowfade Gold Antifade reagent (Invitrogen) or Vectashield H-1000 medium (Vector). A Zeiss LSM510 confocal microscope or a Leica inverted fluorescence microscope connected to a digital video camera (Leica DC100) were used to obtain confocal or fluorescence images. Confocal image analysis was performed using the LSM 5 Image Browser (program version 2.8). 4–6 independent experiments were performed and the final data presented selected as the most representative of the results obtained.Cells were grown overnight on sterile glass slides (covered with gelatine 0.1%), with serum-free medium and then stimulated with IGF1 for 15 minutes. The glass slides were washed with PBS and fixed with 2%(w/v) formaldehyde (Sigma-Aldrich) for 30 min. Cells were permeabilized in PBS/Triton x-100 0,1% (Invitrogen) for 30 minutes, incubated with PBS 50 nM NHAfter treatment with IGF1, cells were fixed for 15 minutes in 2% paraformaldehyde/0.1% glutaraldehyde and spun down at 7500 rpm for 5 min. Cells were embedded in 12% gelatine, spun down and fixed over-night at 4°C (with a 2.3 M sucrose solution on top of them). Cell pellets were cut on an ultramicrotome  at 70 nm thickness and sections were placed on carbon-coated hexagonal grids. The sections were incubated with rabbit polyclonal antibody against IGF1R overnight at 4°C, washed with PBS, incubated with ProteinA-Gold-10 nm antibody for 2 h at room temperature, and embedded in methyl-cellulose-0.3% uranyl acetate for 5 min. Photos were taken with a JEOL JEM1011 Transmission Electron Microscope connected to a digital video camera (MegaView III) or a Leica DM/RB microscope with an RCM module and a DFC-420C digital camera. 3–5 independent experiments were performed and the final data presented selected as the most representative of the results obtained.IP and WB studies were performed as previously reported 50 of proliferation (control treatment performed using the vehicle for CPMZ and MCD). The percentage of proliferation inhibition induced by the specific CAV1/clathrin siRNAs was also evaluated. 4–6 independent experiments were performed and the final data are presented as averages of all replicates. To determine the proliferation rate, we used the MTT method, as previously described.The dose-response proliferation levels of the cell lines subjected to drug treatment were analyzed to determine the ICThe apoptosis and necrosis indices were assessed by flow cytometry, as previously described.50 of proliferation as the isoeffective point (using Origin 6.0 to plot the data). Isobolograms were acquired by plotting the IC50 of AEW on the Y-axis and the IC50 of CPMZ or MCD on the X-axis.The effects of the combination of AEW with CPMZ or MCD were analyzed using Loewe's isobolographic analysis, revised by Steel and Peckman, as previously described 2EDTA, 1 mM EGTA, 50 mM NaF, 1%Triton x-100, 5 mM Na3VO4 and protease inhibitor cocktail tablet (Roche). 400 µg of protein was used. The signals from the membranes were detected as described for WB and the levels of chemiluminescence of each spot were evaluated by 2D-densitometry using the AIDA©. software . 3 independent experiments were performed and the final data are presented as averages of all replicates.The effects of the AEW with CPMZ and/or MCD combinations were analyzed at the level of global kinase phosphorylation, using the dot-blot Proteome Profiler™ Array Human Phospho-Kinase Array Kit (R&D Systems), according to manufacturer's instructions. Briefly, cells were treated with 75 nM AEW, 7.5 mM CPMZ and/or 2.5 mM MCD for 1 hour, stimulated with IGF1 for 15 minutes, and lysates were prepared with the appropriate lysis buffer  WB detection of clathrin and Cav1 protein levels. B) Densitometric analysis of the WBs. The siRNAs used effectively reduced CAV1/clathrin protein levels, with a 40–65% reduction, depending on the ES cell line treated. UTF: untransfected; mock: transfected without siRNA. 3 independent experiments were performed and the final data presented selected as the most representative of the results obtained -panel A and as averages of all replicates-panel B.(TIF)Click here for additional data file.Figure S2Densitometric analyses of WB shown in  of effects of CAV1 or clathrin inhibition/interference on the IGF1R signaling pathway. The inhibition of phosphorylation reached levels of over 60% reduction (p<0.05). The results obtained with the A673 and A4573 cell lines were identical to those described for TC71 (data not shown). Data presented is representative of 3–6 independent experiments.(TIF)Click here for additional data file.Figure S3Effects of CAV1 or clathrin inhibition/interference on the IGF1R signaling pathway. Ewing's sarcoma TC71 cells were starved overnight, stimulated with IGF1 (50 ng/ml) for 15 minutes, and then proteins were extracted and WB was performed, as described in the Material and Methods section. A) Effects of clathrin inhibition/interference. B) Effects of Cav1 inhibition/interference. The phosphorylation of AKT and MAPK proteins in siRNA- or drug-treated cells without IGF1 treatment showed that siRNA or drug treatment alone do not interfere with protein phosphorylation. 3 independent experiments were performed and the final data presented selected as the most representative of the results obtained.(TIF)Click here for additional data file.Figure S4Effects of CAV1 or clathrin inhibition on apoptosis in the ES cell lines A673 and A4573. Ewing's sarcoma cells were grown until they reached 30–40% confluence, treated with MCD/CPMZ for 72 hours, and then apoptosis was measured by flow cytometry, as described in the (TIF)Click here for additional data file.Figure S5Effects of CAV1 or clathrin inhibition on an IGF-1-independent ES cell line. Ewing's sarcoma TTC466 cells were grown until they reached 30–40% confluence, treated with MCD/CPMZ for 72 hours, and then proliferation was measured with the MTT assay, as described in the A) Characterization of the IGF1 proliferation-driven signaling dependence of the TTC466 cell line, as already published by Martins el al. Cancer Res. 2008; 68(15):6260–70. B) CPMZ or MCD treatment. CAV1 or clathrin inhibition hardly affected ES cell line proliferation. The IC50 of proliferation was much higher than with the 3 other cell lines studied (5–10x), showing that the toxicity of CPMZ and MCD is related to IGF1R endocytosis inhibition and not due to non-specific secondary effects of these drugs. Data presented as the average of 4-6 independent replicates.(TIF)Click here for additional data file.Figure S6Effects of IGF1R tyrosine kinase inhibition combined with CAV1 and/or clathrin inhibition on the ES cell phospho-proteome. Images of the p-array membranes of all samples studied. A: AEW, C: CPMZ and M: MCD treatments; + control: IGF1 stimulation without drug treatment; - control: basal conditions, without IGF1 stimulation. Data presented is representative of 3 independent experiments.(TIF)Click here for additional data file.


Deficits in inhibitory control, the ability to suppress ongoing or planned motor or cognitive processes, contribute to many psychiatric and neurological disorders. The rehabilitation of inhibition-related disorders may therefore benefit from neuroplasticity-based training protocols aiming at normalizing inhibitory control proficiency and the underlying brain networks. Current literature on training-induced behavioral and brain plasticity in inhibitory control suggests that improvements may follow either from the development of automatic forms of inhibition or from the strengthening of top-down, controlled inhibition. Automatic inhibition develops in conditions of consistent and repeated associations between inhibition-triggering stimuli and stopping goals. Once established, the stop signals directly elicit inhibition, thereby bypassing slow, top-down executive control and accelerating stopping processes. In contrast, training regimens involving varying stimulus-response associations or frequent inhibition failures prevent the development of automatic inhibition and thus strengthen top-down inhibitory processes rather than bottom-up ones. We discuss these findings in terms of developing optimal inhibitory control training regimens for rehabilitation purposes. Inhibitory control refers to the ability to suppress ongoing or planned motor or cognitive processes and enables adapting to rapidly changing situations , the pre-supplementary motor area (preSMA), and the basal ganglia  to “Go” and “NoGo” stimuli between the beginning vs. the end of a 40 min auditory spatial Go/NoGo training and during passive listening of the same stimuli before vs. after the training session , which in turn lead to the (re)engagement of controlled forms of inhibition. During inhibitory tasks, post-error shifts to more cautious response modes translate into switches from fast automatic to slow, top-down controlled forms of inhibition  responses as in, e.g., impulsivity (Marteau et al., Empirical support for these assumptions comes from Thorell et al. , who shoSeveral other studies demonstrate that when the domain-general inhibitory control network is modified by training, the effects of the training will influence subsequent complex behavior involving the same inhibitory control component (Houben and Jansen, The reciprocal effect has also been confirmed by studies showing that practice on complex tasks involving an inhibitory control component improves performance on basic inhibitory control tasks. For instance, Di Russo et al.  showed tEvidence for training-induced improvement in inhibitory control and for the modification of the underlying fronto-basal network suggest that practicing inhibition tasks might help recovering for inhibition-related pathologies (e.g., Johnstone et al., Many questions remain open within the field of inhibitory control plasticity. For instance, the long-term effects of inhibitory control training, and the effects of long training sessions, remain unresolved. Houben et al.  showed eWe have proposed a few task parameters favoring the reinforcement of controlled rather than automatic inhibitory processes, which in turn would promote the generalization of the effect of inhibitory control training to other conditions and tasks. These proposals remain, however, mostly speculative and need further investigations. In this regard, the relevance of inhibitory control training for the rehabilitation of inhibition-related disorders also needs to be further established.Collectively, current literature on inhibitory control plasticity suggests that training inhibitory control may constitute a promising tool for the rehabilitation of many inhibition-related psychiatric and neurological disorders.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.


Aptenodytes forsteri was discovered breeding on Emperor Island , in the Dion Islands, close to the West Antarctic Peninsula (Stonehouse 1952). When discovered, the colony comprised approximately 150 breeding pairs; these numbers were maintained until 1970, after which time the colony showed a continuous decline. By 1999 there were fewer than 20 pairs, and in 2009 high-resolution aerial photography revealed no remaining trace of the colony. Here we relate the decline and loss of the Emperor Island colony to a well-documented rise in local mean annual air temperature and coincident decline in seasonal sea ice duration. The loss of this colony provides empirical support for recent studies  that have highlighted the vulnerability of emperor penguins to changes in sea ice duration and distribution. These studies suggest that continued climate change is likely to impact upon future breeding success and colony viability for this species. Furthermore, a recent circumpolar study by Fretwell & Trathan (2009) highlighted those Antarctic coastal regions where colonies appear most vulnerable to such changes. Here we examine which other colonies might be at risk, discussing various ecological factors, some previously unexplored, that may also contribute to future declines. The implications of this are important for future modelling work and for understanding which colonies actually are most vulnerable.In 1948, a small colony of emperor penguins  Emperor penguins breed in coastal locations around the Antarctic with most colonies assembling on stable fixed or ‘fast’ ice, with just three occurring on land. All colonies show a similar breeding schedule regardless of colony location. Birds gather at traditional sites in autumn, with the development of the stable fast ice, usually from April onwards. Courtship, egg laying and incubation take place as winter proceeds, while hatching, brooding and crèche formation occur as winter abates and spring and early summer approach. Chicks are tended by both parents until fledging occurs in mid-summer, usually during November or December coincident with the breakup of stable fast ice into ‘pack’ (i.e. ice floes that drift with the winds and currents); however, chicks may still also be fed while taking refuge on drifting ice floes. Adults moult in late summer, during February, again usually on fast ice or on consolidated pack. Thus, emperor penguins depend upon stable fast ice for approximately eight months of the year, so late fast ice formation in winter and/or early breakup in spring can strongly reduce the chances of successful breeding at any given colony location.Recently, changes in sea ice duration and distribution, associated with climate change, have been reported as important factors affecting emperor penguin population processes inter alia, data on prey species, predation and local and regionally scaled sea ice indices. Detailed demographic studies are currently only available from just one site Notwithstanding, conclusions about emperor penguin biology and ecology in relation to environmental change are limited by a severe lack of long-term population monitoring data and associated environmental data including, One location where emperor penguin breeding population counts have been made but never previously published is at Emperor Island in the Dion Island group, Marguerite Bay, west of the Antarctic Peninsula. This site is one of the more northerly emperor penguin breeding locations; it was first discovered in 1948 Though studies At Emperor Island, emperors used to breed on the low rock and shingle isthmus at the southeast end of the island, one of only three colonies known to breed on land. This colony therefore also offers opportunities to speculate about how a range of other environmental drivers might also have affected the population decline.In this study, we combine breeding count data from an irregularly visited emperor penguin colony with locally recorded air temperature data, locally observed fast ice duration data and remotely sensed circumpolar sea ice duration data to evaluate the impacts of changing environmental conditions on breeding population size. Information on breeding counts, local air-temperature and local fast ice conditions were obtained from unpublished Falkland Island Dependency Survey (FIDS) and British Antarctic Survey (BAS) reports archived at BAS. Circumpolar sea ice duration data were obtained from the literature Our study colony  was located on Emperor Island in the Dion Island group, in Marguerite Bay, west of the Antarctic Peninsula . EmperorThe numbers of adults, eggs and chicks at Emperor Island  have beewww.antarctica.ac.uk/met/READER/surface/Adelaide.All.temperature.html; accessed on 7 October 2010). This is the geographically closest (∼12 km) air temperature record to the Dion Islands for the 1960s and 1970s.No surface air temperature records exist for the Dion Islands . The continuous temperature record has missing values for July and August 1999, but is analysed here from April 1977 until April 2010.After the base at Adelaide Island was closed, a BAS base at Rothera Point  was opened and this now provides the geographically closest (∼40 km) air temperature record for the most recent decades  on fast ice  and it mMacronectes giganteus) were predatory on the group; their nearest known breeding site is at Avian Island, 12 km away. With warmer, earlier springs we speculate that giant petrels or other raptorial seabirds could be more active at some of the more northerly emperor colonies. Future monitoring at emperor colonies should therefore also include monitoring of nearby raptorial seabird species.Warming and reduced sea ice duration may also have led to changes in a number of important trophic interactions that might have contributed to the decline of the colony. Changes in fish, krill and squid communities on which emperor penguins feed are likely to be important drivers of population change The number of ecological processes affected by climate change is extensive, including for processes operating at different times of year and across all life history stages. In The rate of climate change in the West Antarctic Peninsula region has been extremely rapid The ability of emperors to accommodate changes in patterns of climate and environmental variation is likely to be critically important Recent studies indicate that reductions in sea ice associated with climate change will have profound consequences for emperor penguin populations Current assessments suggest that the observed di-pole in sea ice between the Antarctic Peninsula and the Ross Sea will alter as the ozone hole heals in the second half of this century Of fundamental importance to future forecasts of emperor penguin colony viability will be an increased ecological understanding of how they utilise their available habitat and therefore how environmental changes will affect both their use of habitat and their behaviour. For example, the wider regional pack ice, in terms of the level of convergence/divergence, seasonality and concentration, will impact on the well-being of non-breeding birds and breeding birds both during and outside the breeding season. Changes in pack ice extent and duration may affect predictable foraging grounds that are within foraging range of each breeding or moult location; similarly changes in polynya extent and persistence will almost certainly be an important factor. Also, knowledge about fast ice production is a potentially crucial factor for breeding and possibly moult. At present there are fundamental gaps in our knowledge about the types of sea ice features that emperor penguins rely upon. Similarly, current climate models do not represent most of these sea ice features to the extent required for future habitat modelling. Coupled atmosphere-ocean models are the foundation for most climate initiatives. However, the models have many problems in simulating sea ice extent, as small errors in the atmospheric circulation or oceanic conditions can give large errors in the sea ice extent and area In this paper we have considered the demise of the emperor penguin colony on Emperor Island in relation to climate change. However, alternative plausible explanations might be used to interpret the reduction in numbers at the colony. Such alternatives are essential to consider if we are to avoid incorrectly diagnosing threats to potentially vulnerable species, or indeed individual colonies, and if we are to understand the influence of regional change on marine ecosystems Firstly, increased snowfall consequent on increased warm, wet conditions may have contributed to population declines Secondly, short, but extreme weather events could also have led to the demise of the colony. Such extreme events have been reported for the West Antarctic Peninsula region www.ats.aq/documents/recatt/Att178_e.pdf; accessed 7 October 2010) and apart from difficulties of access during the breeding season, tourism and other visits (except for purposes of specific scientific research) are not permitted.Thirdly, as the numbers of tourists visiting Antarctica have increased, concerns have been expressed about the potential disturbance caused by visitors. Disturbance may be caused by a number of activities, including by visitors approaching too close to penguin colonies Fourthly, the demise of the emperor colony could have been because of disease. For example, disease was implicated in a die-off of Adélie penguins in the vicinity of Mawson Station  during 2001 Finally, marine harvesting has been shown to have important impacts on penguins; commercial fisheries have reduced the carrying capacity of the Benguela ecosystem for penguins to only 10–20% of what it was in the 1920s In summary, we believe that tourism (or other visits), and fishing can be discounted as causes leading to change at the emperor colony on Emperor Island. However, the other alternative hypotheses remain feasible and cannot be ruled out entirely, although on examining each hypothesis in turn we have no evidence that increased snowfall, extreme events, or disease played any part in the decline of this colony.We suggest that the Emperor Island colony might be a sentinel for those other colonies in the West Antarctic recently discovered The location of this colony suggests that ecological models, even simple conceptual models such as shown in our partial ecogram , are impOur more specific conclusions include:inter alia, penguin breeding parameters, prey species, predation and fast ice coverage. However, the available data suggest that changing sea ice conditions (plausibly both pack ice and fast ice) have had a major impact on a colony that was already vulnerable and towards, but not at, the edge of the species geographic range.Our study is severely limited by a lack of consistent long-term monitoring information documenting, The principle factors leading to the decline are now difficult to disentangle and might involve factors that operate either at the breeding colony  and/or within the seasonal foraging ambit of the population .Extreme weather events may have contributed to the colony decline, but records are inadequate to determine whether this was an important factor.The Emperor Island colony now apparently no longer exists and any future analysis will continue to be bound by existing (and sparse) data.Given their lack of agility, emperors would be limited by terrain and elevation, nevertheless, shifting their breeding habitat to low-lying accessible land is not necessarily a long term survival option for the species.


To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces. ► The CLEC-2-PDPN interaction promotes DC migration to LNs ► CLEC-2 activation induces actin-rich protrusions and motility in DCs ► CLEC-2 activation reduces actomyosin contraction and promotes actin polymerization Cell motility is crucial for trafficking of leukocytes between tissues and for their interaction with one another and their microenvironment. As the quintessential antigen-presenting cell, dendritic cells (DCs) rely on migration via stromal networks to carry antigens long distances from parenchymal tissues to lymph nodes (LNs), where they initiate adaptive immunity and tolerance . DCs genTo reach LNs, tissue-resident DCs must first crawl to and enter afferent lymphatics, which begin as blind-ended vessels within most parenchymal tissues and convey lymph toward the thoracic duct . Upon arPdpn−/− mice die shortly after birth due to severe defects in lymphatic-vessel formation leading to fatal edema  as a candidate molecule that migratory DCs encounter en route to LNs because of its high expression by both LECs and FRCs . Interesal edema . PDPN haal edema .Clec1b) is a member of the dectin-1 subfamily of C-type lectin receptors (CLRs) and is the receptor for PDPN  scaffolds and molecular cues that coordinate leukocyte migration and compartmentalization within lymphoid organs . En routPdpn mRNA and protein and the localization of PDPN on lymphatics were unchanged during inflammation , which binds specifically to CLEC-2, we confirmed surface expression of CLEC-2 on BMDCs , and peritoneal macrophages . Consistby BMDCs A. We als and LNs A. LN andon BMDCs B and LN on BMDCs C. Thus, Next, we examined whether CLEC-2 surface expression by DCs changed upon exposure to an inflammatory mediator. We found that exposure to lipopolysaccharide (LPS) caused CLEC-2 surface expression to increase ∼2-fold D. HoweveClec1b−/− mice exhibit neonatal lethality, fetal liver chimeras (FLCs) were generated from wild-type (WT) and Clec1b−/− embryos for evaluation of CLEC-2 function. Six weeks after reconstitution, CLEC-2 expression was analyzed in CD11c+ and CD11c− LN cells from WT or Clec1b−/− FLCs. As expected, Clec1b mRNA signal was detected in CD11c+ cells from WT FLCs, but not Clec1b−/− FLCs  entered the draining LNs at 24 hr; however, the percentage and total numbers of Clec1b−/− DCs were significantly reduced compared with WT DCs  skin painting to examine migration of skin-resident DCs to LNs in mice lacking CLEC-2. Because −/− FLCs A. Notablc+ cells A, suggesnditions B and 3D.nditions . Consist WT FLCs E. This dClec1b−/− BMDCs were performed. WT and Clec1b−/− DCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and far-red dyes, respectively, mixed in equal numbers (2 × 105 of each), and coinjected into the same footpad. Donor DCs arriving in the draining  and distal LNs were enumerated 24 hr later via flow cytometry. The numbers of Clec1b−/− DCs were significantly reduced compared with their WT counterparts, and WT DCs migrated 3-fold more efficiently from skin to LN . As a third approach to confirm the role of CLEC-2 in migration, DCs were treated with rPDPN-Fc protein prior to injection to occupy surface CLEC-2 -peptide-loaded DCs from WT or Clec1b−/− donor mice via footpad injection, followed by intravenously administered naive, CFSE-labeled OT-1 T cells 1 day later. OT-1 T cell proliferation was significantly impaired in mice that received Clec1b−/− DCs compared with their WT counterparts  and then cocultured with naive, CFSE-labeled OT-I T cells. As shown in Clec1b−/− DCs served as antigen-presenting cells. We then examined whether endocytic activity might explain the different abilities of WT and Clec1b−/− DCs to stimulate T cell responses in vivo and found that WT and Clec1b−/− DCs exhibited equal antigen-uptake activity  compared with their WT counterparts, despite equal starting numbers of WT and Clec1b−/− DCs. The directionality, displacement, and velocity of DCs that penetrated the tissue were determined over a 120 min period. WT DCs exhibited greater displacement  reticular structure, reminiscent of the collagen network of the LN paracortex D–4F. HigOnce the DCs adhered to and spread along the FRCs, they began migrating while maintaining close contact with the FRCs. The DCs generally migrated relatively long distances on the FRC network: between 50 and 300 μm in any one direction over 60 min  and S5. Ccr7−/− DCs migrated as efficiently as WT DCs along the scaffold . Furthermore, consistent with a requirement for CLEC-2 activation by PDPN in this niche, we found that DC migration was severely diminished in networks formed by Pdpn−/− FRCs compared with WT FRCs  culture. A375 cells expressing GFP-CLEC-2 spread rapidly upon exposure to rhodocytin and within 5 min had spread to cover nearly twice the surface area of neighboring untransfected cells  phosphorylation and increase actomyosin contractility through its interaction with Rho kinases . We wonded cells E and 7G,ed cells . The phoed cells . Thus, wed cells H and 7I.ed cells I. CollecClec1b−/− DCs completed the passage from skin to LN, as measured by flow cytometry. This could suggest a preferentially important role for CLEC-2 in a DC subset that efficiently cross-presents antigens such as the CD103+ dermal DC. Alternatively, this could suggest that CLEC-2 deficiency not only reduces DC migration but also prevents DCs from properly interacting with the FRC network and T cells. Consistent with this latter possibility, we found that Clec1b−/− DCs were severely impaired in spreading and crawling on the FRC network in 3D.Here we describe a role for CLEC-2 signaling in DC motility. The CLEC-2 ligand PDPN is highly expressed by LECs and FRCs lining structures that DCs encounter during migration from tissues to LNs. CLEC-2 is expressed by skin and LN DCs, but surface levels of this protein appear to be tightly regulated. Engagement of CLEC-2 by PDPN coordinately reduces actomyosin contractility and promotes actin polymerization in DCs, thereby allowing them to spread along stromal cell scaffolds, extend protrusions, and migrate. The CLEC-2-PDPN interaction promotes DC migration to LNs at all stages of this journey: from leaving peripheral tissues and entering lymphatic vessels, to crossing the subcapsular sinus, and finally to migrating through LN parenchyma to the T cell zone. T cell activation in LNs was markedly reduced despite the fact that some Analysis of the downstream signaling components revealed that CLEC-2 activation in DCs, as in platelets, required the adaptor protein and exchange factor Vav and led to alterations of actin dynamics via activation of Rac1. Our study shows that CLEC-2 activation by PDPN induces F-actin-rich protrusions conducive to cell migration. Additionally, we find that RhoA activity is coordinately decreased upon CLEC-2 activation. Rac and Rho have been shown to be antagonistic in a variety of contexts , and thiClec1b+ DCs and Pdpn+ stromal cells, we propose the following model. Initially, CLEC-2 and PDPN interact and cluster on the respective cell membranes. CLEC-2 oligomerization then induces tyrosine-phosphorylation events via Syk, PLCγ, and Vav. Vav promotes the activity of Rac1, which indirectly and coordinately reduces RhoA activity. These events are spatially restricted to domains on the DC surface that are in direct contact with PDPN. Therefore, specifically at the contacts between DCs and FRCs, the activity of RhoA effectors, such as ROCK (Rho-associated protein kinase), are reduced, leading to reduced phosphorylation of MLC  for 5–6 days. Unstimulated BMDCs were used in functional experiments unless otherwise specified. For experiments in which matured BMDCs were used, the cultures were treated with 10 ng/ml LPS for 24 hr.LN FRCs were isolated and expanded as previously described . For BMD2 in minimum essential medium alpha containing 10% fetal calf serum (FCS) during imaging. For experiments involving CLEC-2 activation in 3D culture, CLEC-2 ligands  were added to the gel as all components were mixed.DCs were seeded either alone or at a 5:1 ratio with FRCs into a collagen/matrigel matrix   for 2 hr at 37°C with 5% CO2. Noninfiltrating DCs were then gently washed away, and ears were fixed in 1% paraformaldehyde (PFA) for 4 hr. Ear sheets were imaged with an inverted Zeiss 510 confocal microscope.DC entry to lymphatic vessels was carried out as previously described . In brieClec1b−/− BMDCs were labeled with cell-tracking dyes, washed with PBS, then incubated with the LN slices for 90 min at 37°C. After this time, LN slices were gently washed with RPMI containing 10% FCS, laid into MatTek dishes, and imaged with an inverted Zeiss 510 confocal microscope for 2 hr at 37°C with 5% CO2. Z stacks of 30 μm were projected with ImageJ Z Project. Time-lapse sequences were analyzed with the ImageJ manual-tracking plugin to measure cell velocity and to track individual cells.For DC migration in LNs, LNs were dissected from Ubiquitin-GFP chimeric mice (WT BM > Ub-GFP host) and embedded in 4% low-melting-temperature agarose at 36°C. Blocks were then cooled on ice. Vibratome sections of LNs 300 μm thick were cut and incubated with RPMI containing 10% FCS for 30 min at 37°C. WT and Statistical tests in this paper were performed with Prism software (GraphPad). Data sets were first subjected to an F test to establish whether data to be compared exhibited comparable SD. Following this, the appropriate tests were performed as indicated in each figure legend. In general, comparison of multiple groups was performed with the use of a Kruskal-Wallis ANOVA and, if significant, followed by Dunn’s post test between individual data sets. Comparisons of two data sets were most commonly performed with the use of a Mann-Whitney U test.


Binding of Syk to phosphorylated immunoreceptor tyrosine-based activation motifs (p-ITAM) of the receptor subunits results in conformational changes and tyrosine phosphorylation at multiple sites that leads to activation of Syk. The phosphorylated tyrosines throughout the molecule play an important role in the regulation of Syk-mediated signaling. Reconstitution of receptor-mediated signaling in Syk−/− cells by wild-type Syk or mutants which have substitution of these tyrosines with phenylalanine together with in vitro assays has been useful strategies to understand the regulation and function of Syk.The protein tyrosine kinase Syk plays a critical role in Fc PLCγ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane to produce diacylglycerol, which activates protein kinase C (PKC), and inositol 1,4,5-triphosphate (IP3) [3 to its receptor in the endoplasmic reticulum (ER) membrane rapidly induces the first stage of calcium (Ca2+) mobilization, which is the transient release of Ca2+ from ER stores. The depletion of calcium in the ER induces formation of multimers of Stim1in the ER membrane, which move and interact directly with Orai1, a pore subunit of the Ca2+ channel, in the plasma membrane. Together with calcium released from ER, this induces prolonged Ca2+ influx through store-operated calcium release-activated calcium (CRAC) channels in the plasma membrane [γ is essential to generate signals for sustained Ca2+ influx. All these events culminate with mast cell degranulation, phosphorylation of MAP kinases, and activation of nuclear factor for T-cell activation (NFAT) and nuclear factor κB (NFκB), which turn on cytokine synthesis.Tyrosine-phosphorylated Syk leads to downstream propagation of signaling with phosphorylation of linker for activation of T cells (LAT), Vav, BTK, phospholipase CSLP-76) , 13. Pho , 13. Phte (IP3) . The binmembrane , 16. SykSyk gene was first cloned on the basis of partial sequenced information of the 40 kDa kinase from a porcine spleen. The product of this gene was described as a member of a new nonreceptor-type protein tyrosine kinase of 72 kDa (p72syk). Interestingly, unlike src family protein kinases, p72syk has a second SH2 instead of an SH3 domain [ζ chain of the T-cell receptor, termed ZAP-70 [syk. Those findings indicated that Syk and ZAP-70 belonged to a distinct subfamily of nonreceptor protein tyrosine kinases [2-terminal SH2 (SH2-N) and COOH-terminal SH2 (SH2-C), while interdomain B or linker region is between the SH2-C and the kinase domains; following the kinase domain there is a short COOH-terminal extension (tail) [3 domain . Moleculd ZAP-70 . This al kinases , named S kinases , 20. Strn (tail)  2-L/I-(X)6/7-Y-(X)2-L/I, where the two tyrosines residues are phosphorylated upon receptor activation. The β- and γ-subunits of FcεRI complex contain ITAM on their cytoplasmic tails. Phosphorylated ITAM (p-ITAM) serves as the docking site for the two SH2 domains of Syk [2-terminal phosphotyrosine [In vitro experiments using synthetic peptides corresponding to the β- or γ-ITAM of FcεRI showed that Syk binds preferentially to the biphosphorylated γ-ITAM [γ-ITAM than the SH2-N. The binding of Syk to the p-ITAM results in conformational changes of Syk exposing its COOH-terminal region [ITAM-based signaling has been extensively characterized and serves as a model for receptor-mediated signal transduction , 9. The s of Syk –34. The tyrosine , 32 ; the last two of which are also conserved in human ZAP-70 [mulation , 62, 63.εRI aggregation, and this phosphorylation is independent of Syk. These phosphatases are found associated with FcεRI in mast cells [In vitro experiments show that both SHP1 and SHP2 dephosphorylate β- and γ-subunits of the FcεRI [−/− mice are hyperresponsive to TCR stimulation as a result of increased activation of ZAP-70 [The regulation of Syk function also includes the downregulation of its activity after receptor activation; this process involves the participation of protein phosphatases and ubiquitin ligases. Cbl-b-mediated Syk ubiquitination and degradation has been the most characterized mechanism to downregulate Syk function in receptor signaling (previously discussed). The phosphatases SHIP1, SHP1, and SHP2 are tyrosine phosphorylated after Fcst cells , 65. In he FcεRI . TULA-2,he FcεRI , 68. It he FcεRI . Additiohe FcεRI , 69, 70.f ZAP-70 . These fεRI-mediated signaling. Morphological studies show that resting RBL-2H3 cells are normally spindle shaped with small microvilli on their surface; these cells become flattened and spread and their surfaces are ruffled and plicated after receptor activation [2+ dependent [εRI induces minimal or no morphological change in RBL-2H3 Syk−/− variant mast cell line; but this is reconstituted by stable expression of Syk in these cells. Additionally, receptor-mediated redistribution of actin is observed only in cells expressing Syk after transient transfection of Syk−/− cells (The RBL-2H3, a mast cell line, has been widely used as a model to study Fctivation . These mependent . For exa/− cells . Therefoα is phosphorylated upon BCR activation which inhibits signaling [α, indicating a dual-specificity kinase activity for Syk.Receptor-induced Syk tyrosine phosphorylation has been well characterized in hematopoietic cells , 38, 73.ignaling . Surpris2- or COOH-terminal seems to be more active than wild-type Syk [Studies have shown that Syk is a key player in mast cells and other hematopoietic cells. Several studies have also suggested the mechanisms that regulate Syk function based on its structure and phosphorylated tyrosines. Mutating Syk has been the strategy most frequently used to understand its function but, surprisingly, this could affect the autoinhibitory structure of the molecule resulting in secondary effects. For example, mutations of the three conserved tyrosines in the COOH-terminal region of Syk decreases its binding capacity to the p-ITAM due to decreased autoinhibitory structure of mutated Syk and not because of the mutation itself . Also, Stype Syk , 63. Thi


In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation  was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies.The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable  Drosophila . Characterization of this in vivo TMHT mouse model revealed increasing soluble, but not insoluble total tau and ptau (Thr231) levels over age and increased human ptau at residues Thr181, Ser199, Thr231, and Thr235. Furthermore, the TMHT mouse model showed a progressive increase in human tau protein in the amygdala over age and strong spatial learning deficits as early as 5 months of age as well as olfactoric deficits.The microtubule-associated protein tau is an abundantly represented protein in the central and peripheral nervous system, especially concentrated in axons of neurons Goedert . The mosin vitro system comprising a novel stable cell line and phosphorylation site assays that is applicable for medium throughput screening of early drug candidates modulating tau phosphorylation, selected for pre-clinical development. The combination of stable transgenic cell lines for tau with bespoke mass spectrometry assays opens a new window of opportunity to successfully combat tauopathies.In summary, we have established a new l-glutamine (Lonza), non-essential amino acids  (HyClone), and 10 mg/ml gentamycin (Invitrogen) at 37°C and 5% CO2 in a humidified atmosphere.SH-SY5Y cells, a clone of the human neuroblastoma cell line SK-N-SH, were purchased from LGC Standards. Cells were cultured in DMEM (Lonza) supplemented with 10% fetal calf serum (Lonza), 200 mM 5 per well. Upon 24 h, cells were transfected with FugeneHD transfection reagent (Roche) according to the manufacturer’s protocol. After 24 h incubation, the culture medium was changed. Following another 24 h, cells were transferred to 10 cm dishes in selective medium containing 300 μg/ml gentamycin-disulfate G418 (Roth) and 1 × 106 untransfected SH-SY5Y cells as feeder cells that died after a few days on the selective medium. After 3 weeks of G418 selection, single clones were isolated and cultured in 96-well plates. Only one clone showed TMHT441 expression. Stable TMHT transfected SH-SY5Y cells were named SH-SY5Y-TMHT441 cells.For stable transfection of SH-SY5Y cells, a pcDNA3-TMHT441 (V337M/R406W) construct was used. The TMHT441 gene contains the longest isoform of human tau regulated by a CMV promoter and carries two well-characterized mutations known to promote tau aggregation and/or hyperphosphorylation. The construct also contains an ampicillin and neomycin resistance gene. Before stable transfection, cells were seeded in six-well plates at a density of 5 × 10d-Lysine-coated chamber slides. After 2 days in culture, cells were washed, fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.2% Triton/PBS for 30 min, blocked with 20% horse serum/0.2% BSA/0.1% Triton/PBS for 1 h, and labeled with primary antibody HT7 (Pierce Endogen) in 0.2% BSA/0.05% Triton/PBS for 1 h followed by blocking with 20% horse serum/0.2% BSA/0.1% Triton/PBS for 20 min. Cells were then labeled with secondary fluorescent antibody Cy3 (Jackson ImmunoResearch) in 0.2% BSA/0.05% Triton/PBS for 1 h. DAPI solution (25 mg/ml in A. bidest.) was added 1:2,000 to the secondary antibody solution. After primary and secondary antibody incubations, cells were washed three times with 0.05% Triton/PBS. Samples were mounted with Mowiol and analyzed by fluorescent microscopy.For immunofluorescent labeling, SH-SY5Y-TMHT441 cells were seeded on Poly-For all phosphorylation-related experiments, differentiated SH-SY5Y-TMHT441 cells were used. Therefore, cells were treated with 10 μM retinoic acid for 7 days. Differentiated cells were treated with 0.1–50 μM kinase inhibitors SP600125 (Sigma), AR-A014418 (Sigma), RP106 , IC261 , or 0.2% DMSO as vehicle for 4 h. Each kinase inhibitor was tested in two autonomous experiments, and in each case cell viability was determined using a standard MTT assay (Promega).After treatment, cells were washed twice with cold PBS and harvested for Western blotting, Mesoscale Discovery, and mass spectrometry analyses. For all measurements, cells were harvested at a confluency of 90% in RIPA buffer, containing 1× protease inhibitor cocktail  as well as 1× phosphatase inhibitor cocktail 3 (Sigma) and sonicated on ice. Total protein amount of lysates was determined with a BCA Protein Assay Kit (Pierce Endogen). For Western blotting, the following antibodies were used: Tau-5, β-actin (Abcam); AD2 (BioRad), AT270, AT180, AT8 (Thermo Scientific).Total tau and phosphorylated tau at residues Thr181, Thr231, and Ser396 of vehicle only and inhibitor treated SH-SY5Y-TMHT441 cells were analyzed by Multi Array® Phospho-Tau (Thr 231)/Total Tau immunosorbent Assay and two prototype assays, Phospho-Tau (Thr181) and Phospho-Tau (Ser396), .To increase the range of individual sites covered by precise quantitative assays, we adopted two SRM-based assays   transgenic mice over-expressing the longest human tau isoform tau441 (2N4R) carrying two mutations, V337M and R406W, under control of the neuron specific murine Thy-1 (mThy-1) promoter was used as positive control .g and the supernatants were used for the analysis of tau. Immunoblots were performed with cortical and hippocampal protein lysates of 12-month-old hemizygous TMHT transgenic and corresponding non-transgenic brain tissue. For a detailed characterization of the TMHT transgenic mouse model, see Flunkert et al. (unpublished data).Protein was extracted from the hippocampus and cortex as described previously  was too small.All data received from immunosorbent assay (MSD) were analyzed using unpaired in vitro AD model by stably transfecting SH-SY5Y cells with the TMHT441 construct which has already been used successfully for the generation of an in vivo model . To allow pre-screening of drug candidates and prioritization for transfer into advanced pre-clinical development, we established an del Fig. , later rTo determine the expression levels of the TMHT construct, SH-SY5Y-TMHT441 cells were labeled with a human specific anti-tau antibody (HT7) by immunofluorescence staining. All SH-SY5Y-TMHT441 cells strongly over-express the TMHT construct compared to non-transfected control SH-SY5Y cells Fig.  vs. 1d, Next, we analyzed whether different kinases, including JNK, GSK3-β, Cdk1/5, and CK1 kinase, which are known to contribute to tau phosphorylation, are also involved in tau phosphorylation of SH-SY5Y-TMHT441 cells. In all experiments, two negative controls were used, untreated and vehicle treated SH-SY5Y-TMHT441 cells, and all presented data were normalized to the corresponding vehicle control. Additionally, normalization of data to cell toxicity did not change the outcome of the assay (data not shown).50 values for SP600125 of 100 nM and 91 nM for phosphorylation at Thr181 and Thr231, respectively. Since the applied SP600125 concentrations were in the upper range of the sigmoidal dose response curve (data not shown), the calculated IC50 values should be seen as estimates only. Tau phosphorylation at residue Ser396 was almost reduced to half as analyzed by Phospho-Tau SRM when using 50 μM SP600125 and at lower concentrations of 1 μM SP600125 decreased phosphorylation to 62% as measured by Mesoscale Discovery . Only lower concentrations of AR-A014418 caused a decrease of Ser396 phosphorylation. When using 10 μM of inhibitor, AR-A014418 phosphorylation at Ser396 was reduced to 71% compared to control samples , the calculated IC50 value should be seen as estimate only. The CK1 kinase inhibitor consequently caused inhibition of the same phosphorylation sites as the GSK3-β kinase inhibitor.Treatment of differentiated SH-SY5Y-TMHT441 cells with increasing concentrations of the Cdk1/5 kinase inhibitor RP106 decreased phosphorylation of tau at residues Thr181 and Ser396 to 74% and 68% at 0.1 μM RP106, respectively, but had no effect on phosphorylation at residue Thr231 as measured by Mesoscale Discovery Fig. . Due to In summary, our data demonstrate that SH-SY5Y-TMHT441 cells express high levels of human transgenic total tau and phosphorylated tau at residues Thr181, Ser202, Thr231/235, and 396/404. Tau phosphorylation was modifiable by four different kinase inhibitors, suggesting that tau phosphorylation of SH-SY5Y-TMHT441 cells is dependent on the JNK, GSK3-β, Cdk1/5, and CK1 kinase pathway. All these pathways were previously shown to be involved in tau phosphorylation . Measurement of total and ptau Thr231 by Mesoscale Discovery confirmed Western blotting results.Using immunocytological and biochemical approaches, we first verified if the SH-SY5Y-TMHT441 cells over-express tau protein. Furthermore, we analyzed tau phosphorylation at verified disease relevant residues, including Ser181, Ser199, Thr231, and Ser396 . This discrepancy might depend on the use of different site-specific antibodies or TMHT mutation-specific conformational changes of tau. The CDK1/5 protein kinase is also known to be involved in microtubule assembly and disassembly  caused decreased phosphorylation at residues Thr181 and Ser396 but had no effect on Thr231 in SH-SY5Y-TMHT441 cells. Previous studies observed phosphorylation of all three residues by CDK1/5, but we were unable to see this reproducibly in the SH-SY5Y-TMHT441 cell line using Mesoscale Discovery assays  involved in tau phosphorylation events in vivo and, therefore, represent a suitable cell culture system for the fast screen of novel phosphorylation modifying compounds directed against tau hyperphosphorylation, building the foundation for follow-up in vivo proof-of-concept studies in TMHT mice that express virtually the same gene construct.By using the specific kinase inhibitors SP600125, AR-A014418, RP106, and IC261 (Bennett et al. in vitro and in vivo examinations of drug efficacy.Combined analyses of new tau-phosphorylation modifying drugs in the SH-SY5Y-TMHT441 cell line and the TMHT mouse model will enable fast and reliable


Understanding the mechanisms by which signal transduction pathways mediate changes in RNA abundance requires the examination of the fate of RNA from its transcription to its degradation. Evidence suggests that RNA abundance is partly regulated by post-transcriptional mechanisms affecting RNA decay and this in turn is modulated by some of the same signaling pathways that control transcription. Furthermore, the translation of mRNA is a key regulatory step that is influenced by signal transduction. These processes are regulated, in part, by RNA-binding proteins (RBPs) which bind to sequence-specific RNA elements. The function of RBPs is controlled and co-ordinated by phosphorylation. Based on the current literature we hypothesize that RBPs may be a point of convergence for the activity of different kinases such as phosphoinositide-3-kinase and mitogen-activated protein kinase which regulate RBP localization and function. The response of cells to environmental stimuli frequently involves changes in gene expression. This may be controlled at multiple levels including the production of new RNA by transcription. Post-transcriptional regulation at the RNA level includes nuclear RNA processing , as well as RNA export, decay, localization, and translation. These processes are integrated with changes in protein stability and function. Signaling pathways are a major mechanism for co-ordination of these distinct mechanisms .Substantial evidence obtained over two decades has highlighted the importance of mRNA stability in gene regulation . The halThe difference in mRNA half-lives can lead to significant changes in the abundance of mRNA . This wacis-acting) and trans-acting factors present in the nucleus and cytoplasm , mTOR , and p38 MAPK. Experimentally determined phosphorylation sites on the RBPs we will focus on are depicted in Figure 1. A more extensive list derived from phospho-proteomics studies is given in Table 2.Diverse stimuli  phosphorylates PKB at hydrophobic motif  and phosphoinositide-dependent kinase 1 (PDK1). PKB is then activated following phosphorylation by PDK1 at Thr Ser473; . PKB, wh Ser473; . Genetic Ser473; . PI3K al60 and Ser113 in human TTP are predicted to be PKB phosphorylation sites  phosphorylation site and the function of this phosphorylation is presently unknown.In a phospho-proteomic study the Blenis Group reported SerTable 2; . The sam202 as a phosphorylation site for mTOR and this finding was substantiated using the mTOR inhibitors rapamycin and KU-0063794 in MEFs  phenylhydrazone, an uncoupler of mitochondrial oxidative phosphorylation. The interaction between TTP and Protor2 was suggested to be necessary for enhancing TTP-mediated turnover of mRNAs such as IL-3, GM-CSF, COX-2, and TNF . The siRMitogen-activated protein kinases are major regulatory hubs where inflammation and stress responses are regulated. Three major MAPK pathways are p38, JNK, and ERK. We discuss below the findings implicating p38 and its substrate MAPK activated protein kinase 2 (MK2) in regulating RBP activity.52 and Ser178 which stabilizes TTP protein .In NIH 3T3 fibroblasts, the stability of TPA induced IL-3 mRNA is regulated by the p38 and PI3K pathways . The latin vitro  studies 92 and Ser203) and inhibit its mRNA decay activity  has been shown to regulate the function of the protein. Similarly, p38-mediated phosphorylation of HuR at Thr118 targets a site previously shown to be targeted by Chk2 .HuR provides a further example of a point of convergence. In this case the circumstances under which the mTOR would affect HuR have not been studied but the phosphorylation site  is an RBP reported to regulate alternative splicing of mTOR . In Sam6Gene expression is controlled at the post-transcriptional level by RBPs. However, regulation of the effector function of RBPs on RNA decay and translation is controlled by signals from protein kinases. These effects include inhibition of function by uncoupling from the RNA decay machinery and in some instances reassignment of function. Kinase-dependent relocation of RBP into different compartments of the cell seems to be a common theme amongst structurally diverse RBPs. RBPs represent a class of proteins upon which signaling by the PI3K and p38 pathways converge. Existing literature on this might indicate a potential redundancy of kinases phosphorylating the same serine or threonine amino acid in different cellular functions. A fuller understanding of the interplay between kinases, RBPs and target RNAs may provide important new insights into the dynamic regulation of gene expression.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.