We have used cellular reconstitution to systematically study multisite phosphorylation of the TCR ζ-chain. We find that multisite phosphorylation proceeds by a nonsequential random mechanism, and find no evidence that multiple ITAMs modulate a switchlike response but do find that they alter receptor potency and maximum phosphorylation. Modulation of receptor potency can be explained by a reduction in molecular entropy of the disordered ζ-chain upon phosphorylation. We further find that the tyrosine kinase ZAP-70 increases receptor potency but does not modulate the switchlike response. In contrast to other multisite proteins, where phosphorylations act in strong concert to modulate protein function, we suggest that the multiple ITAMs on the TCR function mainly to amplify subsequent signaling.Multisite phosphorylation is ubiquitous in cellular signaling and is thought to provide signaling proteins with additional regulatory mechanisms. Indeed, mathematical models have revealed a large number of mechanisms by which multisite phosphorylation can produce switchlike responses. The T cell antigen receptor (TCR) is a multisubunit receptor on the surface of T cells that is a prototypical multisite substrate as it contains 20 sites that are distributed on 10 conserved immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR  Protein phosphorylation is a ubiquitous mechanism of signal transduction that is regulated by the opposing actions of kinases and phosphatases , 2. FailA well-known regulatory mechanism is a switchlike response, whereby the phosphorylation state of a protein can be highly sensitive to the concentrations of its modifying kinase and phosphatase. A large body of work has focused on understanding whether multiple phosphorylation sites on a single substrate can confer switchlike responses. Mathematical models have shown that switchlike responses can be produced by a number of mechanisms that rely on multiple phosphorylation sites, such as substrate sequestration , nonesseThe T cell antigen receptor (TCR) on the surface of T cells is a prototypical multisite substrate . T cells6–9YXX[L/I], where X is any amino acid . These sites are phosphorylated by the membrane-anchored tyrosine kinase Lck and Fyn and dephosphorylated by the transmembrane phosphatases CD148 and CD45. When both tyrosines in an ITAM are phosphorylated they generate docking sites for the tandem SH2 domains of the cytosolic tyrosine kinase ZAP-70. Bound ZAP-70 can phosphorylate tyrosines on other substrates that initiate the signal transduction that may lead to T cell activation  with consensus sequence YXX[L/I]Xtivation .Given that both kinases and phosphatases are constitutively active in resting T cells , 21, it ζ-chain. The ζ-chain has been shown to be sequentially phosphorylated . For each ITAM mutant, both ITAM tyrosines were mutated to phenylalanine. Lck was cloned into the mammalian expression vector pEF3 as a constitutively active Y505F-variant (∗) containing the SH2-binding mutations, R41K and R190K, as well as the catalytically dead mutation, K368A, was generated by QuikChange PCR (Agilent Technologies).All sequences used in this study were of murine origin unless otherwise stated. TCR i et al. . Briefly-variant . An SH2--variant , and clo2 in DMEM containing 10% fetal bovine serum. A day before transfection, cells were seeded to reach 70% confluence, and a single 175 cm2 flask was transiently transfected for each experimental condition. For experiments including only Lck, CD148, and TCR ζ-chain variants, 8 μg of each plasmid  was complexed with 24 μg of branched-chain PEI  in 0.9 mL serum-free medium for 10 min, before dropwise addition to cells in serum-free medium. An additional 25 μg of ZAP-70 DNA and 25 μg of PEI was included in transfection mixtures in experiments containing ZAP70. Cells were incubated at 37°C for 4 h before transfection medium was replaced with complete medium supplemented with fetal bovine serum. Cells were harvested after 24 h by gentle shaking, centrifuged, and resuspended in fresh serum-free medium at an appropriate density for assay.HEK 293T cells were cultured at 37°C and in a 10% CO2O2 at room temperature for 10 min. H2O2 was inactivated by the addition of 20 μg/mL catalase at room temperature for 10 min. A 24-step, 1.25-fold serial dilution of pervanadate was made up in serum-free medium starting at 1 mM. Harvested transfectants were plated at 300,000 cells per well in 96-well assay plates in a volume of 60 μL/well and treated with 60 μL/well pervanadate serial dilutions for 30 min at 37°C in a tissue culture incubator. Reactions were terminated and cells were lysed by adding 120 μL/well lysis buffer containing 2% Nonidet P-40 substitute , Mammalian Protease Inhibitor Cocktail (Sigma-Aldrich), and 250 μM sodium orthovanadate in TBS . Cellular debris was removed by centrifugation and lysates were transferred to wells of previously prepared immunosorbent capture plates.Basal kinase/phosphatase balance was disrupted by inhibition of phosphatase activity with a serial dilution of pervanadate. Pervanadate was generated immediately before the assay by incubating 10 mM sodium orthovanadate with 0.7% Hζ-chain phosphorylation was quantified in immunosorbent assay by capturing chimeric TCR ζ-chain with anti-rat CD2 antibody and detecting phosphorylation with anti-phosphotyrosine antibody. A day before assay, MaxiSorp flat-bottom 96-well plates  were coated overnight at 4°C with 100 μL/well anti-rat CD2 antibody (AbD Serotec) at 0.7 μg/mL in carbonate-bicarbonate buffer, pH 9.6. On the day of the assay, capture antibody was decanted and wells were blocked with 200 μL 1% bovine serum albumin in TBS for 1 h at room temperature. Plates were washed three times with TBS-T buffer (TBS containing 0.05% TWEEN-20). Cleared saturating amounts of lysate was transferred to plates and TCR ζ-chain captured for 2 h at 4°C. Lysate was decanted and plates were washed 6 times with TBS-T. Phosphorylation was probed by addition of 100 μL/well of biotinylated anti-phosphotyrosine antibody  at 1 μg/mL, and plates were incubated for 1 h at room temperature. Plates were subsequently washed 3 times with TBS-T. Biotinylated antibody was probed with 100 μL/well of 0.1 μg/mL IRDye 800CW-streptavidin  for 1 h at room temperature. Plates were washed three times with TBS-T, dried, and fluorescence-detected by scanning plates in an Odyssey Sa Infrared Imaging System (LI-COR).TCR 6 cells for 15 min on ice in 100 μL 1% PBS-A . Surface antigens were stained by incubating for 1 h on ice with 1 μg/ml FITC-conjugated anti-FLAG antibody (Sigma-Aldrich) and 1 μg/mL phycoerythrin-conjugated anti-rat CD2  in 100 μL PBS-A. Cells were washed three times in PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were washed three times in PBS and permeabilized in 0.1% saponin (Sigma-Aldrich) in PBS-A. Intracellular antigens were stained by incubating for 1 h at room temperature with 1 μg/mL Alexa Fluor 647-conjugated anti-mouse Lck  and 1 μg/mL Pacific Blue-conjugated anti-mouse ZAP-70  in 100 μL PBS-A. Cells were washed three times with PBS and analyzed using a FACScan flow cytometer .Transfectants were blocked by incubating 1 × 10Emin and Emax, which correspond to the minimum and maximum values, respectively, log10(EC50), which is the logarithmic value of the kinase/phosphatase that yields half of the maximal response (otherwise known as potency), and n, which is the Hill number that determines the degree of the switchlike response .The steady-state phosphorylation profiles were fit to a logarithmic Hill function in Prism , that produces estimates of ζ 123) as our standard curve so that experimental data can be averaged across different days. This normalization was performed by (1) dividing the response for each module by the value of Emax for the index module and (2) by subtracting the pervanadate concentration series on a log-scale (x axis) by the value of EC50 for the index module. Fitted Hill numbers were normalized to the value of the index module.We used an index module (wild-type t-test in Prism (GraphPad Software). Given that the Hill number and log10(EC50) of the index module was 1 and 0, respectively, we used a t-test to determine whether the corresponding values for the group of single ITAM mutants  or the double ITAM mutants  differed from these values. An unpaired t-test was used to compare between the group of single and double ITAM mutants.All statistical analyses were carried out on normalized averaged quantities using a ζ-chain, we reconstituted four components in various combinations (A). The wild-type substrate was a chimera consisting of the ζ-chain transmembrane and cytosolic domains but with the extracellular domain of rat CD2 (previously used as a simplified receptor (To study the effects of multiple phosphorylation sites on the T cell receptor inations A. The wreceptor ). The wireceptor . Lastly,B), which comprise a nonhematopoietic human cell line that is not expected to express any of the endogenous T cell receptor proximal signaling proteins . Moreover, component expression in the positive population was correlated . We found limited phosphorylation without pervanadate treatment and moreover, treatment with the Lck inhibitor PP2 did not lead to detectable reduction in phosphorylation (not shown), suggesting that CD148 dominates over Lck basally. Given that HEK293 cells are expected to have endogenous tyrosine kinases and phosphatases, we determined their contribution by transfecting in the substrate with only Lck or only CD148. Although we observe dephosphorylation in the absence of exogenous CD148 or phosphorylation in the absence of exogenous Lck, a large range exists where phosphorylation is regulated primarily by exogenous Lck and CD148 . Moreover, a timecourse revealed that by ∼15–20 min, the system has come to steady state (D).To determine the total phosphorylation profile of the antibody C. We fody state D.ζ-chain ITAMs is modified. We reconstituted Lck, CD148, and either the wild-type TCR ζ-chain, ζ-chain with one ITAM mutated , or ζ-chain with two ITAMs mutated , where X indicates that the two ITAM tyrosines have been mutated to phenylalanine (A).To determine the effects of multiple phosphorylation sites, we reconstituted signaling modules where the number of lalanine A.ζ-chain phosphorylation follows an unstructured random modification mechanism.As expected, a reduced ITAM number led to a reduced maximum phosphorylation by ∼1/3 per mutated ITAM. The observation that the maximum phosphorylation depends on the number of ITAMs, but not their identity, suggests that a strict sequential mechanism of phosphorylation is unlikely to be operating. Rather, it is more likely that EC50 (potency) and the Hill number (switchlike response). We found no significant difference in the Hill number between the wild-type ζ-chain, which as a dimer contains 12 phosphorylation sites, and ζ-chains with ITAM mutations (B). This observation is also consistent with a random mechanism of phosphorylation because, as discussed in the introduction, a sequential mechanism predicts that the switchlike response will be modulated by the number of sites  by reporting normalized parameter values as the number of phosphorylation sites is modified see .C). This effect is not predicted by the standard models of multisite phosphorylation where potency often remains unchanged as the number of sites changes  is similar, implying that both enzymes are equally efficient at modifying each of the three ITAMs. We conclude that it is the juxtaposition of multiple phosphorylation sites (or ITAMs) in the wild-type ζ-chain configuration that elicits a cooperative effect that increases potency.We observed a positive correlation between receptor potency and the number of sites C. This  changes . A simplD and To rule out that the differences in the phosphorylation profiles are a result of differences in molecular expression, we performed flow cytometry for every reconstituted signaling module. We found that the percent of cells positive for the three components and the expression levels of the three components in the seven reconstituted modules were similar D and S4ζ-chain, we wondered whether this domain could explain the observed changes in potency. This recruitment could increase the local concentration of Lck in the vicinity of the ζ-chain leading to enhanced efficiency of phosphorylation, which would be greatest for wild-type ζ that contains the largest number of sites. We introduced a point mutation to the Lck SH2 domain that is known to abolish binding (Lck-R154K) and performed the reconstitution experiments on all seven ζ-chain mutants. We found that the observed changes in potency persisted, suggesting that the SH2 domain of Lck is not responsible for increased potency  (ZAP-70∗). These two domains are known to favor phosphorylation; the SH2 domains can bind with high affinity to phosphorylated ITAMs protecting them from dephosphorylation and the catalytic domain may be important to directly phosphorylate ITAM tyrosines. We compare ZAP-70 with ZAP-70∗ to control for the total protein load imposed on the HEK293 cells by reconstitution.We first compared the effects of ZAP-70 on the wild-type ∗ revealed the expected increase in potency but no change to the switchlike response was observed (A–C), which is consistent with a recent report from Hui and Vale  and observed, as before, no trend in the Hill number but a significant increase in potency with the number of phosphorylation sites. Importantly, we did not observe any differences in potency within the ζ-chain group that contained two ITAMs or within the ζ-chain group that contained one ITAM, suggesting that ZAP-70 binds all ζ-chain ITAMs in vivo with similar affinities. As before, we examined the expression of all components to ensure that any differences in the phosphorylation profiles were not a result of differences in molecular expression  and three double (ζXX) ITAM mutant ζ-chains exhibited similar profiles indicates that Lck and CD148 modify each ITAM with similar efficiencies ruling out a trivial explanation for the enhanced potency. We therefore hypothesized that the number of phosphorylated ITAMs, independent of their specific identity, determines the rate of enzymatic modification.We have found a correlation between the number of phosphorylation sites or ITAMs with receptor potency that cannot be explained by standard multisite phosphorylation models see . Moreoveζ-chain dimer (A). Because we do not have data relating to the effect of individual tyrosines in an ITAM we have coarse-grained the two tyrosines of each ITAM into an effective single site. We model a phosphorylation-dependent enhancement (or priming) of enzymatic efficiencies by enhancing kon for both enzymes with increasing levels of ζ-chain phosphorylation. To do so, we introduce a new parameter, λ, which is the fold increase in the on-rate per ITAM phosphorylation so that after all ITAMs are phosphorylated the on-rate is increased by λmax = λ6. Because all calculations are in the steady state, we found similar results if enhancement is applied to koff or kcat (not shown).We formulated a mathematical model that includes the six ITAMs on the in dimer A. Becauλ = 3 (or λmax = 729) produced changes in potency without large differences in the Hill number (B). The value of λ that can reproduce these results was not unique because a parameter scan revealed a region of λ − kon space that is consistent with changes in potency without changes in the Hill number . Therefore, we conclude that a phosphorylation-dependent enhancement is the most likely explanation for the correlation between the number of ITAMs and receptor potency.Computations with the mathematical model for l number B. The vl number . We fountal data A. ThereWe next sought a plausible molecular mechanism that can be responsible for the phosphorylation-dependent enhancement. It has been previously proposed that molecular entropy may be reduced by phosphorylation of disordered amino-acid chains . In thisζ-chain is predicted to be disordered based on the sequence of its cytoplasmic chain . The model includes the ζ-chain dimer anchored to the plasma membrane with each polymer in the dimer, modeled as a freely jointed chain with variable number of segments but a fixed overall length. The length of each segment is known as the Kuhn length  to a hypothetical situation where the ζ-chain is completely ordered  as a result of maximum phosphorylation (A).Given that the dimeric gorithms , 35), wen length . To calcrylation A.λmax in the model: the catalytic domain size and the Kuhn length. Larger catalytic domains will incur a larger entropic penalty (increasing λmax) and smaller Kuhn lengths increase the basal number of configurations (increasing λmax) (A and B). We therefore generated a heat map showing λmax when these parameters are varied (C) and also show a specific slice of the heat map for clarity (D). When the Kuhn length is 0.3 nm (or approximately the length of the Cα–Cα bond), we find that λmax ≈ 8000; whereas λmax ≈ 700 (used in the phosphorylation model above) can be found when the Kuhn length is 0.5 nm, which is consistent with the flexibility previously reported for polypeptide chains (There are two factors that determine ng λmax) , A and Be varied C and al clarity D. When e chains .ζ-chain, finding only a weak dependence on λmax (D).These calculations are based on the enzyme catalytic domain interacting with a test tyrosine at the center of the chain, but we have also performed the calculations with a test tyrosine at position 152, which is the most distal tyrosine for murine  on λmax D.In summary, a phosphorylation-dependent enhancement in the binding interactions can produce the observed correlation between the number of phosphorylation sites and receptor potency, and this enhancement is consistent with a disorder-to-order transition upon phosphorylation that reduces the molecular entropic penalty to binding.ζ-chain. In contrast to model predictions, we have found no evidence that multiple phosphorylation sites are able to modulate the switchlike response but have found that multiple sites can modulate receptor potency and maximum phosphorylation.Using cellular reconstitution, we have performed systematic experiments to test predictions of mathematical models for multisite phosphorylation as applied to the T cell receptor ζ-chain with different concentrations of Lck and CD45 on liposomes using purified proteins and found only a modest Hill number of 2 that did not change when ZAP-70 was included, which is consistent with our finding. James and Vale . Previous work has suggested differences in ZAP-70 binding affinities ranging from 3- to 30-fold based on measurements with the isolated tandem SH2 domains , which ζ-chain ITAMs are not sequentially phosphorylated, they may be involved in kinetic proofreading, which relies on a delay  between initial ligand binding and downstream signaling for antigen discrimination (ζ-chain is dispensable for many T cell responses (What is then the purpose of multiple ITAMs in the TCR complex? Although the TCR mination , 45, 46.mination  and moreesponses . This leesponses  for an aThe observation that phosphorylation of one ITAM can enhance the phosphorylation of other ITAMs is related to the phenomenon termed “phosphorylation priming”. Phosphoproteomic analysis revealed that phosphorylation of one site can enhance the phosphorylation of neighboring sites within ∼10 amino acids . Moreoveζ-chain, and indeed many noncatalytic tyrosine-phosphorylated receptors (The mechanism underlying phosphorylation priming is incompletely understood. A possible mechanism is a disorder-to-order transition that phosphorylation may induce in intrinsically disordered regions of proteins . The T ceceptors , have cyeceptors , 7, 13.E subunit of the TCR complex (ζ-chain (Another mechanism that may explain phosphorylation priming across ITAMs is the electrostatically mediated association of the cytoplasmic tails of transmembrane receptors with the membrane. Such interactions have been demonstrated for the CD3 complex , 54, the(ζ-chain , and the(ζ-chain . Because(ζ-chain , 14, 15.(ζ-chain .ζ-chain. For example, phosphorylation has been shown to generate a switchlike response in the membrane localization of the scaffold Ste5 by bulk electrostatics, whereby phosphorylation increases the negative charge of the substrate and therefore leads to reduced membrane association (ζ-chain with the membrane is thought to be regulated by bulk electrostatics (Previous experimental work has reported switchlike responses by multisite phosphorylation that can apply to unstructured proteins such as the TCR ociation , 57. As ostatics , 53. A kζ-chain does not modulate the switchlike response but can modulate the potency and maximum phosphorylation of the receptor. A key difference between the TCR and other multisite substrates known to exhibit switchlike responses is that phosphorylation sites in the latter case are thought to act in concert. For example, in the case of Ste5, Sic1, and NFAT, multiple phosphorylation sites collectively regulate association with the membrane (Although frequently responsible for switchlike responses in other proteins, we have found that multisite phosphorylation of the TCR membrane , 57, assmembrane , or a comembrane , 60, resmembrane . In thisH.M., B.d.W., P.A.v.d.M., and O.D. designed research; H.M., B.d.W., L.C., J.A., and O.D. performed research; H.M., B.d.W., L.C., J.A., P.K.M., P.A.v.d.M., and O.D. analyzed data; and H.M., B.d.W., P.A.v.d.M., and O.D. wrote the article.


Fibroblast growth factor receptor 1 (FGFR1) is correlated closely with the occurrence and development of lung cancer. FGFR1 kinase inhibitors have exhibited significant therapeutic effects against non-small-cell lung cancer. Recently, non-ATP competitive FGFR1 inhibitors have attracted extensive attention due to their low side effects.in vivo.Caliper Mobility Shift Assay was used for FGFR1 inhibition test and kinase inhibitory mode study. Hoechst staining and Annexin V/PI staining were used to evaluate the cell apoptosis induction. Western blot were then performed to confirm the intracellular FGFR1 inhibition and apoptotic protein expression. Finally, the anti-tumor effect and mechanism of Af23 and Ad23 was evaluated 50 values of 0.6 μM and 1.4 μM against FGFR1 kinase, respectively. The kinase inhibitory assay carried at different ATP concentrations showed that the FGFR1 inhibition mode of both Ad23 and Af23 was non-ATP-competitive. Further, Af23 and Ad23 significantly suppressed FGFR1 phosphorylation and cell proliferation in non-small-cell lung cancer (NSLCLC) H460 cells and induced cell apoptosis. Af23 and Ad23 also showed significant anti-tumor activity in the H460 xenograft mouse model, accompanied with the inhibition of FGFR1, ERK, and AKT phosphorylation without exhibiting toxicity.In this study, we designed, synthesized and discovered two novel non-ATP competitive FGFR1 inhibitors, Af23 and Ad23, using NDGA as a leading compound. They had ICThese results indicate that Ad23 and Af23 are potential agents for the treatment of non-small-cell lung cancer. This work also provides a structural lead for the design of new non-ATP-competitive FGFR1 inhibitors. The non-ATP-competitive inhibitors, called type II or type III inhibitors, offer the possibility of overcoming these problems  + X}, where X is the ATP concentration, and n is the Hill coefficient. Specific details of this method were presented in a previous report [This experiment was performed to test the relationship between the compounds and ATP in which the concentration of the substrate was constant, while the concentrations of ATP were set at 4192, 2096, 1048, 524, 262, 131, 66, and 33 μM. The global competitive inhibition fit for the compounds was performed based on percent conversion = (Vmax*X)/{km*[(1 + I/Ki)s report .50 values were calculated by GraphPad software.The anti-proliferative activities of compounds were detected by MTT assay. H460 cells were seeded in a 96-well plate with RPMI-1640 medium that contained 0.1% FBS for 24 h. Then, the cells were treated with the compounds at the indicated concentrations  for 72 h. The proliferation of the H460 cells was detected through MTT assay, and the ICBio-Rad, Hercules, CA). The lysates were separated by SDS-PAGE electrophoresis, and electro-transferred to a 0.22-μm polyvinyldene difluoride membrane. After blocking with TBS that contained 5% non-fat milk for 1.5 h at room temperature, the membranes were incubated with different primary antibodies overnight at 4°C. Following the TBST wash, immuno-reactive bands were detected by incubating with respective secondary antibody conjugated with horseradish peroxidase for 1 h. Immuno-reactive bands were visualized by using an ECL kit .Cells or homogenated tumor tissues were lysated. Protein concentrations in all the samples were determined by using the Bradford protein assay kit . The cells were imaged under fluorescent microscope , and the pictures were taken at 200× objective.After the H460 cells were incubated with the compounds for 72 h, cells were stained with Hoechst 33342 dye according to the protocol provided with the kit .H460 cells were placed in 60-mm plates for 12 h and then treated with NDGA, Ad23, or Af23 at the indicated concentrations for 24 h. Then, the cells were harvested and stained with annexin V and propidium iodide (PI) in the presence of 100 mg/mL of RNAse and 0.1% Triton X-100 for 30 min at 37°C. Flow-cytometric analysis was performed using FACS calibur (6 cells in 200 μL of PBS) of 7-week-old, BALB/cA nude mice. Two days after the H460 cells were injected, the mice were injected intraperitoneally (i.p.) with a water-soluble preparation of either compound Ad23 or compound Af23 in PBS at a dosage of 5 mg/kg/day for 28 days, whereas the control mice were injected with the liposome vehicle in PBS (n = 10 in each group). The volume of the tumors were determined by measuring their length (l) and width (w) and calculated using the formula; V = 0.52 × l × w2. The weight of the tumors were recorded on the day the mice were killed.All animal experiments complied with the Wenzhou Medical College Policy on the Care and Use of Laboratory Animals . Five to six-week-old athymic nu/nu female BALB/cA mice (18–22 g) were purchased from the Animal Center of the China Pharmaceutical University . The animals were housed at a constant room temperature with a 12:12-hr (light: dark) cycle and fed a standard rodent diet and water. H460 cells were harvested and mixed with Matrigel at proportions of 1:1. Then, the cells were injected subcutaneously into the right flank  followed by staining with primary antibodies. The signal was detected by biotinylated secondary antibody and developed with 3,3-diaminobenzidine (DAB).On day 30 after tumor induction, the mice were killed in a COin vitro experiments were repeated at least three times. Data were presented as means ± SD or mean ± SEM. The statistical significance of differences between groups was obtained by the student’s t test or ANOVA multiple comparisons in GraphPad Prism 5 . P values less than 0.05 (p < 0.05) were considered to be significant.All in vitro and in vivo [50 values of NDGA against FGFR1 and FGFR3 were 24.5 and 72.4 μM, respectively, indicating that NDGA exhibits better inhibitory activity against FGFR1 than FGFR3  , the kinase activity was decreased by more than 90%, and further increases in the concentration of ATP, even up to 4190 μM, had no effect on the inhibitory potency of the compounds  of Af23 and Ad23 were much greater when compared to NDGA. Further, Western blot analysis showed that the cleavage of caspase-3 and caspase-9 increased after treatment with Ad23 or Af23, indicating that these compounds could induce apoptosis in H460 cells after a 12-h treatment  and Af23-treated group (48.2 ± 11.12%) induced a greater rate of cell apoptosis than NDGA (27.2 ± 5.27%). We have also tested the growth inhibition of Ad23 and Af23 against human liver cells HL7702 and human fetal lung cell line MRC-5 which expresses low level of FGFR1. The results showed that all the IC50 values of Ad23, Af23, and NDGA against HL7702 or MRC-5 cells were greater than 30 μM (Figure Next, the growth inhibition of H460 cells by NDGA, Ad23, and Af23 was studied by MTT assay. Our data showed that Af23, Ad23, and NDGA exhibited marked inhibitory effects against H460 cells (Figure in vivo, we analyzed the expression of p-FGFR1 in the tumor tissues. As shown in Figure In order to further assess the anti-tumor activities of the NDGA analogs, we tested the efficacies of Af23 and Ad23 in the H460 xenograft mouse model. Treatment with Af23 or Ad23 for 28 days resulted in significant reduction in tumor volume (Figure Prior studies have revealed that phosphorylation of FGFR1 can lead to the activation of downstream signaling cascades including ERK and AKT, which plays an important role in the proliferation and survival of cancer cells. Thus, the phosphorylation of ERK and AKT in tumor samples was tested by immunohistochemical assays Figure E. SimilaFGFR1 is highly related to the development of lung cancer -15,17,2450 values of 0.84 and 1.23 μM, respectively [50 values of Ad23, Af23, and ARQ069 are at micromolar concentration levels, the observation that the inhibition of FGFR1 autophosphorylation (Figure 50 values with that of classic ATP-competitive inhibitors for the inhibition of both kinase autophosphorylation and downstream signaling pathways [NDGA was reported previously to inhibit FGFR3 kinase . In thisectively . Althougpathways ,24. Our in vitro. As shown in Figure in vivo anti-tumor activity. In the present study, the data revealed that the compounds Ad23 and Af23 suppressed the phosphorylation of FGFR1 in H460 tumor tissues, thereby inhibited the downstream phosphorylation of ERK and AKT, and reduced the expression of BCL-2, COX-2, and Cyclin D1 (Figure in vivo (Figure in vivo. However, continued research is needed to examine the underlying FGFR1-binding mechanism and preclinical evaluation of these two compounds. In spite of the predicted “DFG-OUT” docking model, it is unclear how Ad23/Af23 exactly binds to the FGFR1 kinase domain or other domains, which needs to be demonstrated by X-ray diffraction-based structural biology study. Also, it is important to test the RTK-inhibitory selectivity of these two compounds at both molecular and cellular levels in the future.Figure in vitro and in vivo. These two compounds were shown to have the potential to be developed as novel agents for the treatment of NSCLC. The results of the present study indicate that Ad23 and Af23deserve further studies both in the pre-clinical evaluation and in the field of medicinal chemistry for developing structurally different and more effective bisaryl-1,4-dien-3-one-containing FGFR1 inhibitors.Given the critical importance of FGFR1 in the pathogenesis and development of NSCLC, this study identified two novel, non-ATP-competitive inhibitors of FGFR1 kinase, i.e., Ad23 and Af23, both of which exhibited good anti-tumor activity


Ursus maritimus) have been identified as vulnerable to changes in sea ice conditions. We use sea ice projections for the Canadian Arctic Archipelago from 2006 – 2100 to gain insight into the conservation challenges for polar bears with respect to habitat loss using metrics developed from polar bear energetics modeling.Sea ice across the Arctic is declining and altering physical characteristics of marine ecosystems. Polar bears (st century with projected warming. Each polar bear population in the Archipelago may undergo 2–5 months of ice-free conditions, where no such conditions exist presently. We identify spatially and temporally explicit ice-free periods that extend beyond what polar bears require for nutritional and reproductive demands.Shifts away from multiyear ice to annual ice cover throughout the region, as well as lengthening ice-free periods, may become critical for polar bears before the end of the 21Under business-as-usual climate projections, polar bears may face starvation and reproductive failure across the entire Archipelago by the year 2100. Ursus maritimus) are inextricably linked to Arctic sea ice and are sensitive to sea ice loss Pusa hispida), the primary prey species of polar bears Observed changes in global climate have influenced Arctic sea ice cover more than most models have predicted st century, with significant losses in the Hudson Bay and peripheral Arctic seas Optimal polar bear habitat is predicted to decline in the 21st century The CAA and Greenland were thought to have the greatest likelihood of sustaining polar bears to the end of the 21www.gfdl.noaa.gov/coupled-physical-model-cm3). This simulation includes a realistic spatial distribution of sea ice extent and thickness and simulates the trend in observed minimum sea ice extent during the observational record (1979–2013). This pilot simulation was dynamically downscaled using the ice-ocean Massachusetts Institute of Technology General Circulation Model (MITgcm) simulation in regional mode over the Arctic at a resolution of 18km (available from http://mitgcm.org). The 3-hourly atmospheric forcing fields from GFDL-CM3 were bias-corrected at the monthly scale using differences for variables  or ratios for variables  between the Japanese 25 year Reanalysis (JRA25) st century with large trends in many of the Arctic climate forcing fields. MITgcm parameters were provided by Nguyen Global climate simulations contributed to the Coupled Model Intercomparison Project Phase 5 (CMIP5) are too coarse to effectively resolve the narrow channels of the CAA. Although numerically challenging, one solution is to dynamically downscale a global simulation onto a finer grid using a regional climate model. A comparison between over 30 different CMIP5 models led us to select one simulation from the Geophysical Fluid Dynamics Laboratory Coupled Physical Model (GFDL-CM3) driven by radiative scenario RCP8.5 to pilot the regional model  and central regions  may be entirely ice-free for 5 months, and may no longer reach 100% SIC at maximum ice extent. In the north , the simulation estimates a 2–4 month ice-free season by the end of the 21st century, and maximum concentrations <100% in 2080–2090. Ice thickness and snow depth exhibit similar declines throughout the CAA. Ice thickness in the late 20th century was twice to nearly five times the thickness of the projected thickness in the same month of the late 21st century.While multiyear ice is not good hunting habitat due to its low prey abundance eptember . By the th and late 21st centuries in the western regions . Furthermore, using a conservative estimate of a minimum 20 cm snow depth requirement for seal habitat Snow depth declines in part due to the reduction in sea ice surface and dates of formation, but also due to a predicted shift in precipitation from snow to rain Polar bears fare poorly when sea ice is absent for prolonged periods, losing body mass without the opportunity to hunt st century with ice-free seasons reaching critical duration, and early break-ups occurring in parts of all populations we examined. Similarly, to the east of the CAA, the west coast of Greenland and much of Baffin Bay may no longer be suitable habitat for polar bears before 2050, though ice should persist along the east coast of Baffin Island until much later. Early break-up in the narrow channels of the central CAA may become critical in 2060–70s, whereas the adjacent coastlines of the open Arctic Ocean remain largely non-critical until near 2100.We find that sea ice conditions may become unsupportive of polar bear population persistence in the CAA and its surroundings by the late 21st century.It is important to consider that what we deem a critical point-of-no-return occurs once the ice-free period crosses our critical threshold and remains critical for the remainder of the modeled period. Nevertheless, it is feasible that single seasons, or clusters of seasons, may become critically ice-free before that point, with subsequent seasons being non-critical. As such, we examined the cumulative number of critical events for of the aforementioned critical periods (A–D) by population . We founst century.Without exception, our simulation projects the sea ice habitat in all polar bear populations of the CAA may change from a multiyear to an annual ice system before the end of the century, and the remaining annual ice might not persist sufficiently long each year to allow hunting opportunities for polar bears as we currently understand them. Our model suggests that, by 2070, over 80% of the CAA might experience break-up in July, forcing pregnant females to retreat to land early, with possible negative effects on their reproductive output. Given that our study area comprises approximately one quarter of the world's polar bears, and nearly one-tenth of the total current habitat, our analyses project significant habitat loss and alteration under the business-as-usual model scenario used to estimate sea ice conditions over the coming 21Conservation efforts to protect polar bear habitat in the Canadian Arctic should focus on regions that are slower to experience change in sea-ice concentration and ice-free period. The Queen Elizabeth and Norwegian Bay populations retain multiyear ice the longest, and their northerly fjords and channels consistently exhibit the fewest critical ice-free events. Nevertheless, by 2100 all regions of the study area may cross the critical point-of-no-return, putting the persistence of the CAA polar bear populations in jeopardy.


Ice seals overwintering in the Bering Sea are challenged with foraging, finding mates, and maintaining breathing holes in a dark and ice covered environment. Due to the difficulty of studying these species in their natural environment, very little is known about how the seals navigate under ice. Here we identify specific environmental parameters, including components of the ambient background sound, that are predictive of ice seal presence in the Bering Sea. Multi-year mooring deployments provided synoptic time series of acoustic and oceanographic parameters from which environmental parameters predictive of species presence were identified through a series of mixed models. Ice cover and 10 kHz sound level were significant predictors of seal presence, with 40 kHz sound and prey presence (combined with ice cover) as potential predictors as well. Ice seal presence showed a strong positive correlation with ice cover and a negative association with 10 kHz environmental sound. On average, there was a 20–30 dB difference between sound levels during solid ice conditions compared to open water or melting conditions, providing a salient acoustic gradient between open water and solid ice conditions by which ice seals could orient. By constantly assessing the acoustic environment associated with the seasonal ice movement in the Bering Sea, it is possible that ice seals could utilize aspects of the soundscape to gauge their safe distance to open water or the ice edge by orienting in the direction of higher sound levels indicative of open water, especially in the frequency range above 1 kHz. In rapidly changing Arctic and sub-Arctic environments, the seasonal ice conditions and soundscapes are likely to change which may impact the ability of animals using ice presence and cues to successfully function during the winter breeding season. Histriophoca fasciata) and bearded seal (Erignathus barbatus) vocalizations are salient vocalizations recorded seasonally in the Bering Sea from January-June when sea ice is present Phoca largha) in navigating under ice to locate breathing holes Ribbon  since 1995 and 2004, respectively for the M2 and M5 moorings Active and passive acoustic sensors were incorporated into subsurface acoustic moorings deployed at two locations on the 70-m isobath of the eastern Bering Sea shelf at sites M2  and M5  in 2009 and 2007, respectively. Moorings at these locations have been deployed and maintained as part of the NOAA Ecosystems and Fisheries-Oceanography Coordinated Investigations (EcoFOCI) Program  scientific echosounder system of Acoustic Water Column Profilers , Passive Aquatic Listener (PAL) recorder, and an AURAL (Autonomous Underwater Recorder for Acoustic Listening) (Multi-Électronique (MTE) Inc., Québec, Canada). The mooring was constructed in the following order from top (approximately 60–62 m) to bottom (approximately 70 m): 36″ floatation, 300 kHz RDI ADCP, 30″ floatation, AURAL, AWCP system, PAL, and acoustic release. The AWCP system was mounted in an upward-looking direction 15° off vertical to eliminate interference from flotation and instruments in the mooring line directly above the active acoustic system.This study utilized data from the PAL and AWCP system. AWCPs record acoustic backscatter to monitor the presence and location of acoustic scatterers such as zooplankton and fish within the water column The PAL is an event detector, or adaptive sub-sampling acoustic recorder, with a temporal sampling strategy designed to allow the instrument to record data for up to one year Field data was collected under Observational Institutional Animal Care and Use Committee (IACUC) #36003 “Characterizing Biological Scatter and Its Implications for Marine Mammals in the Bering Sea” from The Pennsylvania State University. There was no direct interaction with any vertebrates in this study, as all data from marine mammals were obtained remotely through passive acoustic listening.http://pafc.arh.noaa.gov/ice.php. Ice conditions surrounding the mooring locations were estimated within an approximate 20 km2 around the mooring.Daily mean ice cover (or percent cover in this specific region) and ice thickness data were obtained from the images produced by the NOAA Ice Desk at the National Weather Service in Anchorage, Alaska. The images are posted on Balaena mysticetus), gray whale (Eschrichtius robustus), killer whale (Orcinus orca), beluga whale (Delphinapterus leucas), walrus (Odobenus rosmarus), ribbon, and bearded seals). Ribbon seal grunts, roars, and downsweeps were used to indicate presence Ribbon and bearded seal presence was determined from the PAL soundbites with the understanding that detection of vocalizations indicates seal presence, and the lack of acoustic detection does not imply animal absence. Soundbites were reviewed by a human classifier and verified by a second independent human classifier blind to the results of the first reviewer. Sound sources present in the soundbites were identified from spectrograms  made from the original 100 kHz recordings downsampled to 48 kHz using Adobe Audition 3.0 (Adobe Systems Incorporated). These settings provided a bandwidth of 61 Hz, with a frequency resolution of 47 Hz, and a time resolution of 2.7 ms. Marine mammal vocalizations were classified aurally and visually from the spectrograms by species  with units of dB re 1 µPa2/Hz.Analysis of PAL spectra included examination of spectral shape and levels. Temporal clusters of similarly distinctive sound spectra lasting tens of minutes to hours were manually identified and classified. Sound levels were computed from the time series of spectra. Each spectrum was computed from 1024 point samples of the 4.5 s time series. This resulted in a 513 point power spectral density with each of the bins covering 97 Hz of the 50 kHz usable bandwidth. The spectra were then reduced from 513 points to 64 points by averaging spectra levels over two bins below 3 kHz and over ten bins from 3 to 50 kHz. The resulting power spectral density, relative to µ1Pav in units m2/m3) was calculated from 24 hour integrations over each 5 m depth layer using EchoView software . Targets within each depth and time bin were classified as to the likely source of the scattering based on differences in scattering amplitude between the three frequencies. Analyses using this dB-difference approach Pseudocalanus spp., Acartia longiremis, Oithona spp. and Calanus), 2) medium scatterers (lengths of 5–15 mm) which includes juvenile krill, chaetognaths, and amphipods, 3) large scatterers such as adult euphausiids (lengths of 15–30 mm), 4) resonant scatterers which represents an organism with a gas-inclusion such as a swim-bladdered fish or siphonophore, and 5) unknown. The acoustic system was not able to detect the weak scattering strengths of scatterers less than approximately 5 mm in length unless they were present in extremely dense aggregations. Aggregations were classified as belonging to one of the five categories  by determining the shortest geometric distance between the three dB differences calculated for the aggregation and that of the theoretical scatterers. If the closest geometric distance was more than 12 dB , then the aggregation was classified as unknown.To assess prey parameters related to zooplankton/fish abundance and community composition, the AWCP data were processed in 5 m vertical depth bins. Daily mean volume backscatter coefficient  designed to identify predictor variables of ice seal presence . There idrop1 command from the basic stats package in R  and generalized additive models (GAMs) allow model fitting to describe relationships between variables without constraints of linear regression models GLMM and GAMM techniques are on the “frontier of statistical research” and as such model selection and validation for generalized models on absence-presence response data is difficult Seasonal ice was present at both mooring locations in the Bering Sea during each winter of the study . The ice2/Hz) above 5 kHz. The internal noise floor of the recorder was likely a limiting factor for sound levels below approximately 32 dB.The grouping of PAL spectra identified four general sea surface conditions  . ValidatThe initial models to determine predictors of ice seal presence included ice thickness, % ice cover, 200 kHz Sv, four size categories of % prey composition , and five sound levels . The final GLM and GLMM model both included ice cover, 10 kHz sound, 40 kHz sound, and an interaction between ice cover and large crustaceans as significant predictors of ice seal presence . Ice seaParameters estimated by both the GLM and GLMM are nearly identical , suggestGAMM models showed primarily linear relationships between seal presence and ice cover and 10 kHz sound, with the exception of ice cover at M5 , Table 2Phoca largha), ring seal (Pusa hispida), harbor seal (Phoca vitulina) Sea ice and 10 kHz sound levels were the strongest predictors of ice seal vocal presence during the winter breeding season in the Bering Sea. The results indicate that as 10 kHz (and to a lesser extend 40 kHz) sound levels increased, the detection of ice seal vocalizations decreased. Neither ribbon seals nor bearded seals have a significant amount of energy in their vocalizations above 10 kHz The modeling results directed a more detailed examination of the acoustic environment that ice seals encounter on an annual basis. Open water conditions are the loudest up to approximately 8 kHz . Above 8This observational study was not able to establish a cause-effect relationship or identify a specific threshold or optimal sound level range that ribbon and bearded seals may employ to navigate under ice. Long-term tagging studies with acoustic dosimeters and GPS location capabilities will be needed to confirm this theory and provide direct evidence of the mechanisms of under-ice navigation in ice seals. It will also be useful to investigate this relationship across locations and regions in both the Arctic and Antarctic to assess whether this concept can be generalized to all ice-dependent species required to navigate under ice. This work presents a particularly timely observation, as the sea ice and acoustic conditions of the oceans, the strongest predictors of ice seal vocal presence during the winter breeding season, are changing due to climate change and industrialization related to shipping and energy exploration/production Table S1Response and predictor variables used in the GLM and GAM modeling at the M2 (A) and M5 (B) locations. Bearded and ribbon seal acoustic presence is a binary response: 0 for absent, 1 for present. The 200 kHz Sv are daily mean values, and the scatterer percent composition is reflective of the daily mean values within each size category. Ice cover % and ice thickness are daily assessment values.(DOCX)Click here for additional data file.Table S22/Hz.Daily mean sound levels at M2 (A) and M5 (B) used in the GLM and GAM modeling. Sound level units are dB re 1 μPa(DOCX)Click here for additional data file.


In the invitro experiments, PDE5 inhibition resulted in activation of cGMP-dependentprotein kinase 2 and consequent inhibition of glycogen synthase kinase3β phosphorylation, destabilization of cytosolicβ-catenin and the ultimate suppression of canonical Wnt signaling andreduced osteoblastic differentiation in HEK293T and C3H10T1/2 cells. In animalexperiments, systemic inhibition of PDE5 suppressed the activity of canonical Wntsignaling and osteoblastogenesis in bone marrow-derived stromal cells, resulting inthe reduction of bone mass in wild-type adult C57B/6 mice, significantlyattenuated secreted Frizzled-related protein-1 (SFRP1) deletion-induced activation ofcanonical Wnt signaling and excessive bone growth in adultSFRP1−/− mice. Together, these results uncover ahitherto uncharacterized role of PDE5/cGMP/PKG signaling in bone homeostasisand provide the evidence that long-term treatment with PDE5 inhibitors at a highdosage may potentially cause bone catabolism.Inhibitors of phosphodiesterase 5 (PDE5) are widely used to treat erectiledysfunction and pulmonary hypertension in clinics. PDE5, cyclic guanosinemonophosphate (cGMP), and protein kinase G (PKG) are important components of thenon-canonical Wnt signaling. This study aimed to investigate the effect of PDE5inhibition on canonical Wnt signaling and osteoblastogenesis, using both  The stabilizedcytosolic β-cat enters the nucleus and consequently activates itsdownstream target genes via lymphoid enhancer-binding factor-1 (Lef-1) and T-cellfactors.2 This signaling is fine-tuned in part via a negative feedbackmechanism involving secreted and transmembrane Wnt inhibitors and activators, secretedFrz-related proteins (SFRPs), and Dickkopf-1 (Dkk1).4In the canonical Wnt , resulting in a decrease inCa2+ influx and consequent relaxation of smooth muscles, whichproduces the therapeutic effects in clinical erectile dysfunction (ED) and pulmonaryhypertension (PH).14 Currently, little isknown regarding the involvement of PDE5 in Wnt signaling regulation of bone formationand homeostasis. The objective of this study was to determine the effect of PDE5inhibition on canonical Wnt signaling and bone mass.Phosphodiesterases (PDEs) are a large family of enzymes that cleave cyclic nucleotides.To date, 11 PDE subtypes have been identified, among which PDE5 has been mostextensively studied. PDE5, cyclic guanosine monophosphate (cGMP), and cGMP-dependentprotein kinase (PKG) are among the major components of the non-canonical Wnt signalingpathway and are involved in the regulation of intracellular CaμM, tadalafil and vardenafildecreased the reporter activity by 58 and 30%, respectively, in thepresence of control L cell-conditioned medium (L), and by 42 and 60%,respectively, in the presence of Wnt3a-expressing cell-conditioned medium (Wnt3a) used inhibited the Lef1-luciferase activity in adose-dependent manner; at 10  (Wnt3a). In contd medium .Neitherd medium . Tofurtd medium . PDE5a sd medium . PKG1 and medium . Silencid medium . Thus, iβ-cat is the central signal transducer of the Wntsignaling pathway, we evaluated the effect of PDE5 inhibition onβ-cat messenger RNA (mRNA) abundance and protein content, andstabilization in 293 T cells, to further assess the possible involvement of PDE5in Wnt signaling. PDE5 inhibitor tadalafil did not significantly changeβ-cat mRNA levels at either 5 or 10 μM butattenuated Wnt3a-induced increases in β-cat protein levels inwhole-cell lysates, cytosolic, and nuclear fractions by 58, 60, and 65%,respectively, at 10 μM  and atThr41, Ser37, and Ser33 by GSK3β regulates its stabilization in thecytosol, and the phosphorylated β-cat is recognized by E3 ubiquitinligase and undergoes proteolytic degradation.16 To determine the role of PDE5 in the stabilization ofβ-cat, we assessed changes in β-cat phosphorylationin 293T cells treated with tadalafil at 10 μM, in either thepresence or absence of Wnt3a. Wnt3a slightly decreased the phosphorylation ofβ-cat at Ser45, whereas tadalafil showed no significant effect but induced by tadalafil (1.4-fold), and tadalafil was capable ofreversing the negative effect of Wnt3a on β-cat phosphorylation(3-fold)  (Phosphorylation of t effect. Phospho(3-fold) . In conton (60%) .β-cat. We observed that the cGMP analog, 8-Br-cGMP, increasedβ-cat phosphorylation by 40% at Thr41, Ser37, and Ser33,but decreased GSK3β phosphorylation by 50% at Ser9 in 293Tcells (β-cat phosphorylation (80%) but an increase inGSK3β phosphorylation (2-fold) (β-cat and 70% for GSK3β)  andpotentiated Wnt3a's induction of GSK3β phosphorylation(50%)  . These er GSK3β) . We furtn by 60% . Moreoveon(50%) .β signaling as a downstream event of PDE5/cGMP/PKG2signals in the regulation of β-cat stabilization.GSK3β inhibitor, SB216763, increased Lef1-luciferase activity butshowed no significant effect in the presence of tadalafil in 293T cells (β siRNA (GSK3βsi) where GSK3βprotein content was decreased by 60~80%  C57BL/6mice. Orally administered for 2 months at 45 or75 mg/kg daily that was20- and 32-fold higher than the clinical dosage for ED (10 mg daily) and 5-and 8-fold higher than the clinical dosage for PH (40 mg daily),18 tadalafil robustly decreased the mass of cancellous bonebut not of cortical bone as revealed by morphological and histological analyses(μCT) confirmed thattadalafil at both 45 and 75 mg/kg resulted in decreases in bone mineraldensity (BMD) by 30 and 35%, in trabecular bone volume (BMTV) by 42 and48%, in trabecular number (TbN) by 35 and 50%, but increases intrabecular separation (TbSp) by 58 and 97%, respectively, while it had noeffect on trabecular thickness (TbTh), when compared with the vehicle treatment mice. At thebaseline level, SFRP1−/− mice had a significantlyhigher mass of cancellous bone but not cortical bone as compared withSFRP1+/− mice, as revealed by histological analysisof the longitudinal sections of the distal femur. At an oral dose of75 mg/kg daily for 2 months, tadalafil robustly decreased the mass ofcancellous bone but not in cortical bone in SFRP1−/−mice (μCT further confirmed that there weresignificant increases in BMD (2.1-fold), BMTV (2.3-fold), and TbN (1.5-fold) butnot in TbTh and TbSp in SFRP1−/− mice as compared withSFRP1+/− mice, and that tadalafil treatment resultedin significant decreases in BMD (38%), BMTV (28%), and TbN(47%), but not in TbTh and TbSp as compared with the vehicle treatment, and tadalafilattenuated SFRP1 knockout-associated excessive formation of mineralized nodules by~25% over vehicle  than thosefrom SFRP1+/− mice, whereas systemic treatment withtadalafil in SFRP1−/− mice robustly attenuated thetranscription of these genes  than those from SFRP1+/− mice,and systemic treatment with tadalafil in SFRP1−/− micerobustly attenuated the transcription of these markers .In one of our previous studies, we have demonstrated that Wnt3a through LRP5/6,Dvl, and most likely Frz receptors, activates a signaling module composed ofGαq/1115 suggesting that Wnt3a fails to activate Frz2, and thatPDE5/cGMP/PKG signals regulate canonical Wnt signaling independently ofWnt3a. The current study has shown that PDE5/cGMP/PKG2 signals regulate thestabilization of β-cat at the protein but not the mRNA level.Inconsistent to this observation, Li et al.22 and Tinsley et al.23 have shown that both non-steroidal anti-inflammatory drug,sulindac sulfide (SS), and PDE5 inhibitor, tadalafil, inhibit PDE5 activity andincrease cGMP levels, resulting in PKG activation, decreased proliferation, andapoptosis through transcriptional suppression of β-cat, but not itsproteolytic degradation in colon cancer cells, and more recently, Tinsley etal.24 have demonstrated that SS maytrigger β-cat protein degradation and suppress β-cat genetranscription through increased phosphorylation in human breast cancer cells. Theinconsistency is possibly due to the use of different cell types in the previousstudies  and this study .A previous study has observed that activation of Frz2 induces the cGMP-dependentphosdiesterase and decreases the intracellular cGMP levels,β-cat at Thr41, Ser37, and Ser33 but not at Ser45, suggesting thatGSK3β but not CK1 may be downstream of the PDE5/cGMP/PKG2signaling module. This notion is further supported by the findings that knockdown ofPKG2 is sufficient to activate the phosphorylation of GSK3β and thestabilization of β-cat. Finally, PKG2 and GSK3βco-existing within one immunoprecipitated complex prompts us to speculate that PKG2directly phosphorylates GSK3β in the complex. In line with thisspeculation, several recent reports have shown physical interactions between PKG2 andGSK3β. In ATDC5 cells, PKG2 directly phosphorylatesGSK3β to promote the hypertrophic differentiation.25 In UMR106 cells, PKG2 also phosphorylates theGSK3β at Ser9, and the apparent Km of PKG2 forGSK3β is about 0.15 xM.26 Thus, the present study has identified a mechanism in whichactivation of cGMP/PKG2 by inhibition of PDE5 results inGSK3β-mediated destabilization of β-cat and consequentsuppression of canonical Wnt signaling.Inhibition of PDE5 and activation of cGMP/PKG2 induce the phosphorylation of28 With regard to non-canonical Wnt signaling, little is knownregarding its role in bone development and homeostasis in both embryogenesis andadulthood.11 Consistent with the roleof PDE5/cGMP/PKG2 signaling in Wnt3a-induced osteoblastogenesis invitro, systemic administration of a PDE5 inhibitor, tadalafil, reduces bonemass not only in normal but also in SFRP1−/− knockoutadult mice in this study. The severe phenotype after tadalafil treatment reflects theimportance of PDE5 activities in the maintenance of bone homeostasis. Endogenous Wntsignaling plays an important role in bone formation through stimulating theosteoblastogenesis and suppressing the adipogenesis andosteoclastogenesis.29 Our invivo analyses show that: (1) inhibition of PDE5 reduces the trabecular bonebut not the cortical bone, which is identical to the phenotypes of SFRP1 or LRP5gain- and loss-of-function;32 (2)inhibition of PDE5 leads to the robust inhibition of Lef1 and Dkk1transcription in BMSCs; (3) inhibition of PDE5 almost abolishes the SFRP1knockout-producing overgrowth of bone; and (4) neither inhibition of PDE5 nor SFRP1gain-of-function affects the osteoclastogenic activities. Together, these invivo observations imply that inhibition of PDE5 attenuates not only theosteoblastogenic differentiation in response to Wnt3a but also the activities ofcanonical Wnt signaling, suggesting that inhibition of PDE5 reducesosteoblastogenesis possibly through suppressing canonical Wnt signaling.The role for canonical Wnt signaling in the embryonic development of bone is wellestablished, but the function in maintaining the bone homeostasis in adulthood isless clear.14 Consistent with ourfindings, previous studies have demonstrated that mice lacking endothelial NOSexhibit profound abnormalities in bone formation and a significant delay inosteoblastic differentiation,34 and that mice lacking either PKG2 or PKG1 maydevelop severe phenotypes; however, analyses of the skeletons of PKG2 but not PKG1mutants have revealed the obvious defects in chondrogenesis but not inosteoblastogenesis.39 It is likely that PKG2 and PKG1 play a functionalredundancy in osteoblastogenesis.Nitric oxide synthase (NOS) is the enzyme responsible for producing NO and NOactivates soluble guanylyl cyclases to increase the intracellular cGMP levels andactivate PKG.β-cat through acGMP/PKG2/GSK3β signaling-dependent mechanism. Given thatPDE5 inhibitors are commonly used in patients with ED and PH, our findings may havesignificant clinical implications in alerting physicians of putative adverse effectof PDE5 inhibitors.In summary, the present study has demonstrated that PDE5 inhibition may cause bonemass loss involving non-canonical Wnt pathway, which operates interactively withcanonical Wnt signaling in a way to destabilize β-actin, and protein A/G PLUS-Agarose were from Santa CruzBiotechnology . Antibodies against p-β-cat(Ser33/37/Thr41), p-β-cat (Ser45), β-cat,p-GSK3β (Ser9) and GSK3β were from Cell Signaling. Antibodies against AP and RunX2 were purchased from Abcam, and the antibody against Lef1 was from Proteintech . The IRDye 680 and 800 second antibodies were from LI-COR Bioscience and the Alexa 555-conjugated secondary antibodies were fromLife Technologies . Cyclic GMP Direct Immunoassay kits wereobtained from Abcam and TRAP staining kits were from Sigma.Tadalafil and vardenafil were obtained from Selleckchem .KT5823, 8-Br(Bromo)-cGMP, Alizarin Red S, and SB216763 were from Sigma . Recombinant murine Wnt3a protein was purchased from R&D System. Antibodies against PKG1, PKG2, Lamin B, Dkk1, IgG,11 Conditional media were prepared from Wnt3a-expressing andcontrol L cells, respectively, as described previously.16C3H10T1/2 cells, Wnt3a-expressing and control L cells, and HEK293T cells wereall obtained from ATCC . For maintenance, C3H10T1/2 cellsand HEK293T cells were cultured, respectively, in Basal Medium Eagle (LifeTechnologies) and Dulbecco's modified Eagle's medium (LifeTechnologies) supplemented with 10% fetal calf serum . Primary bone marrow stromal cells (BMSCs) were isolated from thefemur and tibia, and cultured in alpha Minimum Essential Media (Life Technologies)with 15% FCS as described previously.μg Lef1 reporterconstruct using Lipofectamine 2000 reagent and 0.02 μg Renillaluciferase construct  for 6 h in the absence ofserum.40 In some cases, the cellswere co-transfected with siRNA oligonucleotides targeting the test genesPDE5a, PKG1, PKG2, and GSK3β (seeSV40 large T-antigen-expressing HEK cells (293T cells) were plated onto 24-wellplates. Next day, they were transfected with 2 4 and 1.5 ×104 cells/cm2 overnight, after a variety oftreatments. Cytosolic and nuclear fractions of cells were prepared by using NE-PERNuclear and Cytoplasmic Extraction Reagents as per the manufacturer's instructions. β-Actin and Lamin-Bwere used as the internal standards for the cytosolic and nuclear fractions,respectively. Western blot analysis and immunoprecipitation were performed usingstandard protocols and the immunoreactive signals for the proteins of interestwere quantitated using the LI-COR Odyssey Infrared Imaging System (LI-CORBioscience) and ImageJ (http://rsb.info.nih.gov/ij/). 293T cells seeded on chamber slides at 0.75 ×104/cm2 and cultured in regular medium overnight andin serum-free medium for 12 h. After pre-treatment with tadalafil for2 h, serum-starved cells were further stimulated with recombinant murineWnt3a protein at 100 ng/ml in fresh serum-free medium for30 min. Cells were then immunostained with primary antibodies againstβ-catenin and subsequently counterstained with4',6-diamidino-2-phenylindole. Immunoreactive protein signals were examinedby confocal microscopy.293T and C3H10T1/2 cells were plated 1 × 1041. For mineralization assays, confluent cells wereincubated in the presence of 50 mg/ml ascorbic acid and 50 mMβ-glycerophosphate for 21 days. Total RNA was extracted usingTRIzol reagent  Co., Ltd., Dalian, China). mRNAlevels of osteoblast markers and target genes of canonical Wnt signaling includingDkk1, Lef1, AP, Runx2, and Osxwere determined by real-time PCR as previously described.43 GAPDH was included asan internal control and the relative levels of mRNA species of interest werecalculated by the 2−ΔΔCt method.44 The primers used in our PCR analysis arepresented in the C3H10T1/2 cells were seeded onto six-well plates. At confluence, cells werestimulated with conditioned medium prepared from control L or Wnt3a-expressingcells for 48 h in the presence of vehicle or tadalafil. AP activity wasdetermined and expressed as nanomoles of p-nitrophenol formed per minute permilligram of protein as previously described.+/− mouse strainswere gifted by Dr Akihiko Shimono.45Two-month-old mice were orally administrated with tadalafil daily at 0, 45 or75 mg/kg for 2 months. We used μCT  for three-dimensionalreconstruction and quantification of bone parameters, and reconstructed each imagefrom one hundred 16-μm slices immediately below the growth plate, witha threshold of 200.46 Histologicalexamination and immunohistochemistry staining were performed on paraffin sections(4 μm) after decalcification. Immunohistochemistry stainingwas performed by using the Histostain-Plus Kit as described previously.47 Aftersequential treatments, tissue sections were sequentially incubated with normalserum for 30 min, control IgG and primary antibodies against AP, Runx2,Lef1, or Dkk1 for 2 h, and then HRP-conjugated secondary antibody for30 min. The diaminobenzidine solution was used for development of browncolor and sections were counterstained with hematoxylin. The quantitativehistomorphometry was performed using the Osteomeasure Analysis System. The Animal Studies Committee at ZhejiangUniversity approved all mouse procedures.Male C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co. and founder SPRP1P<0.05.The SPSS statistical package  was used.Experiments were repeated at least three times. Numerical data were expressed asmeans±S.D. and analyzed by one-way ANOVA and Tukey–Kramer multiplecomparisons test. Differences were considered significant when


The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at ∼50 kDa, whereas its calculated molecular mass is ∼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His–Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser250, which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser250 substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser250 phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys268 in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of ∼80 amino acids (aa) at the C-terminus, BCNT-C . Whereas the yeast Swc5 and  Chromatin dynamics are pivotal aspects of gene regulation mechanisms in eukaryotes. A main issue is how the canonical histone H2A-H2B dimer is converted into various histone H2A variant-H2B dimers accompanied by common and/or unique chromatin-related components ,2. TheseDrosophila homologue, Yeti, is essential for development by providing chaperon-like activity in higher chromatin organization  and BCNT-C and is a typical intrinsically disordered protein  and by a Qiagen-tip for transfection of HEK cells. DNA sequences were confirmed by a Big Dye terminator mix and an ABI 3130 Sequencer (Applied Biosystems) and later by Fasmac Co Ltd. The primers for PCR are listed in Supplementary Table S1.Human eviously . Two var2 at 37°C. For the generation of HEK-TR cell lines that constitutively express His–BCNT, the cDNA in pCMV6-AN–His was transfected with GenJet (SignaGen Laboratories) and the cells were subjected to limited dilution after 48 h and cultured in the same medium supplemented with 250 μg/ml G418 (Enzo Life Sciences) in a 96-well dish. Among three generated clones, the G11 (HEK-TR clone which constitutively expresses His–BCNT) clone was used for further studies based on the advantages of its growth rate. The clone was routinely sub-cultured in G418-containing medium using Trypsin/EDTA (Sigma).The HEK (human embryonic kidney)-293T cell line  and its derivative T-Rex293 , were cultured in a MegaCell Dulbecco's Modified Eagle's Medium (Sigma) with 3% FBS, 2 mM glutamine, 50 μg/ml gentamycin in an atmosphere of 95% air/5% COE. coli were expressed by shifting the temperature to 15°C, letting the cultures stand for 30 min, supplementing with 0.1–1 mM IPTG and then shaking for 19–23 h at 15°C. Target proteins were isolated at 4°C basically according to the manufacturer's instructions (Qiagen). The cells were lysed in lysis buffer  supplemented with lysozyme  at 4°C for 30 min in a rotary shaker and then sonicated in an ice-water bath for 2.5 min . The extract was centrifuged for 30 min at 4°C at 17000 g and the supernatant was mixed with Ni-NTA (Ni2+-nitrilotriacetate) agarose beads or its magnetic beads (Qiagen) in a siliconized tube and incubated for 1 h in a rotary shaker. Following the removal of the unbound fraction by brief centrifugation, the beads were suspended in lysis buffer and transferred to a spin column using a wide-bore tip. After washing two times with the lysis buffer and then two times with washing buffer containing 20 mM imidazole, the bound proteins were eluted with 150 mM imidazole and then with 250 mM imidazole-containing buffer without any inhibitors.Various Bcnt in pCold II in 7 cells per 150 mm dish (Sigma) and cultured for 3 days with one medium change. To detect acetylation sites, the cells were incubated with 4 mM nicotinamide and 50 nM trichostatin A for 2 h prior to extract preparation. The cell layers were washed once with chilled PBS on ice and harvested in 1 ml of lysis buffer per dish  on ice. The extract was harvested using a scraper, frozen immediately in liquid nitrogen and stored at −80°C until use. For the isolation of His–BCNT, the stored extract was thawed, sonicated and centrifuged as described for the preparation from E. coli. The supernatant was incubated with anti-His antibody-linked beads (MBL) in a siliconized tube for 1 h in an ice-water bath. The tube was tipped several times during incubation. The bound fraction was transferred to a spin column in lysis buffer containing all inhibitors. The column was washed three times with lysis buffer with all inhibitors, followed by one wash with PBS without inhibitors, after which the adsorbed fraction was eluted with 50 μl of 2 mM His-tag peptide (MBL) at room temperature according to the manufacturer's protocol. Alternatively, the cell extracts were lysed in lysis buffer containing 1% Nondet P-40 (Sigma) and the supernatant was obtained as described above. The extract was fractionated by 20%–45% ammonium sulfate precipitation, after which the pellet was resuspended in lysis buffer with all inhibitors and dialysed overnight against 20 mM Hepes/NaOH (pH7.4), 150 mM NaCl, 1 mM EDTA in a cold chamber. His–BCNT was isolated from the dialysates as described above. Both the N- and the C-terminal regions of BCNT were routinely confirmed by mass analysis using Asp-N  digests to identify m/z=MHHHHHHAIAMEEF (m/z=1763.6) or MHHHHHHAIAM (m/z=1358.5) and DLRLSKMKP (m/z=1087.6).The G11 clone was seeded at a density of 1.1 × 10E. coli and acetylated using recombinant CREB-binding protein  and 14C-radioactive acetyl-CoA  as described previously . The 1H-15N HSQC spectrum [Production of a  length 2–257, wasspectrum  was recoSDS/PAGE was performed essentially as described by Blattler et al.  except tAchromobacter protease I  [http://proteomecentral.proteomexchange.org) with the dataset identifier PXD002228.The protein bands were excised and digested with trypsin  or endoproteinase Asp-N (Roche Diagnostics) as described previously . AchromoIbaraki)  was alsoIbaraki) . The digIbaraki)  via the 1H-15N-HSQC spectrum shows that most of the 1H frequencies of the backbone amides are clustered within a ∼1 ppm area with extensive resonance overlap. The result indicates that the fragment is largely unstructured [E. coli and examined the relationship between the estimated mass on SDS/PAGE and the calculated mass . A total of seven phosphorylation sites were identified without any pre-treatment B. Mass qrylation .2+. Incubation with these two phosphatases causes Upper band to converge with Lower one and okadaic acid, a PP2A-specific inhibitor, blocks this shift  sequence in BCNT-C. We replaced the serine with alanine or glutamic acid and expressed the two mutants in either E. coli or HEK-293T cells. All of the mutant proteins show distinctively different mobilities on SDS/PAGE, indicating that Ser250 phosphorylation contributes significantly to the mobility shift  and/or undergo dimerization that would block access to the N-terminal His-tag for affinity binding, although we are unable to exclude the possibility of a general expression problem concerning the production of intrinsically disordered proteins.Under these limiting conditions, we identified seven phosphorylation sites without pre-treatment with phosphatase inhibitors, in contrast with the detection of acetylation of exogenous BCNT expressed in HEK-TR cell lines for which pre-culture with histone deacetylase inhibitors is required. Secondary PTMs of exogenous proteins is unavoidable from either transiently or permanently expressing cells. For the transiently expressed BCNT, we were able to detect its acetylation but not phosphorylation. This sample is tagged with a C-terminal Myc and isolated from HEK293 cells pre-cultured for 24 h with sodium butyrate, a histone deacetylase inhibitor, prior to extract preparation and prepared in the presence of sodium vanadate, a phosphatase inhibitor . It is interesting to compare the differences in PTMs between transiently and constitutively expressed proteins more precisely taking into account the differences in the epitope tags and their locations at either the N- or the C-terminus.250 phosphorylation is apparent only in Upper band, whereas the phosphorylated state of Lower band, including the missing Ser250 phosphorylation, is exactly the same as that of the dialysed protein. The substitution of Ser250 with alanine or glutamic acid causes significant changes in the SDS/PAGE mobility. However, S250E BCNT expressed in E. coli runs slightly faster than Lower band from HEK-293T cells, suggesting that another phosphorylation(s), such as at Ser21 and/or Ser26, might also influence mobility. Ser250 resides in a region between disordered and ordered regions, rather than in a purely disordered region. This type of phosphorylation site accompanying a SDS/PAGE mobility shift is frequently observed such as in superoxide dismutase 1 (SOD1) [250 in the unique WESF motif in mammalian Bcnt is asparagine in the chicken CENP-29 homologue [250 phosphorylation, it may play a crucial role in mammalian Bcnt-specific signalling modes.Among seven phosphorylated sites detected in BCNT, the Ser1 (SOD1) , p56Lck 1 (SOD1)  and p35 1 (SOD1) . We specWe previously reported that rat and bovine brain Bcnt appear as doublets at ∼43 kDa , which p