D-pinitol is a cyclitol present in several edible plant species and extensively investigated for the treatment of metabolic diseases in humans, as food supplement, and demonstrated protective effects in the cardiovascular system. For these reasons, the present work aimed at investigating the mechanisms involved in the vascular effects of D-pinitol in mouse mesenteric artery. Mesenteric arteries from male C57BL/6 mice were mounted in a wire myograph. Nitrite was measured by the 2,3-diaminonaphthalene (DAN) method. Protein expression and phosphorylation were measured by Western blot. The systolic blood pressure (SBP) was measured by tail-cuff plethysmography. D-pinitol induced a concentration-dependent vasodilatation in endothelium-intact, but not in endothelium-denuded arteries. Nω-Nitro-L-arginine methyl ester (300 μM) abolished the effect of D-pinitol, while 1H-oxadiazoloquinoxalin-1-one  shifted the concentration-response curve to the right. KN-93 (1 μM) blunted the vasodilator effect of D-pinitol, but H-89 (0.1 μM) did not change it. 1-[2-(Trifluoromethyl) phenyl]imidazole (300 μM), indomethacin (10 μM), celecoxib (5 μM), wortmannin (1 μM), ruthenium red (10 μM), tiron (10 μM), MnTMPyP (30 μM), MPP (0.1 μM), PHTPP (0.1 μM), and atropine (1 μM) did not change the effect of D-pinitol. D-pinitol increased the concentration of nitrite, which was inhibited by L-NAME and calmidazolium (10 μM). D-pinitol increased the phosphorylation level of eNOS activation site at Ser1177 and reduced the phosphorylation level of its inactivation site at Thr495. In normotensive mice, the intraperitoneal administration of D-pinitol (10 mg/kg) induced a significant reduction of the SBP after 30 min. The present results led us to conclude that D-pinitol has an endothelium- and NO-dependent vasodilator effect in mouse mesenteric artery through a mechanism dependent on the activation of eNOS by the calcium-calmodulin complex, which can explain its hypotensive effect in mice. Inositols are cyclohexane polyols also known as cyclitols. They are arranged in nine stereoisomers: scyllo, myo, neo, chiro, cis, mucus, and allo . InositoD-pinitol (3-O-methyl-D-chiro-inositol) is a cyclitol present in several edible plants, including soybean and carob .2PO4 0.4; NaHCO3 14.9; MgSO4.7H2O 1.17; CaCl2.2H2O 2.5; and glucose 5.5. A segment of the second branch of the mesenteric artery was dissected, and the adipose and connective tissues were removed. The arteries were sectioned into rings (1.6–2.0 mm long) with an internal diameter ranging from 150 to 250 μm. The rings were mounted in a wire myograph , kept in carbogen aerated PSS at 37°C. After mounting, the artery was stretched to a length that yielded a circumference equivalent to 90% of that given by an internal pressure of 100 mmHg; this required a load of approximately 200 mg. The vessel was maintained for an equilibration period of 60 min. The mechanical activity was recorded isometrically as previously described  in mesenteric arteries in the presence and the absence of a functional endothelium pre-contracted with phenylephrine (3 μM). The participation of nitric oxide synthase (NOS) was investigated in arteries pretreated with Nω-nitro-L-arginine-methyl-ester , a non-selective inhibitor of NOS, and 1-(2-trifluoromethylphenyl) imidazole , a selective inhibitor of neuronal NOS (nNOS). The activation of guanylate cyclase was investigated with 1H- -oxadiazoloquinoxalin-1-one . The involvement of cyclooxygenase (COX) 1 and 2, phosphatidylinositol-3-kinase (PI3K), Ca2+/calmodulin-dependent kinase II (CaMKII), and non-selective cationic channels was verified in arteries pretreated with indomethacin (10 μM), celecoxib (5 μM), wortmannin (1 μM), KN-93 (1 μM), and ruthenium red (10 μM), respectively. Tiron (10 μM) and MnTMPyP (30 μM), a cell-permeable analog of superoxide dismutase, were used to investigate the action of antioxidant drugs on the vasodilator effect of D-pinitol. The participation of muscarinic receptors and α and β estrogen receptors was investigated in arteries pretreated with atropine (1 μM), MPP (0.1 μM), and PHTPP (0.1 μM), respectively.Mice were euthanized by decapitation, the abdomen was cut, and the mesenteric bed was quickly removed and placed in a dissecting plate with physiological salt solution (PSS) with the following composition (mM): NaCl 119.0; KCl 4.7; KHescribed . The fun2-) using the fluorescence method with 2,3-diaminonaphthalene (DAN), according to 2 atmosphere. Samples were collected in the absence  or the presence of D-pinitol (20 μM) or ACh (10 μM). The involvement of NOS and calmodulin in the production of nitrite was evaluated in the presence of L-NAME (300 μM) and calmidazolium (10 μM), respectively. 150 μl samples were collected, added to 150 μl of purified water, followed by the immediate addition of 15 μl fresh DAN solution (0.05 mg/l in 0.62 M HCl) in 96-well opaque black plates . The reaction proceeded for 10 min at room temperature and protected from light. After this period, the reaction was stopped with 5 μl of NaOH (2.8 N) and the absorbance determined using a spectrofluorometer  at 365 and 415 nm, as respective excitation and emission wavelengths. The nitrite concentration in the samples was calculated using a standard curve with predetermined concentrations of sodium nitrite in each experiment and normalized by the amount of protein in the branches. The results were expressed in [nitrite] nM/μg of protein.The assessment of NO production in the mesenteric artery was performed indirectly by the measurement of nitrite (NO2 and 5% CO2) at 37°C. D-pinitol (20 μM) was added and after 5, 15, or 30 min, the arteries were collected, and frozen at -80°C. Arteries that were not stimulated by D-pinitol were used to determine the basal level of phosphorylation (time zero). After, the tissues were homogenized in the presence of lysis buffer  plus 0.3% Triton X-100, 0.5% SDS, and protease inhibitors cocktail , supplemented with a cocktail of protease inhibitors . 30 μg of protein were applied on the SDS-PAGE gel (sodium dodecyl sulfate polyacrylamide) 7.5%. Then, proteins were transferred to a PVDF membrane  and the membranes were blocked  before overnight incubation with the specific primary antibody: anti-eNOS , anti β-actin , anti-phospho-eNOS-Ser1177 , and anti-phospho-eNOS-Thr495 . The immunocomplexes were detected by a chemiluminescence assay  and the densitometry analyses were done using the software ImageJ 1.48v.Western blot analysis was performed as previously described , with soD-pinitol (10 mg/kg) or saline. The basal SBP was measured 10 min before the administration of D-pinitol.The systolic blood pressure (SBP) was measured by the tail-cuff plethysmography  using MRD-pinitol. One-way ANOVA was used for the statistical analysis of nitrite dosage and Western blot data. All results were expressed as mean ± standard error of the mean (SEM) and found to be significant if P < 0.05.Statistical analyses were performed using GraphPad Prism 4 program. Vasodilator data were represented as the percentage of reduction in the sustained contraction induced phenylephrine (3 μM). Two-way ANOVA followed by Bonferroni post-test was used to analyze the cumulative concentration-response curves and the reduction in the SBP induced by D-pinitol induced a concentration-dependent vasodilator effect. The maximal vasodilator effect (Emax) achieved was 21.4 ± 2.4% . However, in the absence of a functional endothelium, the vasodilator effect of D-pinitol was abolished . The non-selective inhibition of eNOS with L-NAME blunted the vasodilator effect of D-pinitol  while the selective inhibition of nNOS with TRIM did not alter its vasodilator effect . The inhibition of guanylate cyclase with ODQ induced a significant shift to the right on the concentration-response curve of D-pinitol . The inhibition of COX-1 with indomethacin , COX-2 with celecoxib , PI3K with wortmannin , and non-selective calcium channels blockade with ruthenium red  did not change the vasodilator effect of D-pinitol. KN-93, a selective inhibitor of CaMKII, blunted the vasodilator effect of D-pinitol , while H-89, a selective inhibitor of PKA, did not change it . Antioxidant drugs such as Tiron  and MnTMPyP , a cell-permeable analog of SOD, did not alter the concentration-response curve to D-pinitol in endothelium-intact mesenteric arteries. Preincubation of arteries with atropine, MPP, and PHTPP did not change the concentration-response curve to D-pinitol . L-NAME blunted the increase induced by D-pinitol while calmidazolium induced a significant reduction . A similar effect was observed with ACh (10 μM), which was also inhibited by L-NAME and calmidazolium .D-pinitol increased the phosphorylation level of the activation site of eNOS at Ser1177 5 and 15 min after the stimulation of endothelium-intact mesenteric arteries . Besides, the level of phosphorylation of the inactivation site of eNOS at Thr495 was decreased 15 and 30 min after the stimulation of the arteries with D-pinitol .D-pinitol induced a significant reduction in the SBP of normotensive mice . The significant reduction in the SBP was observed 30 min after the administration of D-pinitol in comparison to the saline SBP at 30 min and to the SBP observed before the administration of D-pinitol.D-pinitol has been extensively investigated for the treatment of metabolic diseases in humans, as food supplement, and demonstrated protective effects in the cardiovascular system. The present study demonstrated that D-pinitol induces a vasodilator effect in mice mesenteric arteries by a mechanism dependent on the production of endothelium-derived NO through the activation of the calcium-calmodulin complex.D-pinitol have demonstrated that this cyclitol has vascular endothelium protective effects  (D-pinitol.Previous studies with  effects . The prein (1–7)  and subsin (1–7)  that plain (1–7) . Therefo2O2 and epoxyeicosatrienoic acids (D-pinitol, demonstrating the involvement of the EDRFs in the vasodilator effect of this drug. The involvement of eNOS in the formation of EDRF participating in the control of the vascular tone is already well accepted. However, several studies have demonstrated the presence of nNOS in the endothelium of mice mesenteric arteries and aorta, validating its participation in the control of the vascular tone (D-pinitol by L-NAME and the absence of inhibitory effect by TRIM, suggest the endothelium-dependent effect of D-pinitol occurs through the activation of eNOS, and that nNOS is not involved. The reduction of the vasodilator effect of D-pinitol by ODQ, a selective inhibitor of guanylate cyclase, supports the participation NO as the eNOS-derived EDRF in the vasodilator effect of this cyclitol in mice mesenteric arteries. In addition, the absence of effect of COX inhibitors rules out the participation of prostacyclin in the vasodilator effect of D-pinitol.The endothelium plays an important role in the cardiovascular system. It functions as a semipermeable barrier between the blood and the vessel wall, regulating vital functions such as vascular tone, response to inflammatory stimuli, and blood coagulation . The conic acids . In thislar tone . The inhD-pinitol, suggesting that the PI3K/Akt pathway is not involved in the activation of eNOS by this cyclitol in mesenteric arteries. The calcium-dependent mechanism involves the increase in intracellular calcium concentration resulting from the release of intracellular stores of calcium or the influx through the plasmatic membrane of endothelial cells (D-pinitol in the presence of ruthenium red suggests that the TRPVs are not involved in the vasodilatation induced by this cyclitol. Thus, the calcium-dependent mechanism of eNOS activation induced by D-pinitol can be related to the release of the intracellular calcium stores or by the influx of calcium through other channels. The calcium-dependent mechanism occurs through the formation of calcium-calmodulin complex (D-pinitol as this drug inhibited the contraction induced by phenylephrine. However, calmidazolium significantly inhibited the production of nitrite induced by D-pinitol, in a similar way as observed with ACh. Therefore, D-pinitol seems to activate eNOS by a calcium-dependent mechanism.eNOS can be activated by calcium-independent and -dependent mechanisms . The calal cells . The traal cells . In the al cells . The abs complex . This me complex . In the 1177 is associated with the activation of eNOS (D-pinitol. The inhibition of the vasodilator effect of D-pinitol by KN-93 confirms the participation of CaMKII in the activation of eNOS and supports a calcium-dependent mechanism for D-pinitol. In order to confirm the activation of eNOS, the level of phosphorylation of Ser1177 was investigated in the presence of D-pinitol by Western blot. The significant increase in the level of phosphorylation of Ser1177 at 5 and 15 min after the stimulation with D-pinitol confirms the ability of this cyclitol to activate eNOS. Additionally, the activity of eNOS is also regulated by inactivation sites, such as Thr495 (D-pinitol was able to reduce the level of phosphorylation of Thr495 significantly, once more confirming the ability of this cyclitol to activate eNOS. This is an important point since D-pinitol in arteries from diabetic animals was related to its antioxidant property, while the direct vasodilator effect of D-pinitol was not demonstrated in this previous report. The absence of effect of antioxidant drugs such as tiron and MnTMPyP on the vasodilator effect of D-pinitol, associated to the profile of phosphorylation discussed above, demonstrate that D-pinitol increases the production of NO and induces its vasodilator effect by activation of eNOS rather than by its antioxidant action.The phosphorylation of Ser of eNOS . Kinases of eNOS . The abss Thr495 . SubstanD-pinitol is able to reduce the SBP in normotensive mice. This observation is in line with the vasodilator effect of D-pinitol described above, which suggests a significant reduction in the systemic vascular resistance and blood pressure. A previous report demonstrated that the administration of D-pinitol reduced the cardiovascular risk factors in patients with type 2 diabetes mellitus, including the reduction in the SBP and diastolic blood pressure (D-pinitol has a significant effect on the cardiovascular system and may be investigated as an antihypertensive drug. However, attention should be taken if D-pinitol is used as food supplement, considering that high amounts of this cyclitol can significantly reduce the blood pressure and may produce hypotension in normotensive subjects.Finally, the present study also demonstrates that pressure . These oD-pinitol has an endothelium- and NO-dependent vasodilator effect by a mechanism of action involving the calcium-calmodulin complex activation of eNOS, which might be responsible for its hypotensive effect.The present study allows us to conclude that LM, JS, and GS performed research and contributed to the writing of the manuscript. VL and SC designed research and reviewed the manuscript to the submission version.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.


Avian infectious bronchitis virus (IBV) primarily replicates in epithelial cells of the upper respiratory tract of chickens, inducing both morphological and immune modulatory changes. However, the association between the local immune responses induced by IBV and the mechanisms of pathogenesis has not yet been completely elucidated. This study compared the expression profile of genes related to immune responses in tracheal samples after challenge with two Brazilian field isolates (A and B) of IBV from the same genotype, associating these responses with viral replication and with pathological changes in trachea and kidney. We detected a suppressive effect on the early activation of TLR7 pathway, followed by lower expression levels of inflammatory related genes induced by challenge with the IBV B isolate when compared to the challenge with to the IBV A isolate. Cell-mediated immune (CMI) related genes presented also lower levels of expression in tracheal samples from birds challenged with B isolate at 1dpi. Increased viral load and a higher percentage of birds with relevant lesions were observed in both tracheal and renal samples from chickens exposed to challenge with IBV B isolate. This differential pattern of early immune responses developed after challenge with IBV B isolate, related to the downregulation of TLR7, leading to insufficient pro-inflammatory response and lower CMI responses, seem to have an association with a most severe renal lesion and an enhanced capability of replication of this isolate in chicken. Avian infectious bronchitis virus (IBV) is a highly infectious causative agent of avian infectious bronchitis (IB), a disease of high economic impact, which affects poultry worldwide. IBV replicates primarily in tracheal epithelial cells, inducing several mucosal pathological changes, including ciliary loss, degeneration and necrosis of epithelial cells, glandular degeneration, inflammatory cell infiltration and epithelial hyperplasia ,2. AfterPrevention of IBV infection is currently achieved through vaccination, especially by attenuated viral vaccines, suggesting that local mucosal immunity is essential for induction of effective protection against disease ,8,9. In The continuous emergence of new IBV variants in several countries ,11,12,13Despite the great number of IBV strains and variants described over the last years , most ofA recent study has observed differential early immune response after infection with IBV when comparing IBV-susceptible and IBV-resistant chicken lines. Even before infection, specific genes, including genes related to innate immune response, were found to be differentially expressed in each chicken line. Despite these differences, viral loads were similar in tracheal samples of both chicken lines, indicating that IBV resistance might be associated with how the birds respond to the virus and not how they can prevent an initial infection .The innate immune response is the first line of host defense against infections, and several immune and non-immune cells on mucosal surfaces are involved on recognition of pathogen associated molecular patterns (PAMP) of microorganisms. PAMPs are recognized through pattern recognition receptors (PRR), among which toll-like receptors (TLR) are an important group. During a RNA-virus infection, activation of innate immune responses depends on the interaction of endosome-associated (TLR3 and TLR7) and/or cytosol-located (RIG-I like receptors) PRRs. TLR3 and TLR7 interact with double-stranded (ds) RNA or single-strand (ss) RNA, respectively, and the subsequent activation pathways for TLR3 and TLR7 are mediated by TRIF (TIR-domain-containing adapter-inducing interferon-β) and MyD88 (myeloid differentiation primary response gene 88), respectively, which lead to the production of anti-viral type I interferons (IFNα and IFNβ) and pro-inflammatory cytokines (IL1β and IL6) . ExpressThe inducible nitric oxide synthase (iNOS or NOS2), responsible for the production of nitric oxide (NO) by macrophages in response against microbial infections, is often associated with synergic effects combined with acute phase proteins and cytokines, as IFNs and TNFs, leading to enhanced phagocytosis. Alternatively, an excessive production of iNOS and consequently NO may have negative effects by promoting excessive inflammation or apoptosis ,19. AlthThe important role of pro-inflammatory cytokines on IBV pathogenicity was demonstrated in a previous evaluation of the kinetics of the inflammatory and cell-mediated immune responses in tracheal mucosa of birds infected with the M41 strain of IBV. TNFSF15 and TGFβ transcripts were found to peak at 1 day post infection (dpi), followed by higher upregulation of IL6, IL1β and IFNγ. These increased levels of pro-inflammatory cytokines were associated to highest scores of lesions and viral load at 3dpi, whereas later, at 7dpi, highest increases in CD8 and Granzyme homolog A mRNA expression were detected and associated to a significant decrease in lesions and viral load .It has been recognized that IL6 is associated with development of tissue damage in chickens challenged with IBV, including nephritis . Levels During the development of adaptive immunity against IBV infection, the local cell-mediated immune (CMI) response was associated with CD3+, CD8+ and CD4+ cells influx in the trachea from 3 to 7 dpi with IBV ,9. FurthMechanisms underlying immune-pathogenesis caused by IBV and those relating to viral clearance have begun to be elucidated ,17,22,23The aim of this study was to investigate and compare the pathogenesis and the innate and adaptive immune responses induced by the infection with two Brazilian field isolates of IBV classified in the BR-I genotype.50) according to the Reed & Muench method [Two Brazilian field isolates BI/BR/Embrapa/331/2000  and BI/BR/Embrapa/127/2006 , were isolated from clinical cases of avian infectious bronchitis in poultry flocks located in southern Brazil. Virus isolation and titration were carried out by inoculation of five 10-day-old specific pathogen-free (SPF) embryonated chicken eggs/sample via the allantoic sac route  and viruh method . IBV fieh method . The dedThe chickens were housed and handled at the Laboratory of Animal Health and Genetics—Embrapa Swine and Poultry, Concórdia- SC, Brazil. All procedures have been approved by the Embrapa Swine and Poultry Ethical Committee for Animal Experimentation (CEUA/CNPSA), in accordance with ethical principles and guidelines of animal experimentation adopted by the Brazilian College of Experimentation (process number 016/2012).4.0 EID50/bird of A strain of IBV and 104.0 EID50/bird of B strain of IBV by intra-ocular and intra-nasal routes. One group remained mock infected . During whole experimentation, chickens were monitored twice a day. If animals have presented severe depression and lethargy, they were separated to be euthanized, though, there were no animals in these conditions during experiment. The birds from groups A, B and NC were randomly euthanized by cervical dislocation, at 1, 5 and 8 day post-infection (dpi). Tracheal and renal samples were collected from each group; a portion of the proximal third from each tracheal sample and a fragment of distal left kidney were immediately frozen in liquid nitrogen and kept at -70°C until processing for RNA extraction. Another portion of tracheal and renal samples were subjected to histopathological analysis. Throughout the experimental period, birds received water and feed “ad libitum”, the room temperature was adapted according with the bird´s age, and the monitored ammonia concentration levels remained below 2 parts per million (ppm).Three groups of Specific Pathogen Free (SPF) chicks (White Leghorn lineage) obtained from Valo Biomedia  were housed in separate positive pressure isolators. At 28 days of age, groups A and B were respectively challenged with 10Tracheal samples were divided into three fragments , and a portion of each fragment was prepared for histopathology. The severity of observed lesions scores were determined as previously described . AbsenceThe scores of microscopic lesions in the kidney samples were determined as previously described . The parRNA extractions from the proximal third of tracheal and renal samples of experimentally infected chickens were performed using TRIzol Reagent (Invitrogen) followed by RNA purification using RNeasy Mini Kit (Qiagen). The extracted RNA purity and quantification were estimated by 260nm ultraviolet absorbance and readings at 260/280nm, respectively. RNA quality was verified with Agilent RNA 6000 Nano Kit (Agilent Technologies) in an Agilent 2100 Bioanalyzer instrument (Agilent Technologies) for determination of RIN (RNA Integrity Number) or by RNA analysis in 1% gel electrophoresis.All tracheal and renal samples were tested for IBV viral load by RT-qPCR (hydrolysis probe system) using AgPath-ID One step RT-PCR kit (Ambion), primers and LNA-probe  for amplification of the 3´UTR genome region of IBV, as described . Cq  before cDNA synthesis. Efficiency of each specific Real-time PCR was calculated using two-fold serial dilutions of cDNA pooled from all tested animals, and Cq values plotted into the linear equation.Two-step RT-qPCR was used for the relative quantification of gene expression in the tracheal samples. cDNAs were synthesised according to instructions provided with High Capacity cDNA Reverse Transcription kit (Applied Biosystems) and 1μL (500 ng/μL) of Oligo(dT) primers (IDT). The PCR reactions contained 20 ng of cDNA, 7.5 μL of 2X Quantifast SYBR Green Master Mix (Qiagen), and 3 μM of each primer  in a finThe relative expression of all tested genes  in trachThe comparisons of relative changes in gene expression, viral load and microscopic lesions between the experimental groups were performed using the Kruskal-Wallis test followed by Wilcoxon test.p ≤ 0.05.All analyses were conducted using the SAS 9.4 software (2012), and the probability level for significance was set as The tracheal lesions observed in groups challenged with A or B IBV isolates at 1 dpi were characterized by mild acute tracheitis, consisting of the presence of heterophils and mucus exudation in the tracheal lumen with congestion and heterophilic infiltration in the lamina propria and epithelial cell desquamation . The mosMarked lymphoplasmacytic interstitial nephritis (lesion score 3) was only observed in the challenged groups (A and B), at 8dpi . TubularA higher percentage of birds challenged with IBV B isolate showed relevant tracheal (at 1dpi and 5dpi) and renal (at 8dpi) microscopic lesions  comparedNo positive samples were detected for the presence of IBV genome in tracheal and renal samples from the negative control (NC) group in any of the post-infection intervals analysed .High percentages (87.5% and 100%) of tracheal samples were positive at 1 dpi for the presence of IBV genome from chickens challenged with IBV A and B strains, and differed significantly from tracheal samples from NC group . At 5dpiThe renal samples from chickens of A group showed 88.89% of positivity for IBV genome in kidney samples while all the renal samples from chickens of B group were positive for IBV genome, . The preThe viral load decreased at 8dpi in tracheal samples from all IBV challenged groups (A and B) . DespiteSignificantly higher means of IBV genome load (10 to 1000 times higher) were detected in group challenged with IBV B isolate compared to the group challenged with IBV A isolate, at 5dpi interval in tracheal samples (P = 0.0041) and at 8dpi in renal samples (P = 0.0041). .The mRNA expression data for TLR3, TLR7, MyD88, iNOS, IFNα and IFNβ in tracheal samples are illustrated in TLR7 transcripts were downregulated (5 times) at 1 dpi only in the tracheal samples from B-challenged group, but their levels of expression rose at 5dpi and maintained at high levels at 8dpi in all challenged groups (A and B) .MyD88 mRNA transcripts were upregulated in tracheal samples from all challenged groups (A and B) and at all post-infection intervals. At 8dpi, the samples from A group presented higher levels than those from B group (P = 0.0176) .IFNα was upregulated at 5dpi and 8dpi in tracheal samples from chickens of B group, and only at 8dpi in the samples from A group chickens. IFNβ transcripts were upregulated in chickens from B group at 1dpi and 5dpi, while in chickens from the A group, only at 5dpi .The iNOS gene showed a significant increase in mRNA expression at 1dpi in challenged groups. At 5dpi and 8dpi, these transcripts dropped to basal levels .IL1β transcripts were significantly upregulated in tracheal samples from all challenged groups at 1, 5 and 8 dpi and the highest levels of IL1β expression were reached at 8dpi. .IL6 mRNA transcripts were significantly upregulated in tracheal samples from challenged groups at 1 and 5 dpi. At 8 dpi, only the samples from group A showed upregulation in the expression of this cytokine gene .The TNFSF15 mRNA was only significantly upregulated at 8dpi, in the group challenged with IBV A isolate .The CD3 and CD4 mRNA transcripts were downregulated at 1dpi only in the tracheal samples from the B group. At 5dpi and 8dpi, all tracheal samples from challenged groups showed upregulation of CD3 and CD4 expression, and the highest levels of expression of these genes were observed at 5dpi .At 5dpi and 8dpi, all challenged groups presented upregulation of CD8β transcripts, and the highest levels of expression of this gene were detected at 5dpi .The levels of IFNγ gene expression started to increase at 1dpi in group challenged with IBV A isolate. All challenged groups, presented upregulation of IFNγ transcripts, at 5dpi and 8dpi and the highest levels were found 5dpi .The transcripts of Granzyme homolog A mRNA were upregulated in all challenged groups at the three post-infection intervals evaluated, except for the group challenged with IBV B isolate, at 1dpi .The host-virus interactions that result in more severe pathogenesis induced by different pathotypes of IBV are not fully elucidate, but the IBV pathogenesis has been associated with high virus replication rates, induction of exacerbated inflammatory responses and delayed activation of anti-viral effector mechanism of innate and adaptive immune responses ,16,31. IIBV B isolate demonstrated an enhanced ability for viral replication in chicken host, as we observed significantly higher IBV genome loads in tracheal samples (at 5dpi), and in kidneys (at 8dpi) of birds challenged with this isolate, compared to the group infected with IBV A isolate. Furthermore, a higher percentage of positive birds for IBV genome and the presence of pathological alterations were observed in the IBV B infected group, especially in renal samples, that were IBV positive in birds infected with this isolate at 8dpi, while only 17% were positive in the group infected with IBV A isolate.This enhanced viral replication of IBV B isolate compared to A isolate, may have played a role as one of the determinants for the higher percentage of chickens with relevant microscopic tracheal and renal lesions in birds from the IBV B challenged group. This could result in increased pathology of the IBV B isolate. However, the reason for the differences observed in tissue tropism and pathogenicity of different IBV strains remains an open question and cannot be associated in the present study to differences in S1 protein sequences, as A and B IBV isolates have an identity of XX % for this protein. Similarly, a recent study demonstrated that despite the same efficiency of S1 protein binding to the epithelial cells of oviduct, in a sialic-acid dependent manner, observed for Qx IBV strain (associated with nephropathogenicity and reproductive tract disorders) and B1648 IBV strain (associated with nephropathogenicity), a high susceptibility to infection was observed for QX strain in contrast to the resistance to infection with B1648 strain . The autIn view of the differences observed here for these two Brazilian IBV isolates (A and B) in terms of viral replication and pathological alterations induced, the current study investigated how innate and adaptive immune responses are developed after challenge with each one of these viruses, aiming to better understand the virus-host interactions and the putative immune-related mechanisms that might determine the course of pathogenicity induced by IBV isolates with different virulence profiles.Several studies on innate immune responses elicited by viruses and mediated by PRRs have demonstrated that the levels of expression of TLR genes are usually upregulated, in order to activate intra-cellular pathways involved in the production of type I anti-viral IFNs and pro-inflammatory cytokines ,7,16,17.Although other genes related to innate immune responses were also found to be differentially expressed in response to infection with the Brazilian IBV isolates, including TLR3, type I IFNs (IFNα and IFNβ) and iNOS, no significant differences were observed between the group infected with IBV B isolate and that infected with IBV A isolate. These results suggested that the identified differences between the two IBV isolates are not related to the biological activities associated with these innate response genes. However, TLR3 and iNOS genes were significantly upregulated and peaked at 1dpi, while type I IFNs were significantly upregulated and peaked at 5dpi. These results contradict other findings for TLR3, IFNα, IFNβ and iNOS gene expression in the respiratory tract of chickens challenged with a Connecticut strain of IBV , since nAs memory immune responses induced by IBV vaccination could have some delay until reach effective anti-viral activities, the innate immune responses might be crucial to reduce the viral replication and pathological alterations induced by a virus pathogen as well exert a relevant role in the activation and shaping of the anti-viral adaptive immune responses. Thus, we hypothesize that the suppressive effect on TLR7 pathway could be associated with an enhanced ability for viral replication and induction of lesions for both tracheal and renal tissues. However, further analyses of these assumptions are necessary.In addition, a reduction of expression of CMI-genes for adaptive immune responses was also observed only in the group challenged with IBV B isolate, as the transcript levels of CD3 and CD4 were significantly downregulated at 1dpi only in this group. Granzyme homolog A and IFNγ transcripts were also differentially expressed between the challenged groups in this interval, as significant upregulation was observed only in A group. Therefore the downregulation of cell-mediated immune related genes could be a consequence of a primary virus challenge effect and could have negative consequences for the chicken host. In a previous study, we were able to detect an increase in the expression of cell-mediated immune related genes at 1 dpi in tracheal samples from birds vaccinated with Massachusetts attenuated IBV strain and challenged with a virulent homologous strain. This early CMI response, was associated with the level of cellular memory immune responses conferred by immunization, and this effect was dependent on the vaccine dose administered and was negative correlated with the tracheal pathological changes induced by M41 strain of IBV .Although we have not evaluated in this study the gene expression and/or the biological activity of MHC I and II pathways, or other genes related to antigen presentation by dendritic cells, we can speculate that IBV B isolate might have some differentiated properties that confer to this virus a greater ability to decrease antigen presentation and to evade the host adaptive immune responses against this virus. This assumption is supported by the findings that all cell-mediated immune related genes analyzed here have unaltered expression or were significantly downregulated in the group infected by B isolate of IBV at 1dpi, as shown the significantly downregulated levels of expression observed for CD4 and CD3 transcripts, and unaltered expression for IFNγ and Granzyme homolog A genes in birds of this group. In contrast, at 1dpi, the ugroup challenged with IBV A isolate showed unaltered levels of CD3 and CD4 mRNA, while IFNγ and Granzyme homolog A transcripts were significantly upregulated.Natural killer (NK) cells have important roles in the initial mechanisms of innate immunity against IBV, and were also important source of Granzyme homolog A and IFNγ. Although some authors have associated IFNγ production with the activity of NK cells after challenge with IBV , in our In summary, this study has contributed to the better understanding part of host-IBV interactions, and to our knowledge, it is the first study that demonstrate that expression of distinct innate and cell-mediated adaptive immune genes are induced by two IBV isolates that were classified in the same genotype but differing in their virulence activities. Overall, the results point out to the relevance of the involvement of the TLR7 pathway and the associate factors related to the suppression of early innate immune responses as well the early cell-mediated adaptive immune responses and the implications on the pathogenesis of infection by a more virulent IBV isolate. However, further studies are required to confirm this association, and to verify other cellular response pathways that may also be affected by this virus. It remains also to be further investigated the differences in the immune responses triggered by the IBV infection with more distinct genotypes of IBV strains that can differ in pathogenicity for the respiratory, urinary or other tissue targets for IBV infection in chickens. Moreover, it is important for poultry industry and indirectly for human health to better understand how coronaviruses infect the chicken host and which genes and/or pathways are effectively involved in increasing the severity of this disease, especially considering the diseases caused by other coronaviruses such as severe acute respiratory syndrome-associated coronavirus and Middle East respiratory syndrome coronaviruses.S1 FileRed squares are pointing the amino acids changes.(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.


Advances in the understanding of the molecular basis for acute myeloid leukemia (AML) have generated new potential targets for treatment. Fms-like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in AML and mutations in this gene are associated with poor overall survival. AXL plays a role in the activation of FLT3 and has been implicated in the pathogenesis of AML. The studies reported here evaluated the ability of a novel FLT3/AXL inhibitor, gilteritinib, to block mutated FLT3 in cellular and animal models of AML. Initial kinase studies showed that gilteritinib, a type I tyrosine kinase inhibitor, was highly selective for both FLT3 and AXL while having weak activity against c-KIT. Gilteritinib demonstrated potent inhibitory activity against the internal tandem duplication (FLT3-ITD) and FLT3-D835Y point mutations in cellular assays using MV4–11 and MOLM-13 cells as well as Ba/F3 cells expressing mutated FLT3. Gilteritinib also inhibited FLT3-F691 mutations, although to a lesser degree, in these assays. Furthermore, gilteritinib decreased the phosphorylation levels of FLT3 and its downstream targets in both cellular and animal models. In vivo, gilteritinib was distributed at high levels in xenografted tumors after oral administration. The decreased FLT3 activity and high intratumor distribution of gilteritinib translated to tumor regression and improved survival in xenograft and intra-bone marrow transplantation models of FLT3-driven AML. No overt toxicity was seen in mouse models treated with gilteritinib. These results indicate that gilteritinib may be an important next-generation FLT3 inhibitor for use in the treatment of FLT3 mutation-positive AML.The online version of this article (doi:10.1007/s10637-017-0470-z) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) is a rapidly progressing hematologic cancer characterized by loss of normal differentiation and uncontrolled proliferation of hematopoietic progenitor cells in the bone marrow . This dyFms-like tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinase (TK) family that is normally expressed on the surface of hematopoietic progenitor cells . The FLT+ AML in both a subcutaneous xenograft model and a leukemia engraftment model [Similar to FLT3, AXL (a member of the TAM family of receptor TKs) has transforming properties and has been identified as a potential therapeutic target for AML , 17. Prent model –18. Furtnt model . TherefoGilteritinib is a small-molecule FLT3/AXL inhibitor with a structure based on a pyrazine carboxamide scaffold. The research described here aimed at characterizing the activity of gilteritinib against FLT3-driven AML in preclinical models. The inhibitory effects of gilteritinib against FLT3 were assessed in cell-free systems and cellular assays, and its antitumor activity and survival benefits were evaluated in FLT3-driven tumors in mice.Small molecule TK inhibitors (TKI) gilteritinib hemifumarate  and quizartinib were synthesized by Astellas Pharma Inc. . Both gilteritinib and quizartinib were dissolved in DMSO or suspended in 0.5% methylcellulose for in vitro or in vivo experiments. MV4–11 cells were purchased from the American Type Culture Collection ; MOLM-13 cells were purchased from the German Collection of Microorganisms and Cell Cultures . MV4–11-luc cells, which exogenously express firefly luciferase, were prepared at Astellas Pharma Inc. (a detailed protocol for the generation of the constructs is found in the online resource). MV4–11-AXL cells, which exogenously express AXL, were also prepared at Astellas Pharma Inc. Ba/F3 cells expressing FLT3-ITD, FLT3-D835Y, FLT3-ITD-D835Y, FLT3-ITD-F691 L, or FLT3-ITD-F691I mutations  were established by Astellas Pharma Inc. The following antibodies were used for immunoblotting: FLT3 ; phosphorylated pan-tyrosine ; p44/42 MAPK (ERK1/2), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (197G2) (E10), AKT, phospho-AKT (Ser473) (193H12), and AXL (C44G1) ; STAT5 and phospho-STAT5 (Y694) ; phospho-AXL (Y779) ; and β-actin .m value for each kinase in a TK-ELISA or off-chip mobility shift assay (MSA) at Carna Biosciences, Inc. . These assays were conducted according to the manufacturer’s instructions. Initially, two concentrations of gilteritinib (1 nM and 5 nM) were tested to assess each compound’s inhibitory effect on TK activity. Further studies were then conducted using a dose range of gilteritinib to determine IC50 values for kinases in which activity was inhibited by >50% with 1 nM gilteritinib as well as for c-KIT. TK-ELISA and MSA assays were used to conduct IC50 studies for FLT3, LTK, AXL, and c-KIT; the HTRF® KinEASE™-TK assay  was performed according to the manufacturer’s protocol to assess the IC50 value of echinoderm microtubule-associated protein-like 4-ALK .The kinase inhibitory activity of gilteritinib was tested against a panel of 78 TKs (Table S1) using ATP concentrations that were approximately equal to the KThe effect of gilteritinib on MV4–11 and MOLM-13 cells was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay . Subsequent studies were conducted to examine the effect of gilteritinib and quizartinib on Ba/F3 cells expressing either FLT3-ITD, FLT3-D835Y, FLT3-ITD-D835Y, FLT3-ITD-F691 L, or FLT3-ITD-F691I. A detailed protocol is provided in the online resource.FLT3 immunoprecipitation and immunoblotting for all targets were performed following standard protocols. Detailed methods are provided in the online resource.All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Astellas Pharma Inc., and the Tsukuba Research Center of Astellas Pharma Inc. was awarded accreditation status by AAALAC International. Mice were maintained on standard diet and water throughout experimental procedures.Details on the cell engraftment for the mouse xenograft model and the dosing protocols are provided in the online resource.n = 3 per group; five plasma samples showed abnormal results and were not used for data analysis). Tumor samples were also collected from each mouse and tumor weight was measured. The plasma and tumor concentrations of gilteritinib were measured using high-performance liquid chromatography-tandem mass spectrometry at Nemoto Science . Standard pharmacokinetic parameters  were calculated from the mean concentrations of gilteritinib using WinNonlin V6.1 .Nude mice subcutaneously xenografted with MV4–11 cells received gilteritinib suspended in a 0.5% methylcellulose solution as an oral single dose. Blood samples were collected at protocol-specified time points from the inferior vena cava using a syringe with EDTA-2Na, and plasma samples were prepared by centrifugation  and n = 5 mice/group (Ba/F3).Once tumor growth had been confirmed, mice xenografted with MV4–11 cells were monitored for 28 days; mice xenografted with Ba/F3 cells expressing FLT3 mutations were monitored for 7 days. Tumor diameter was measured using a caliper, and tumor volume was determined by calculating the volume of an ellipsoid using the formula: length × width6 cells/mouse) were injected into the bone marrow of the left tibia of female NOD-SCID mice (day 0) following the protocol detailed by Lee et al. [n = 10). Tumor growth was monitored once a week during the dosing period and then every other week until day 100. Survival was also monitored daily until day 168.In the intra-bone marrow transplantation (IBMT) model, MV4–11-luc cells . The coordinate of ligand–protein binding for FLT3 was modeled based on the ATP-binding form of cKIT, which had the most similar kinase domain amino acid sequence in the Protein Data Bank (PDB)  and, therefore, was used as a surrogate for FLT3. The homology modeling was done by the modeling software MOE . Hydrogen atoms were added using the computer program Protonate3D implemented in MOE. The docking mode with the highest docking score was employed. All molecular visualization was produced by MOE.P < 0.05 was considered significant. SAS software , Microsoft Excel , and GraphPad Prism  were used for data processing.For the in vivo subcutaneous xenograft mouse model, values are expressed as mean ± SEM. Tumor volume and body weight on day 28 in the gilteritinib-treated groups were compared with those in the control group using Dunnett’s test. For the IBMT model, the bioluminescence value on day 42 from the gilteritinib-treated group was compared with that from the control group using Student’s t-test. The median survival times were compared using the log-rank test. 50 values were 0.29 nM for FLT3 and 0.73 nM for AXL. Gilteritinib inhibited FLT3 at an IC50 value that was approximately 800-fold more potent than the concentration required to inhibit c-KIT  and 2.9 nM (95% CI: 1.4–5.8 nM), respectively  or 8 h (6 mg/kg and 10 mg/kg) after dosing. Cint Fig. . Gilteriint Fig. .Fig. 2AnPhosphorylated FLT3 decreased by approximately 40% compared with control phosphorylation levels in tumor samples within 1 h after single oral administration of 1–10 mg/kg gilteritinib Fig. c, indicaP < 0.05) and 3 mg/kg/day , and near-complete tumor regression was seen at 6 mg/kg/day  and 10 mg/kg/day  , 1.6 nM (95% CI: 1.1–2.4 nM), 2.1 nM (95% CI: 1.4–3.0 nM), 22 nM (95% CI: 15–33 nM), and 49 nM (95% CI: 29–83 nM), respectively. The growth of parental Ba/F3 cells in the presence of IL-3 was inhibited with an IC50 value of 420 nM (95% CI: 350–500 nM), which represents the off-target inhibition of kinases other than FLT3. We confirmed that phosphorylation of FLT3 and its downstream targets STAT5, AKT, and ERK were inhibited by gilteritinib in a dose-dependent manner in Ba/F3 cells expressing either FLT3-ITD, FLT3-D835Y, or FLT3-ITD-D835Y , 5.7 nM (95% CI: 1.5–21 nM), and 35 nM (95% CI: 9.3–130 nM), respectively.Since mutations within the TKD of FLT3  often confer resistance to FLT3 inhibitors that were previously effective against FLT3-ITD , the eff35Y Fig. . FurtherThe finding that gilteritinib inhibited FLT3-D835Y and FLT3-ITD-D835Y, both of which harbor mutations in the activation loop essential for binding type 2 inhibitors, suggests that gilteritinib is a type 1 FLT3 inhibitor. To further understand the inhibitory activity of gilteritinib against mutated FLT3 at the structural level, computational modeling was performed for FLT3 with gilteritinib Fig. . The mod6 photons/s) on the penultimate day of treatment (day 70). Furthermore, when control mice showed MV4–11 cell infiltration in several areas of the body, gilteritinib-treated mice had no evidence of cell infiltration   Fig. c.Fig. 5GThese data demonstrate that gilteritinib, a FLT3/AXL TKI, shows potent efficacy in preclinical models of FLT3-mutated AML. In vitro kinase assays using gilteritinib revealed strong inhibition of FLT3 and AXL (in the nM range) and an 800-fold weaker inhibition of c-KIT than FLT3. Gilteritinib also inhibited phosphorylation of FLT3 with ITD and/or activation loop D835Y mutations in cellular assays. The inhibitory activity of gilteritinib against the D835Y mutation is further supported by computational modeling that showed gilteritinib binds to FLT3 within the activation loop far from the D835 site, allowing kinase inhibition to occur in the face of FLT3 mutations at this location. This potent FLT3 inhibition translated to a marked decrease in the cell viability of FLT3 mutation-positive cell lines with inhibition of several downstream targets. In vivo, gilteritinib was distributed at high levels in tumors after single oral administration and showed antitumor activity against FLT3-driven tumors in a mouse xenograft model. This antitumor activity was associated with a durable inhibition of phospho-FLT3 and phospho-STAT5, demonstrating the in vivo inhibitory effect of gilteritinib. Furthermore, treatment with gilteritinib decreased the leukemic burden and prolonged survival in a mouse IBMT model. Taken together, these data suggest that gilteritinib blocks FLT3 phosphorylation, impairs downstream signal transduction, and consequently inhibits AML cell proliferation in both in vitro and in vivo models.flt3 and c-kit show a large overall decrease in hematopoietic cell number, whereas mice deficient in only flt3 have normal mature hematopoietic populations with specific deficiencies in primitive B lymphoid progenitors [These preclinical findings regarding the activity of gilteritinib suggest it may be a potential therapeutic treatment option for patients with FLT3-mutated AML. Although several TKIs have been investigated as potential therapies for AML, none have yet to be approved for clinical use , 9. Firsgenitors . Thus, tgenitors .When comparing the next-generation FLT3 inhibitors in development, all three compounds exhibit similar activity against FLT3-ITD in vitro, inhibiting the growth of MV4–11 cells at low nM concentrations  , 25. HowAlthough several similarities exist between gilteritinib and crenolanib , crenolanib treatment showed limited efficacy at the maximum tolerated dose in a xenograft mouse model in which MV4–11-luc cells were inoculated by intravenous injection, resulting in a significant survival benefit without a complete reduction in tumor cells . By contIn addition to the strong preclinical inhibition of FLT3, gilteritinib also targets AXL. In vitro studies have shown that AXL is important for both wild-type and mutant FLT3 activation, suggesting that AXL may have a role in the pathobiology of AML . FurtherAlthough these preclinical studies clearly demonstrate the potent activity of gilteritinib on FLT3 coupled with minimal activity against c-KIT, a few limitations should be noted. While these data are suggestive of a possible enhanced benefit to blocking both FLT3 and AXL, further studies are needed to fully characterize and understand the effect of gilteritinib on AXL in preclinical models thereby helping to fully elucidate the role of AXL in FLT3-mutated AML. In addition, understanding the effect of gilteritinib in cells from patients with AML may help shed light on the clinical implications of treatment with a FLT3-mutant inhibitor because the patient’s cells will be heterogeneous for the FLT3 mutation.Overall, gilteritinib showed consistently potent inhibition of FLT3 in preclinical studies and demonstrated strong efficacy in FLT3-mutant tumor models. Furthermore, initial in vitro assays suggest that gilteritinib effectively blocks AXL activity while having minimal effect on c-KIT. Based on these data, it can be postulated that gilteritinib may lead to a reduced blast number and prolonged survival in patients with FLT3-ITD- or FLT3-D835Y-positive AML. This hypothesis requires confirmation in clinical studies.ESM 1(DOCX 730 kb)


Plasmodium falciparum parasites within human erythrocytes. Parasites invade erythrocytes via a coordinated sequence of receptor-ligand interactions between the parasite and host cell. One key ligand, Apical Membrane Antigen 1 (AMA1), is a leading blood-stage vaccine and previous work indicates that phosphorylation of its cytoplasmic domain (CPD) is important to its function during invasion. Here we investigate the significance of each of the six available phospho-sites in the CPD. We confirm that the cyclic AMP/protein kinase A (PKA) signalling pathway elicits a phospho-priming step upon serine 610 (S610), which enables subsequent phosphorylation in vitro of a conserved, downstream threonine residue (T613) by glycogen synthase kinase 3 (GSK3). Both phosphorylation steps are required for AMA1 to function efficiently during invasion. This provides the first evidence that the functions of key invasion ligands of the malaria parasite are regulated by sequential phosphorylation steps.Central to the pathogenesis of malaria is the proliferation of  Plasmodium falciparum, the causative agent of the most severe form of human malaria, remains an outstanding global health and socioeconomic burden causing more than 200 million clinical cases of disease and over 400,000 deaths per yearThroughout the erythrocytic life cycle, where the clinical manifestations of malaria arise, parasites remain largely protected intracellularly. ‘Merozoites’ produced after each cycle emerge and actively invade new erythrocytes with remarkable coordination and speed2610 by cyclic AMP-dependent protein kinase A (PKA)31617One key player within this invasion machinery is the widely studied invasion ligand Apical Membrane Antigen 1 (AMA1). AMA1 is synthesized during schizogony as an 83 kDa precursor, which is cleaved within specialized secretory organelles called micronemes to yield a 66 kDa mature protein that then is trafficked to the merozoite surface, where it is anchored by a single transmembrane domain exposing a large N-terminal ectodomain and a short C-terminal cytoplasmic domain (CPD)578910111213P. falciparum.Here we reveal key functional phosphorylation events upon AMA1, confirming the primary role of PKA in initiating a hierarchy of phosphorylation events involving a secondary kinase glycogen synthase kinase 3 (GSK3). Multiple phosphorylation appears to impart functional efficiency onto AMA1 during erythrocyte invasion by 588, S590, S601, S610, T612, and T613, and phosphoproteomics on parasite material indicates that all of these are indeed phosphorylated1718610 for red blood cell invasion has been previously reportedet al.588A, S590A, S601A, S610A, T612A, T613A) were ectopically expressed as TY1 fusion proteins in the 3D7 parasite cell line. Importantly, the ectodomain of W2mef AMA1 contains certain amino acid changes that render it resistant to the invasion blocking effects of R1 peptide, which binds 3D7 AMA1 and inhibits RON binding and junction formationWT-TY1) functionally complemented the endogenous protein and, as previously reported610 (AMA1S610A-TY1) decreased invasion efficiency by approximately 70% (613 mutant (AMA1T613A-TY1). This was in contrast to the AMA1S588A-TY1 and AMA1S590A-TY1 mutant, whose complementation capacities were barely reduced, and the AMA1S601A-TY1 and AMA1T612A-TY1 mutants, which displayed intermediate reduction phenotypes , and T613 is absolutely conserved, indicating a key function of these two residues.Six potential phosphorylation sites are present within the cytoplasmic domain of AMA1, Stely 70% . Interesenotypes . This reet al.S610, the evidence is based on the observation that mutation of S610 precludes phosphorylation and is therefore largely indirect. To first demonstrate that PKA phosphorylates S610in vitro, recombinant AMA1 CPD variants were generated as GST fusion proteins for in vitro phosphorylation assays. Wild-type AMA1 (GST-AMA1WT) and phosphorylation-defective S610A (AMA1S610A) were compared with AMA1 with a single acceptor site at S610 left intact and all other phosphorylation sites mutated to non-phosphorylatable alanines (AMA1S610). Phosphorylation of AMA1S610A by purified bovine PKA , H-89 inhibited phosphorylation by 47% and 87%, when considering the Lambda phosphatase dephosphorylated sample as having a baseline signal . There are two MAP kinases in the P. falciparum genome Pfmap1 and Pfmap2 that are expressed in blood stages. The latter kinase is essential and might be required during schizogony as well as for other life cycle stagesP. falciparum also has a cdk5 homolog called protein kinase 5 which appears to have nuclear functionsPfGSK3) is also encoded in the parasite genome and appears to be constitutively expressed throughout the asexual cell cycle based on RNA expression and antibody based protein detection252627610 phosphorylation. This characteristic suggests that PfGSK3 could be involved in T613 phosphorylation subsequent to phosphorylation of S610 by PKA. To confirm PfGSK3 expression in blood stages, the endogenous gene (PF3D7_1316000) was tagged with green fluorescent protein (GFP)   and locain (GFP) .in vitro and found that, indeed, hGSK3β phosphorylated AMA1WT but not AMA1PM in which all the phospho-sites have been mutated  and found that only AMA1T613 was phosphorylated by hGSK3β  were subjected to in vitro phosphorylation assays with parasite extracts, but, while AMA1WT and AMA1S610 were phosphorylated, presumably by residual PKA, AMA1T613 displayed no signal . Recombinant proteins were first incubated with PKA in the presence of non-labeled ATP. Subsequently, these pre-treated AMA1 variants were incubated with either secondary PKA or parasite material in the presence of 32P-γ-ATP, and only incubation with the parasite material resulted in labeling on the substrate in AMA1WT and AMA1S610/T613, but not in the negative control AMA1PM  that also require phospho-priming. This suggests that phosphorylation by PKA is required for further phosphorylation of T613. We then demonstrated that pre-phosphorylation by PKA is required specifically for phosphorylation by recombinant P. falciparum GSK3 protein. After PKA phosphorylation of AMA1WT, AMA1S610 and AMA1S610/T613, only the latter variant could be phosphorylated by PfGSK3β   . Taken tPfGSK3 inhibitor 5v. Although H-89 probably has activity on other kinases we hoped to concentrate its effects against PKA by restricting its use to a two hour window when PKA was maximally expressed. A sample of the culture supernatant, which contained the secreted luciferase that is released during host cell rupture, was collected to quantify levels of egress. To measure invasion, the drug was washed from the cultures. Further invasions were blocked using heparin, and whole culture samples were collected 24h later during peak luciferase expression. Dose-response curves were generated for egress and invasion indicate that the PfPKA inhibitor H-8950 concentration of 4.9 μM  needs to be phosphorylated in order to achieve efficient completion of the invasion processThe invasion of erythrocytes by malaria parasites is a rapid but highly complex process, for which the specific molecular details have been only partially resolved. Several post-translational modifications of the parasite’s invasion machinery, including protein phosphorylation, are thought to act as important checkpoints to facilitate efficient entry into the host cell31617in vivo screen to analyse the contribution of each CPD phospho-site toward the function of AMA1, confirming the importance of S610 and further identifying T613 as a key residue for efficient invasion. We were able to confirm the role of protein kinase A (PKA) in phosphorylation of S610 and revealed GSK3 as a strong candidate for phosphorylation of T613. This was further substantiated by the use of specific inhibitors for PfPKA and PfGSK3 that were able to block invasion. These kinases probably phosphorylate many proteins and at least the H89 inhibitor targets other kinases but by limiting their application to a two hours invasion window we hoped to restrict their major effects to AMA1. Eliminating the possible roles of other proline-directed kinases such as PfMAP2 kinases and PfPK5 would also help validate PfGSK3 as the kinase acting upon T613. Importantly, we found that T613 phosphorylation by parasite lysates and by recombinant GSK3 is dependent upon phosphorylation of S610 by PKA, which is consistent with the documented preference of GSK3 for pre-phosphorylated substrates610 acts as a master switch for phosphorylation.In this study, we used an PfPKA is expressed late in the asexual cell cycle25in vitro substrate of the parasite kinase. Previous findings using cross-reacting antibodies directed against mammalian GSK3 indicate that PfGSK3 associates with Maurer’s clefts in the iRBC cytosol and it was assumed that it might function in cytoskeletal stability or controlling ion channels for the circadian rhythmPfGSK3 cannot be excluded, our data, which are based on GFP-tagged PfGSK3 expressed from the cognate locus, show that the vast majority remains in the parasite either as a nuclear-associated or cytosolic protein. The cytosolic fraction of PfGSK3 is consistent with its role in AMA1 phospho-activation in late stage parasites. Additional nuclear localization of GSK3 is also described for other cell types363739Plasmodium GSK3.PKA is a ubiquitous kinase with numerous roles across many different organisms, and several studies using stimulants and inhibitors of PKA and modulation of gene expression have highlighted the indispensability of this kinase for parasite growth213233610 itself has a functional role in addition to acting to control T613 phosphorylation is yet to be determined. While GSK3 generally responds to phospho-priming, S610 is not located within the GSK3 recognition sequence and thus its phosphorylation may not be a classical phospho-priming step but rather act by opening the conformation of the AMA1 CPD to expose T613 for phosphorylation. Alternatively, the priming mechanism may be conserved but the location of the priming site may have diverged between the mammalian and Plasmodium enzymes. Integrating these findings, we propose the following scenario for activation of AMA1: During schizogony the activity of a bicarbonate-sensitive cytoplasmic adenylyl cyclase increases, which generates the cyclic AMP necessary to activate PKA by dissociation of the active catalytic subunit from the regulatory subunit610 likely defines the rate-limiting step in the hierarchical AMA1 phosphorylation cascade, where phosphorylation of T613, likely by GSK3, follows.Since GSK3 appears to be constitutively expressed with no evident regulation mechanisms, this presents a sophisticated means of controlling AMA1 phosphorylation. PKA signaling, on the contrary, has multiple upstream regulators (described below). Whether phosphorylation of S588, S590, S601, and T612 are not known but individually they do not seem to be as functionally important for invasion since phosphorylation-defective mutations only moderately reduce invasion efficiency. Interestingly, phosphorylation of S610 is likely also important for the phosphorylation of these sites as it is hypothesized to be a master switch for all AMA1 phosphorylationThe kinase(s) that could phosphorylate the other sites present on the CPD, such as SS610A parasites in this complementation assay showed binding and vigorous deformation of the host erythrocyte despite the inability to invadeThe functional specific steps that follow AMA1 phosphorylation are not presently understood. AMA1 appears to be phosphorylated at multiple sites soon after synthesis in late schizonts, as the 83 kDa proprotein form is phosphorylated alongside the processed 66 kDa form, but the purpose of this is currently unknownin vitro and the binding of ligands to aldolase does not even appear to be necessary for invasion in Toxoplasma gondiiP. falciparum ligands promote actin polymerization likely via aldolase, which also binds and polymerises actin for actin-myosin motor function, however AMA1 was not testedWe originally proposed that phosphorylation may facilitate binding of the AMA1 CPD to the actomyosin invasion motor via an aldolase bridge thereby permitting the motor to apply traction to the junction and facilitating merozoite entry into the erythrocyte610 could become rapidly dephosphorylated following T613 phosphorylation if it serves only as a master switch, yielding a singly phosphorylated substrate. Or phosphorylation and dephosphorylation could occur continuously throughout invasion. The dynamics and functional consequences of S610/T613 phosphorylation will be explored further in future studies.The basic structure of AMA1 and the fact that it does not need to be highly expressed for invasion34547Since AMA1 was first recognised as a central invasion ligand 15 years agoama1 gene (PlasmoDB accession number PF3D7_1133400) was amplified from W2mef P. falciparum cDNA. In vitro mutagenesis of ama1 was achieved either by the reverse oligonucleotides containing the mutation site (stop-codon was removed) or by using a two-step primer directed PCR mutagenesis method® High-Fidelity DNA Polymerase (NEB). To ensure correct timing of gene transcription, the ama1 transgenes were controlled by the ama1-promotor using the pARL1-VectorKpnI/AvrII restriction sites into the transfection vector.The PfAMA1 C-terminal tail was cloned via BamHI/XhoI restriction sites and without a stop-codon into the bacterial expression vector pGEX-4T-1 . This construct produces fusion proteins of PfAMA1 tail N-terminally bound to GST. Different mutants of the GST-PfAMA1 tail fusion protein were achieved by site-directed mutagenesis. Purification of GST fusion proteins was performed according to the manufacturer’s protocol .For the generation of GST (glutathione-S-transferase) fusion proteins the DNA sequence of the PfGSK3 as GFP fusion protein were generated by a 3’replacement of the endogenous gsk3 gene. The last 854 bp of the gsk3 coding region (PlasmoDB accession number PF3D7_0321400) was amplified from 3D7 gDNA without the stop-codon and transferred into the transfection vector pARL-GFP via the NotI/AvrII restriction sites. By using NotI restriction site the ama1 promotor was removed which leads, only after integration, to a detectable GSK3-GFP construct. All constructs were confirmed by sequencing. Integration at the right locus was confirmed by PCR analysis of the gDNA.3D7 parasites expressing The probes were collected from ~7–8% synchronized schizont cultures. The parasite material was suspended in SDS sample buffer and incubated at 90 °C for 5 minutes. Proteins were separated on 10–15% SDS-PAGE and subsequently transferred onto a nitrocellulose membrane. Immunoblots were performed using standard procedures and developed by chemiluminescence using ECL . Antibodies used in immunodetection were rabbit monoclonal anti-TY1 (Diagenode) and monoclonal anti-GFP (Roche). Anti-TY1 was used 1:1000, anti-GFP antibody 1:1000 and anti-Aldolase 1:5000 diluted in phosphate-buffered saline (PBS) with 5% w/v skim milk. Secondary antibodies were sheep anti-rabbit IgG horseradish peroxidase (Sigma) and sheep anti-mouse IgG horseradish peroxidase (Roche) used 1:5000 diluted in PBS with 5% w/v skim milk.P. falciparum asexual blood stages were cultured in 0+ erythrocytes. W2mef were derived from the Indochina III/CDC strain. 3D7 and W2mef were transfected with 100 μg of purified plasmid DNA as described previouslydhfr gene. For single cross-over integration of the gsk3-gfp into the endogenous gene locus, after transfection the parasites were alternately grown with and without WR99210 pressure  to promote integration.®16 green fluorescent nucleic acid stain (Invitrogen) for 30 minutes at 37 °C and washed three times with media. For proper cell counting each culture was diluted 1:10 in media and afterwards applied to the FACS for measuring parasitemia. Assays were performed in triplicates on three independent occasions.Parasite erythrocyte invasion assays were performed using 3D7 and transgenic parasites. Parasitemia of sorbitol synchronized parasite culture was measured using a Becton-Dickinson LSR II flow cytometer (FACS). For the experiment a parasite culture with 0.5% parasitemia of trophozoites (4% hematocrit) was incubated in a 96-well Plate (150 μL per well) under standard culturing conditions for 48 hours to allow re-infection in the presence or absence (control) of 100 μg/mL R1. After reinvasion occurred, parasites were stained with 0,1 μM syto50 values.H89 and 5v were serially diluted in DMSO at 500x working concentration to be added to luciferase-expressing parasite cultures with a final concentration of 0.2% DMSO. Synchronised late-stage parasite cultures (~44–48 hour post invasion) were seeded into 96-well microplates at 5% parasitaemia and 2% haematocrit. Compounds were titrated in triplicate and incubated over egress and invasion . Samples of culture supernatant were collected to determine the amount of luciferase released during egress, then the compounds were washed out to prevent inhibition of parasite growth and 100 μg/mL heparin sodium salt (Sigma) was added to prevent any further invasion events. Parasites were returned to culture for a further 24 h and whole culture samples were collected to determine the amount of luciferase expressing parasites present. Whole culture samples were mixed with 0.6x sample volume PBS, 0.4x sample volume Luciferase Cell Culture Lysis 5X Reagent (Promega) and 0.01x sample volume NanoGlo Luciferase Assay Substrate (Promega). Light units emitted from the reaction were measured in a luminometer with the gain reduced by 10% for the sample with the highest signal to prevent saturation. Relative light units were plotted in Prism 6 (GraphPad) and normalized against untreated (100%) and uninfected (0%) samples to generate drug curves and ICPfGSK3 was expressed and purified as a GST fusion protein using pGEX-4-T3 in E. coli BL21 C41PfGSK3 from the flow through was collected and concentrated.2; 2 mM MnCl2, 50 mM NaV, NaF, β-glycerophosphate; EDTA-free complete protease inhibitor cocktail (Roche)) with 200 μM ATP and incubated overnight. The fusion proteins were washed 3 times with ice-cold TBS and incubated again with 1% bovine PKA for 3 h followed by another 3 washes. Phosphorylation assays were performed in 50 μL total volume containing kinase buffer, 1 μCi γ-[32]P-ATP (Perkin Elmer) and 2 μM cAMP or 1 μM CaCl2 (where indicated). Reactions were initiated by addition of 3 μg/100 μL total parasite lysate, 1% bovine PKA, 4 μg/mL human GSK3β (Biaffin) or 25 μg/mL recombinant PfGSK3. The samples were incubated for 45 min at 30 °C, stopped by the addition of Laemmli sample buffer, and analysed by SDS-PAGE. If Thrombin cleavage was necessary, beads were washed three times with ice-cold PBS and GST-tag was cleaved by addition of 0.04 U biotinylated Thrombin according to manufacturer’s protocol (Novagen). SDS-PAGE gels were analysed for total protein by staining with coomassie, and by radiolabeling using a Fuji FLA-7000 or a Typhoon phosphorimager. Two independent experiments were performed in triplicate. To establish background levels of phosphorylation, the density of the GST autoradiograph signal was subtracted from the GST-AMA1 signals. This set the GST sample to 0% and AMA1WT protein was normalised to 100%.Parasite lysates were prepared as describedPfAMA1 (1F9)  or rabbit anti-PfAMA1  and blocked with 1% BSA. Treated parasites were lysed and added to the wells to purify AMA1 . After washing with TBS, the wells were probed with 1 μg/mL rabbit anti-PfAMA1S610p (Genscript [peptide: SFWGEEKRASpHTTPV]) or mouse anti-phosphothreonine (Abcam). The rabbit anti-PfAMA1S610p was detected with 0.2 μg/mL goat anti-rabbit antibody conjugated to horseradish peroxidase (Abcam), and the mouse anti-phosphothreonine antibody was detected with 0.2 μg/mL goat anti-mouse antibody conjugated with biotin followed by streptavidin conjugated with horseradish peroxidase . The wells were developed using tetramethylbenzidine substrate (Thermo Scientific) followed by an equal volume of 2 M HCl, and readouts were performed in a spectrophotometer at 450 nm.Compounds were prepared in DMSO and added to late-stage parasite cultures (44–48 hour post invasion) at a final concentration of 0.2% DMSO then incubated for 2 h at 37 °C. Sample dephosphorylation was carried out with 400U Lambda phosphatase (New England Biolabs) according to the manufacturer’s protocol. Nunc MaxiSorp 96 well ELISA plates were coated with 10 μg/mL mouse anti-Images of unfixed GFP-expressing parasites were captured using a Zeiss Axioskop 2plus microscope with a Hamamatsu Digital camera  with 1 μg/mL DAPI (Roche) for nuclei stain. Confocal microscopy was performed using the Olympus FV1000 confocal microscope. For 3D reconstitution 20–32 z-stacks (0.3 μm step size) were collected using 488 nm laser. All confocal images were analyzed and processed in Imaris 6.2.0. Gauss filters were used with the filter width suggested by Imaris. Single images were processed in Adobe Photoshop CS4.How to cite this article: Prinz, B. et al. Hierarchical phosphorylation of apical membrane antigen 1 is required for efficient red blood cell invasion by malaria parasites. Sci. Rep.6, 34479; doi: 10.1038/srep34479 (2016).


Phosphorylation-dependent YAP translocation is a well-known intracellular mechanism of the Hippo pathway; however, the molecular effectors governing YAP cytoplasmic translocation remains undefined. Recent findings indicate that oncogenic YAP paradoxically suppresses Wnt activity. Here, we show that Wnt scaffolding protein Dishevelled (DVL) is responsible for cytosolic translocation of phosphorylated YAP. Mutational inactivation of the nuclear export signal embedded in DVL leads to nuclear YAP retention, with an increase in TEAD transcriptional activity. DVL is also required for YAP subcellular localization induced by E-cadherin, α-catenin, or AMPK activation. Importantly, the nuclear-cytoplasmic trafficking is dependent on the p53-Lats2 or LKB1-AMPK tumor suppressor axes, which determine YAP phosphorylation status. In vivo and clinical data support that the loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. Our observations provide mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer. Hippo and Wnt pathways are important for cancer development, and they can cross talk; however, the mechanisms behind this connection are unknown. Here the authors show that DVL (a scaffold protein in the Wnt pathway) regulates the shuttling of YAP (a key component of the Hippo pathway) between cytoplasm and nucleus in specific tumor suppressor contexts. Inhibition of the Hippo pathway leads to increased nuclear YAP abundance and TEAD transcriptional activity, resulting in increased organ size as well as overgrowth of cancer4. Conversely, activation of the Hippo pathway induced by cell-to-cell contact leads to phosphorylation and inhibition of nuclear YAP. In mammals, large tumor suppressor (Lats)1/2 serine/threonine kinase phosphorylates at multiple sites on YAP, including Ser127, resulting in cytoplasmic translocation from the nucleus6. Recently, AMP-activated protein kinase (AMPK) has been shown to directly phosphorylate YAP, resulting in cytoplasmic retention and suppression of nuclear YAP activity8. While the phosphorylation-dependent YAP shuttling is critically important in the Hippo pathway and/or in metabolic regulation, the molecular effector of the dynamic intracellular shuttling is not known.The Hippo signaling is an evolutionary conserved pathway that inhibits cell proliferation by contact inhibition, its loss leading to both organ growth and cancer development. The Yes-associated protein (YAP) transcription co-activator is a key regulator of the Hippo pathway9. Highly conserved in metazoan, the Wnt signaling is critically important for coordinative regulation of cell-to-cell adhesion from cell membrane to transcriptional activity in the nucleus. The β-catenin, a key mediator of Wnt signaling, functions both as intercellular adhesion complex through binding to cytoplasmic domain of E-cadherin and as transcriptional co-activator in the nucleus with T-cell factor/lymphoid enhancer factor (TCF/LEF)10. Because the Hippo and Wnt pathways similarly regulate intercellular adhesion and nuclear transcriptional activity11, elucidating a reciprocal link between the two pathways may reveal an important molecular mechanism in human cancer and other diseases. Although the co-activation of Wnt signaling and YAP activity are commonly observed in human cancer, recent findings point to a dilemma in that YAP suppresses canonical Wnt via binding to Dishevelled (DVL) and/or β-catenin15. Although a large body of studies have focused on YAP regulation of canonical Wnt activity in development and cancer14, the upstream function and molecular mechanisms enabling reciprocal regulations between YAP and Wnt signaling are largely unknown16.The canonical Wnt pathway comprises fundamental extracellular signaling involving diverse developmental process, and deregulation of components involved in the Wnt/β-catenin pathway has been implicated in a wide spectrum of diseases, particularly human cancersIn this study, we found that DVL, a scaffolding protein of the Wnt pathway as well as a key regulator of Wnt-independent epithelial polarity, is a molecular effector for nuclear-cytoplasmic shuttling of YAP in a YAP phosphorylation-dependent manner. Furthermore, oncogenic inactivation of p53/Lats2 and the liver kinase B1 (LKB1)/AMPK tumor suppressor axes, two most commonly observed genetic alterations in human cancer, abolish DVL’s function on YAP nuclear export. The loss of tumor suppressor function allows co-activation of the canonical Wnt pathway and nuclear YAP activity by DVL. Our observations demonstrate molecular mechanisms for the dynamic regulation of YAP activity via subcellular trafficking by DVL as well as the importance of p53 and LKB1 tumor suppressor contexts in the reciprocal control between the canonical Wnt and Hippo pathways.13, we focused on roles of enigmatic DVL on YAP activity in this study. As a key scaffolding protein of the Wnt pathway, DVL in mammal consists of three highly similar homolog genes, DVL1, DVL2, and DVL3. Although DVL2 has received close attention in recent developmental studies due to its ubiquitous abundance in various tissues19, differential expression patterns of DVL homologs in human cancer have not been clearly determined20. We first examined the transcript abundance of DVL homologs from the 1093 breast cancer patients and found that DVL3 transcripts were most abundant in clinical samples  YAP interacted with DVL3, whereas phosphorylation-resistant mutants of YAP largely abolished the interaction , kinase-dead AMPK (AMPK-KD) or LKB1 mutant (LKB1-KD) with DVL3. Indeed, the kinase-dead dominant negative mutant of Lats2 or AMPK or LKB1 attenuated DVL-YAP interaction with decreasing YAP phosphorylation as determined by mobility shift on a phos-tag gel and pSer127-specific YAP antibody , YAP homolog and an integral member of the Hippo pathway, binds DVL12. When we examined the effect of DVL on TAZ, the DVL also interacted with TAZ and suppressed nuclear TAZ abundance, TEAD transcriptional activity indicating the critical function of DVL on the Hippo pathway , a specific inhibitor of CRM1 /exportin 1 required for nuclear export of proteins containing a leucine-rich nuclear export sequence (NES). Under confluent contact inhibition state, endogenous YAP mainly localized in the cytoplasm together with DVL . When we examined the subcellular localization of wt or ASA point mutant DVL3, mutational inactivation of NES was sufficient to restrict DVL in nucleus  mouse embryonic fibroblast (MEF) and 293A cells. When we induced DVL3 in these cells, the nuclear YAP abundance in the Lats1/2−/− cells were not decreased by DVL3 -E6 resulted in suppression of Lats2 protein abundance and a subsequent decrease in YAP phosphorylation  of p53 to examine the intersecting role of DVL on YAP and Wnt44. Indeed, ΔTAD of p53 largely abolished DVL suppression of nuclear YAP while preserving DVL’s potential effect on the canonical Wnt activity  and metformin (Met) in MCF-10A cells, then examined the YAP localization with an inducible DVL3 knockdown system. YAP was mainly localized in nuclear space under sparse culture condition and was translocated into the cytoplasm by metabolic stress  is the key upstream activator of AMPKess Fig. . Importaess Fig. , indicatext Fig. .Fig. 7RoTaken together, our results demonstrate that the intact tumor suppressor function of p53/Lats2 or LKB1/AMPK axes is required for inhibition of nuclear YAP by DVL. Conversely, mutational inactivation of p53/Lats2 or LKB1/AMPK axes in human cancer critically allows DVL to become a potent activator of the canonical Wnt pathway without suppression of nuclear YAP  signaling, binds to phosphorylated YAP with the PDZ domain and PY motif, subsequently regulating nuclear-cytoplasmic trafficking of YAP.The Hippo/YAP and canonical Wnt pathways are highly conserved signaling cascades regulating cell-to-cell interaction and contact inhibition, and hyperactive nuclear YAP and Wnt activity are bona fide oncogenes in many types of human cancer30. Intriguingly, loss of APC activates YAP by interacting with Lats1 in a GSK-3 dependent manner50. Further, Axin regulates the Snail-mediated epithelial-mesenchymal transition (EMT) process by acting as a nuclear exporter of GSK-351. The GSK-3 shuttling function of Axin is also required for phosphorylation of the membranous LRP6 Wnt co-receptor and subsequent activation of the intracellular Wnt activity52. Interestingly, we found that soluble Wnt ligands activate YAP and subsequently decrease affinity to DVL and translocation into nuclear space. Considering that DVL is directly regulated by Frizzled (FZD) receptors, our observations provide an interesting mechanistic insight into intracellular dynamics between Wnt and Hippo.Note that other scaffolding proteins of the Wnt pathway have a similar function. The APC (adenomatous polyposis coli) harbors highly conserved NES, whose mutational inactivation in cancer cells results in nuclear accumulation of β-catenin23. While the phosphorylation-dependent YAP translocation by activation of the Hippo pathway and AMPK is well known8, a molecular effector regulating the YAP nuclear-cytoplasmic shuttle has not yet been identified. We show that NES embedded in DVL is responsible for cytoplasmic translocation of phosphorylated YAP. Importantly, DVL is required for nuclear-cytoplasmic trafficking of YAP induced by contact inhibition, α-catenin, E-cadherin, and metabolic stress, indicating that the intracellular shuttling function of DVL is essential to various developmental and oncogenic contexts. Previous observation showed that knockdown of YAP resulted in nuclear accumulation of DVL13, suggesting that the nuclear export machinery of DVL and YAP are mutually dependent. It should be noted that 14-3-3 is required for cytoplasmic retention of phosphorylated YAP5, and that we found DVL interacts with 14-3-3. Although the key role of 14-3-3 in nuclear-cytoplasmic retention of phosphorylated nuclear proteins such as cdc-25, FKHRL1, and p65-IκBα is well known55, 14-3-3 itself does not have a nuclear export function54. These results, together with our observations, suggest that 14-3-3 may facilitate DVL’s nuclear export of phosphorylated YAP. Further study is required to delineate the role of 14-3-3 in DVL-mediated nuclear-cytoplasmic dynamics of YAP and other phosphorylated transcriptional machinery.As a key scaffolding protein of the Wnt pathway, DVL relays extracellular signals from FZD receptors to downstream effectors, interacts with a wide range of proteins, and is involved in diverse signaling pathways including canonical Wnt and non-canonical PCP2. Although DVL binds and exports phosphorylated YAP, increased nuclear YAP by loss of DVL remained unphosphorylated. Interestingly, Ser127 phosphorylation is important for interaction with DVL; however, Ser127 of nuclear YAP is still unphosphorylated in ASA mutant expressing cells, suggesting that Ser127 phosphorylation may be reciprocally coupled to the nuclear export function of DVL. While this study mainly focused on nuclear-cytoplasmic trafficking functions of DVL, the role of DVL in YAP phosphorylation together with the Lats kinase complex in nuclear space (such as phosphorylation at Ser109 and/or Ser381) needs further study to understand in-depth molecular dynamics in the Hippo and Wnt pathways. It should be noted that the modes of interaction and intracellular function of DVL closely resemble Angiomotin (Amot) inhibiting YAP activity26. While the Amot was identified as an angiostatin-binding protein involved in endothelial polarization and migration, recent studies have revealed that Amot is an important regulator of intracellular localization and nuclear YAP activity57. Amot also harbors a conserved PY motif and C-terminus PDZ-binding motif as do DVL and YAP, respectively58. Interestingly, Amot is also phosphorylated by Lats kinase and subsequent interaction with 14-3-358. Similarly to DVL, the Amot family binds and sequesters phosphorylated YAP56, while the nuclear export function of Amot has not yet been determined.The phosphorylation of YAP is important not only for its intracellular localization but also for protein stability and binding to other partnersDsh in Drosophila) and FZD play a central role in PCP signaling, asymmetric relocalization of Dsh and cytoskeletal reorganization of epithelial cells59. Although many recent findings suggest that the Hippo pathway is tightly connected with PCP in development and epithelial polarity in mammal60, the molecular dynamics are largely unknown. Our observations thus provide an interesting molecular link between Hippo and PCP regulation in development and epithelial polarity in human cancer. It should be noted that we could not find typical NES in Drosophila Dsh while there is highly conserved NES at the distal region of the DEP domain in Ascidian and Xenopus23. Further study is needed to identify the evolutionary role of DVL in epithelial polarity.PCP signaling controls polarization of cells in an epithelial sheet via an FZD–DVL complex mediated by non-canonical Wnt signaling. In this process, DVL  and cultured in DMEM medium. The wt and DVL1/2/3-triple knockout 293 T-REx cells were described previously22. Mycoplasma infection was tested regularly with a PCR-based kit . Cell lines were authenticated as described recently68. The transfection was performed by Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen). For the western blot analyses, protein extracts were prepared in Triton X-100 lysis buffer. The nuclear protein abundances of YAP were determined from nuclear-cytosolic fractionation of protein lysates with hypotonic buffer as described previously69. Briefly, the cells (1 × 106) were collected into microcentrifuge tubes and treated with 400 μl of hypotonic buffer  with protease inhibitors) on ice for 5 min. The cell membrane was ruptured by adding 10% NP-40 to a final concentration of 0.6%, then vigorously vortexed for 10 s followed by high-speed centrifuge for 30 s. The supernatant cytosolic fractions were collected separately, and nuclear pellets were washed with ice-cold PBS twice. Nuclear protein was extracted with hypertonic buffer  on ice for 15 min followed by high-speed centrifuge. Relative nuclear YAP abundance compared to loading control HDAC1 was determined by the ImageJ program downloaded from NIH (https://imagej.nih.gov/ij/). Wnt1 extract and Wnt3a conditioned were prepared from RAC311-Wnt1 cells and L-Wnt3a cell as described previously51. Antibodies against YAP , phospho-S127 YAP , DVL1 , DVL2 , DVL3 , α-catenin , Lats2 , LKB1 , AMPK , phospho-AMPK , HA , p53 , E-cadherin , HDAC1 , GFP , Flag , pan-TEAD  and Tubulin  were obtained from the commercial vendors. Phos-tag gel was purchased from WAKO chemicals (AAL-107).MCF-7, SK-BR-3, HCT-116, SW480, A549, and 293 cells obtained from ATCC were routinely cultured in Dulbecco's Modified Eagle's Medium (DMEM) medium containing 10% fetal bovine serum (FBS). MDA-MB-231 cells (a gift from G. Mills) were cultured in a RPMI1640 with 5% FBS. MCF-10A cells (a gift from M. Wicha) were cultured in DMEM/F12 with 5% horse serum, 20 μg ml41. Tetracycline-inducible wt or NES-mutant DVL3 expression vector was generated with the pTRIPZ lentiviral system (Open Biosystems) by replacing RFP.HA-tagged DVL1, DVL2, and DVL3 expression vectors were kindly provided by E. Fearon (University of Michigan). Deletion or point mutants of DVL3  and YAP deletion mutants of PDZ-binding domain, WW domain and K494R were generated by a PCR-based method. The expression vector pCMV-flag-YAP (plasmid number 19045), pCMV-flag-S127A YAP (plasmid number 19050), pCMV-flag-5SA-YAP (plasmid number 27371), 3xflag-pCMV5-TAZ (plasmid number 24809), pLKO1-shYAP (plasmid number 27368 and 27369), pcDNA-HA-Lats2-K655R , pAMPK alpha2 K45R , pBabe-LKB1-K78I , 14-3-3 gamma (plasmid number 52535) and pcDNA3-alpha-catenin (plasmid number 24194) were obtained from Addgene. Reporter construct having 8 × wt TEAD-binding sites (plasmid number 83467), 7 × mutant TEAD-binding sites (plasmid number 83466), 8 × TCF/LEF-binding sites (plasmid number 12456), and mutant-binding sites (plasmid number 12457) were obtained from Addgene. The Tet-pLKO-puro vector  was used for inducible shRNA knockdown. The target sequences of shRNA were 5′-cgacccagctataagttcttcttca for human DVL3-1 and 5′-caatgacacagagacggactctttg for human shDVL3-2. The expression vectors for shRNA against p53 and expression vectors for HPV-E6 and mutant p53 were as described previouslyFor immunoprecipitation analysis, whole-cell Triton X-100 lysates were incubated with Flag-M2 agarose (Sigma) or Ni-NTA beads (Invitrogen) and washed with lysis buffer three times. The recovered proteins were resolved by SDS-PAGE and subjected for immunoblot analysis. For immunofluorescence study, the cells were washed twice with ice-cold PBS and incubated for 15 min at room temperature with 3% formaldehyde in PBS. The cells were permeabilized with 0.5% Triton X-100 for 5 min and then blocked for 1 h in PBS containing 3% bovine serum albumin followed by incubation with primary antibody overnight at 4 °C. Cells were then washed three times with PBS containing 0.1% Tween 20 followed by incubation with anti-mouse-Alexa Fluor-488 (for green) or anti-rabbit-Alexa Fluor-594 (for red) secondary antibody. Cellular fluorescence was monitored using confocal microscopy (Zeiss LSM780). Functional blocking of endogenous E-cadherin complex was performed by directly adding an HECD antibody (10 μg/ml) to normal culture medium for 16 h prior to immunofluorescence study of YAP.n = 3). The expression of ΔCt value from each sample was calculated by normalizing with GAPDH. Primer specificity and PCR process were verified by dissociation curve after PCR reaction. The primer sequences for qPCR were 5′-accagctcctcctcactaacc for DVL1 forward (F), 5′-tcatgtcactcttcaccgtca for DVL1 reverse (R), 5′-catgagaatctggagcctgag for DVL2-F, 5′-atgctcactgctgtctctcct for DVL2-R, 5′-agaaggtttctcggattgagc for DVL3-F, 5′-tgttgagagtgaccgtgatga for DVL3-R, 5′-caaaatctccaagcctatcaagtt for CTGF-F, 5′-actccacagaatttagctcggtat for CTGF-R, 5′-atgggtgtgaaccatgagaag for GAPDH-F, and 5′-agttgtcatggatgaccttgg for GAPDH-R. For TEAD or TCF/LEF reporter assay, the cells were transfected with 50 ng of the reporter vectors and 1 ng of pSV-Renilla expression vector in combination with YAP and/or DVL3 as indicated. Luciferase and renilla activities were measured using the dual-luciferase reporter system kit (Promega), and the luciferase activity was normalized with renilla activity. The results are expressed as the averages of the ratios of the reporter activities from triplicate experiments.Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. The SuperScript III synthesis kit (Invitrogen) was used to generate complementary DNA. Real-time quantitative PCR analysis for CTGF transcripts was performed with an ABI-7300 instrument under standard conditions and SBGR mix . The illuminahiseq_rnaseqv2-RSEM_genes_normalized (MD5) was log2 transformed and the relative transcript abundance of DVL homologs was compared using one-way ANOVA and Tukey’s HSD test. Mutational status of p53 was obtained from Mutation Annotation Format (MAF) from the Firehose website. To generate Kaplan–Meier plots, clinical samples were grouped by p53 status and dichotomized by Axin2 and CTGF transcript abundance. The scatter plots of CTGF and Axin2 abundances were obtained using R. The high and low abundance subsets were determined based on the median transcript abundance of CTGF and Axin2, yielding groups with the most significant differences in 20-year survival based on the log-rank test. The Kaplan–Meier plots were then generated for the respective groups using the R package survival.Publicly available mRNASeq data of 1098 samples of breast cancer (BRCA) including long-term survival information from The Cancer Genome Atlas (TCGA) was downloaded (6) were resuspended in 100 μl of PBS and injected into flank subcutaneous tissue. The tumorigenic capacity was measured twice a week using a digital caliper. The mice were sacrificed after 5 weeks end-point, and the tumor volume was calculated using the equation V (in mm3) = (a × b2)/2, where a is the longest and b the shortest diameter.All animal experiments were performed in accordance with guidelines of the Institutional Animal Care and Use Committee of Yonsei University and approved by the Animal Care Committee of the Yonsei University College of Dentistry and National Cancer Center Research Institute. Female athymic nude mice (6-weeks-old) were used for xenograft assays into flank subcutaneous tissue. The 293 cells were transiently transfected with expression vectors of DVL3 and YAP in combination with shp53 or LKB-KD as indicated prior to 48 h in vivo inoculation. The cells (1 × 10t-tests; data are expressed as means and s.d. The double asterisks denote p < 0.01, one asterisk denoting p < 0.05. Statistical significance of animal experiments was determined using the Mann–Whitney test. No statistical method was used to predetermine sample size.All statistical analysis of reporter assay, RT-PCR, and soft agar assay was performed with two-tailed Student’s The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationPeer Review File