AR signaling is essential for the growth and survival of prostate cancer (PCa), including most of the lethal castration-resistant PCa (CRPC). We previously reported that TGF-β signaling in prostate stroma promotes prostate tumor angiogenesis and growth. By using a PCa/stroma co-culture model, here we show that stromal TGF-β signaling induces comprehensive morphology changes of PCa LNCaP cells. Furthermore, it induces AR activation in LNCaP cells in the absence of significant levels of androgen, as evidenced by induction of several AR target genes including PSA, TMPRSS2, and KLK4. SD-208, a TGF-β receptor 1 specific inhibitor, blocks this TGF-β induced biology. Importantly, stromal TGF-β signaling together with DHT induce robust activation of AR. MDV3100 effectively blocks DHT-induced, but not stromal TGF-β signaling induced AR activation in LNCaP cells, indicating that stromal TGF-β signaling induces both ligand-dependent and ligand-independent AR activation in PCa. TGF-β induces the expression of several growth factors and cytokines in prostate stromal cells, including IL-6, and BMP-6. Interestingly, BMP-6 and IL-6 together induces robust AR activation in these co-cultures, and neutralizing antibodies against BMP-6 and IL-6 attenuate this action. Altogether, our study strongly suggests tumor stromal microenvironment induced AR activation as a direct mechanism of CRPC. Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in American men. AR signaling is essentially required for most human prostate cancers. Therefore, ADT is the standard treatment for advanced and metastatic PCa. Most prostate tumors initially respond to ADT therapy. However, most tumors will relapse and become the castration-resistant PCa (CRPC). Currently, there is no cure for CRPC. Interestingly, AR signaling is re-activated in most CRPC tumors in the presence of castration level of androgen ligands. Hence, AR remains as a major therapeutic target for CRPC.etc [Several molecular mechanisms may count for this AR re-activation in CRPC, including AR gene amplification/overexpression, AR mutation, the presence of AR splice variants, enhanced AR co-regulators signaling, alterations in steroid metabolism, growth factor and/or cytokine induced AR activation etc . Tumor metc . Prostatetc –5. Transetc , 6–9. Ovetc . Subcutaetc , 14. Theetc –18, we hetc . By condetc , 18 and etc , 18. TheInterleukin-6 (IL-6) is a pleiotropic cytokine that play important roles in regulating immune system and inflammation. It is also a key cytokine in regulating human cancers, including PCa . Serum IIn this report, we used direct co-cultures of LNCaP cells with three different human prostate stromal cell lines to show that prostate stroma-specific TGF-β signaling induces AR activation in LNCaP cells in the absence of significant amount of androgens, and that treatment of MDV3100, a second-generation antiandrogen , only pain vitro PCa/stroma co-cultures by plating LNCaP-TGF-β1(a) cells or LNCaP-Ctrl cells on top of the mostly confluent HPS-19I cells, a previously generated human prostate stromal cell line [We have previously shown that stromal TGF-β signaling promotes prostate tumor growth . To furtell line . Since Lell line , only HP12MLP (Smad binding sequence) reporter construct [12MLP reporter activity in the transfected HPS19I cells, and this induction was inhibited by SD-208 compound in a dose-dependent manner  protein kinase-specific inhibitor . To deteonstruct  and a pRr Figure . 400 nM r Figure . Consistr Figure .etc., as well as notably KLK3 (PSA), a prostate epithelial cell specific marker and an AR target gene  control treated LNCaP-Ctrl/HPS19I co-cultures, (2) 400 nM of SD-208 treated LNCaP-Ctrl/HPS19I co-cultures, (3) control treated LNCaP-TGF-β1(a)/HPS19I co-cultures, and (4) 400 nM of SD-208 treated LNCaP-TGF-β1(a)/HPS19I co-cultures. After 6-day treatment, total RNAs were directly extracted from these co-cultures from two independent studies, reverse transcribed, and submitted for microarray analysis using the 4×44K Whole Human Genome Oligo Microarray chip (Agilent Technologies). We found that 187 transcripts representing 136 unique genes were differentially expressed among these groups , including 79 unique genes (in red) that were induced by TGF-β, but blocked or reversed by SD-208 treatment, as well as 23 genes (in green) that were repressed by TGF-β, but blocked or reversed by SD-208 treatment Figure . The mosne Table . The mosc. Table . Controletc.  cells or LNCaP-Ctrl cells cultured in RPMI1640 supplemented with 0.2% FBS. Conditional media, but not TGF-β1 ligand, were used here to more closely simulate the co-culture conditions. After 6 days of treatment, we extracted total RNA from these HPS19I cells and performed microarray. 2349 transcripts representing 1377 genes were differentially expressed among these two treatment groups , a prostate epithelial cell specific and AR regulated gene, was induced in the LNCaP-TGF-β1 (a) / HPS19I cell co-cultures, but not expressed in HPS19I cells when cultured alone. Furthermore, SD-208 treatment blocked this induction Table .Since PSA is a well-established AR target gene in human PCa, our above observation suggested that stroma-specific TGF-β signaling might induce AR activation in PCa cells. To test this, we performed qPCR on the expression of additional well-established PCa epithelia-specific AR target genes including KLK4 and TMPRSS2 in these LNCaP/HPS19I co-cultures. TGF-β induced the expression of all these three AR target genes in the LNCaP-TGF-β1(a)/HPS19I co-cultures, but not in LNCaP cells (data not shown) or HPS19I cells cultured alone Figure . FurtherTo further reduce androgen levels, we also performed LNCaP-TGF-β1(a)/HPS19I co-cultures and LNCaP-Ctrl/HPS19I co-cultures in RPMI1640 media supplemented with 0.2% charcoal stripped FBS deprived of androgen, and treated them with increasing dosages of DHT and/or 10 μM of MDV3100, a second-generation antiandrogen , for sixTo confirm that our observed stromal TGF-β signaling induced PSA expression is AR dependent, we performed knockdown of AR in LNCaP-Ctrl and LNCaP-TGF-β1(a) cells using two independent siRNAs against human AR. Western blots confirmed significant knockdown of AR in both cell lines Figure . These ATo further expand our observation, we also performed co-cultures of LNCaP cells with HTS33B cells and HPS33Q cells, two additional human prostate stromal cell lines that we prepared following a previously described protocol , 38. TheIL-6 is a key cytokine regulating PCa biology, including inducing AR activity and promoting androgen-independent growth –25. BMP-We next examined whether the presence of prostate stromal cells is required for the IL-6/BMP-6 induced AR activation in LNCaP cells. In contrast to our above observations in the co-culture system, neither IL-6 treatment alone nor BMP-6 treatment alone can greatly enhance PSA expression in LNCaP cells cultured alone in RPMI 1640 media supplemented with 0.2% charcoal stripped FBS. However, co-treatments of both IL-6 and BMP-6 induced robust expression of PSA in the LNCaP-only cell cultures, further confirming the cooperative activities of IL-6 and BMP-6 in inducing AR activation in LNCaP cells Figure . We madeFinally, to further evaluate the functional roles of IL-6 and BMP-6 in mediating stromal TGF-β signaling induced AR activation in LNCaP cells, we treated the LNCaP/HTS33B and LNCaP/HPS33Q co-cultures with IL-6 and BMP-6 neutralizing antibodies. Use of IL-6 neutralizing antibody produced an apparent reduction in stromal TGF-β signaling induced AR activation in LNCaP cells, whereas BMP-6 neutralizing antibody attenuated this induction to a less extent Figure . AltogetTumor microenvironment is important for prostate cancer progression. TGF-β is a key cytokine in regulating cancer progression, either by directly regulating tumor cells, such as inducing epithelial to mesenchymal transition (EMT), or indirectly by regulating tumor microenvironment, such as inducing extracellular matrix remodeling, regulating angiogenesis as well as immune responses. We showed here that TGF-β could indirectly induce AR activation in PCa cells through directly modulating stromal cells in tumor microenvironment. Since AR re-activation is requisite for the recurrence of most CRPC tumors, this stromal TGF-β signaling induced AR activation in PCa cells may provide a direct mechanism for CRPC. Importantly, stromal TGF-β signaling greatly potentiated and enhanced DHT-induced AR activation, suggesting that it might be a therapeutic target to inhibit AR activation in CRPC tumors in the presence of castration-levels of androgen.Our current data suggest that IL-6 and BMP-6 might partially mediate stromal TGF-β signaling induced AR activation. Interestingly, although neither IL-6 nor BMP-6 could robustly induce AR activation in LNCaP cells when cultured alone, IL-6 and BMP-6 together greatly enhance AR activity in these cells. Notably, when prostate stromal cells are presented in the LNCaP/stromal cell co-cultures, IL-6 and BMP-6 can each significantly enhance AR activation in LNCaP cells, which may attribute to the basal activation of IL-6 and BMP-6 signaling pathway in these co-cultures. As the expression of IL-6 and BMP-6 are elevated in the tumor microenvironment, these may be important factors in the evolution of castration resistant disease.Consistent with many previous reports, MDV3100 showed robust AR antagonist activities in most of our LNCaP/HPS19I co-cultures studies. However, MDV3100 also exhibited slight apparent AR agonist activity under certain experimental conditions, such as in LNCaP/HPS19I cell co-cultured in 0.2% of charcoal-stripped FBS as described in Figure Scios, Inc. .The Porcine TGF-β1(204-B-002), recombinant human BMP-6 (507-BP), recombinant human IL-6 (206-IL-010), antibodies against human BMP-6 (AF-507), human IL-6 (AF-206-NA), and normal goat IgG (AB-108-C) were purchased from R&D System . MVD3100 was purchased from Selleckchem . SD-208 was obtained from LNCaP cells were purchased from ATCC and cultured in RPMI1640 supplemented with 10% FBS . LNCaP-TGF-β1(a) cells overexpressing an HA-tagged constitutively activated TGF-β1 ligand, HA-TGF-β1(a), and control LNCaP (LNCaP-Ctrl) cells were generated as previously described . Human p5 cells per well and subjected to various treatments in RPMI1640 supplemented with 0.2% FBS or charcoal stripped FBS for 6 to 28 days. The treatments include 50 pM (1.25ng/ml) porcine TGF-β1 (R&D Systems), 400 nM of SD-208, 0.1, 1, and 10 nM of DHT (Sigma), 10 μM of MDV3100 , 25 ng/ml of IL-6, 50 ng/ml of BMP-6, 2 μg/ml of IL-6 neutralizing antibody, 2 μg/ml of IL-6 neutralizing antibody (R&D system), and various vehicle controls. When applicable, LNCaP cells and prostate stromal cells were each similarly cultured and treated as those for the LNCaP/Stroma co-cultures. Total RNA was extracted from these co-cultures using the RNeasy Miniprep kit  or TRIzol® reagent (Invitrogen).HPS19I, HTS33B, and HPS33Q cells were first allowed to grow in Bfs media to confluence on 6 well plates. LNCaP cells were then plated on top of the stromal cells at 1×10Two siRNAs from Invitrogen, AR/HSS179973 (siRNA#1) and AR/HSS179972 (siRNA#2), were used to knock down AR expression in LNCaP-TGF-β1(a) cells and LNCaP-Ctrl cells. The corresponding sequences are CCGGAAGCUGAAGAAACUUGGUAAU (sense), AUUACCAAGUUUCUUCAGCUUCCGG (antisence) for AR/HSS179973, and GAUGAAGCUUCUGGGUGUCACUAUG (sense), CAUAGUGACACCCAGAAGCUUCAUC (antisense) for AR/HSS179972. siRNAs were transfected into LNCaP cells using GenMute™ siRNA Transfection Reagent following manufacture's protocol . Western blots were used to verify knockdown of AR. Antibodies used are anti-AR  and anti-β-actin .12MLP [® Reporter Assay system (Promega). The reporter firefly luciferase activity was normalized to the co-transfected pRL-null Renilla luciferase activity.HPS19I cells were transfected with 0.4 μg of (CAGA)12MLP  and 0.1 12MLP . 24 hour5 of LNCaP-TGF-β1(a) cells or LNCaP-Ctrl cells were then plated on top of the HPS19I cells. The co-cultures were maintained in RPMI1640 supplemented with 0.2% FBS and subjected to treatments with 400 nM of SD-208 or vehicle control for 6 days. Conditioned media from LNCaP-TGF-β1(a) cells and LNCaP-Ctrl cells maintained in RPMI1640 supplemented with 0.2% FBS were also collected and used to treat HPS19I cells for 6 days. Total RNA was extracted from these LNCaP-TGF-β1(a)/HPS19I and LNCaP-Ctrl/HPS19I co-cultures and from the differentially treated HPS19I cells using the RNeasy Miniprep kit (Qiagen). The cDNA reverse transcription and fluorescent labeling reactions were carried out using the SuperScript® Plus Direct cDNA Labeling System with Alexa Fluor® aha-dUTPs kit (Invitrogen). Briefly 3 μg of RNA and 3 μg of Universal Human Reference RNA  were labeled in reverse transcription with Alexa647 and Alexa555 aha-dUTPs respectively for each sample. After labeling, each cDNA was mixed with reference cDNA and purified with SuperScript Plus Direct cDNA Labeling System purification module according to manufacturer's instructions. Eluted sample was mixed with 10xBlocking Solution and 2xHiRPM buffer , incubated for 2 min at 95–98° C and hybridized on 4×44K Whole Human Genome Oligo Microarray chip using SureHyb DNA Microarray Hybridization Chambers in DNA Microarray hybridization oven (Agilent Technologies) at 10 rpm, 65°C for 20 hrs. After hybridization, slides were washed in Gene Expression Wash Buffer 1 and 2 for 1 min and than dried by centrifugation at 2000 rpm for 2 min. Microarrays were scanned with a dynamic autofocus microarray scanner  using Agilent-provided parameters . The Feature Extraction Software v9.1.3.1 (Agilent Technologies) was used to extract and analyze the signals.HPS19I cells were grown to mostly confluence in T75 flasks. 5×10The microarray data were processed using bioconductor , with Combat to adjust for batch effects . Fold ch® 480II (Roche Applied Science). PCR primers used are PSA: 5′ ACCAGAGGAGTTCTTGACCCCAAA 3′ and 5′ CCCCAGAATCACCCGAGCAG 3′, KLK4: 5′ GGCACTGGTCATGGAAAACGA 3′ and 5′ TCAAGACTGTGCAGGCCCAGCC 3′ , TMPRSS2: 5′ CGCTGGCCTACTCTGGAA 3′ and 5′ CTGAGGAGTCGCACTCTATCC 3′, GAPDH: 5′ AGCCACATCGCTCAGACAC 3′ and 5′ GCCCAATACGACCAAATCC 3′, IL-6: 5′ CAAAGATGGCTGAAAAAGATGGA 3′ and 5′ CTGTTCTGGAGGTACTCTAGGT 3′, and BMP-6: 5′ GGACAGCGCCTTCCTCAAC 3′ and 5′ CGTACTCCACCAGGTTCACAAA 3′. Relative expression levels were determined by the ΔΔCt method, and normalized to GAPDH.1 μg total RNA was reverse transcribed with random hexamer primers using the SuperScript III First Strand Synthesis kit (Invitrogen). Reversed transcribed product was then analyzed by real-time PCR using Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) on the ABI Prism PE7700 sequence analyzer  or using the PerfeCTa CYBR Green FastMix  on the Light CyclerP < 0.05 was considered statistically significant.The statistical analyses on microarray data were described above. Statistical analyses (Student's t-tests) for comparing gene expression were generated using Graphpad Prism 5.0 .


Drosophila co-expressing human tau and Aβ, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aβ increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aβ42, and blocking this stabilization of tau suppressed Aβ42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aβ-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD.Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer’s disease (AD). β-amyloid (Aβ) lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic  Alzheimer’s disease (AD) is the most common cause of dementia resulting from progressive neuron loss. Two proteins, β-amyloid (Aβ) and tau, accumulate in AD brains and are involved in AD pathogenesis. In healthy neurons, tau binds to microtubules to regulate its stability; in AD brains, however, tau is detached from microtubules and phosphorylated at multiple sites. Such abnormal tau behavior, which is likely to be triggered by Aβ, results in generation of pathological tau species that mediate neuron loss. However, the detailed mechanisms underlying this event remain incompletely understood. Using transgenic flies expressing human tau and Aβ as a model system, we found that tau phosphorylation at specific AD-related sites stabilized microtubule-unbound tau in the early phase of tau mismetabolism to generate toxic tau species. Moreover, this process is critical for Aβ to promote subsequent tau phosphorylation and neurodegeneration. Our results reveal a critical step in the initiation of tau mismetabolism, and this process may represent a potential therapeutic target for AD. Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by two pathological lesions: deposition of β-amyloid peptides (Aβ) as amyloid plaques and the microtubule-associated protein tau in the form of paired helical filaments in neurofibrillary tangles (NFTs) . GeneticTau proteins are normally enriched in neuronal axons, where they regulate microtubule stability. However, in diseased brains, tau is detached from microtubules and aggregated in the cytosol. The microtubule-binding domain of tau mediates interaction to proteins, including tau itself, which can cause self-aggregation into oligomers, protofibrils, and fibrils –20. In ain vitro and in vivo models of AD [in vitro, in cultured cells, transgenic Drosophila, and transgenic mice [Tau is phosphorylated at more than 40 sites in pathological lesions associated with AD –24, and ls of AD –41. A nuls of AD , and thels of AD , 43–45. ls of AD –49, wherls of AD . Tau phonic mice , 50–52.Drosophila showed that Aβ-induced augmentation of tau toxicity caused by Aβ depends on tau phosphorylation at these sites [Tau phosphorylation at GSK3-target sites, as well as Ser262/356, has prominent effects on tau toxicity –56. In cse sites , 56–58. Drosophila models expressing human tau recapitulate key features of human tauopathies, including progressive neurodegeneration and phosphorylation at AD-related sites via conserved kinases [ kinases , 59–63.  kinases , 59, sug kinases –14.Drosophila co-expressing human tau and Aβ42 in neurons [In this study, we investigated the roles of tau phosphorylation at Ser262/356 by PAR-1/MARK and SP/TP sites by GSK3 in an initial step of tau mismetabolism, using transgenic  neurons , 64. We To understand the mechanisms by which abnormal metabolism and toxicity of tau is triggered by Aβ42, we analyzed changes in human tau induced by co-expression of human Aβ42 in transgenic flies . Becausein vivo microtubule-binding assay [Most tau proteins are normally bound to microtubules in axons, and alterations in the distribution of tau are associated with tau toxicity . To deteng assay . In thisupper) is more highly phosphorylated than tau in the faster-migrating band (taulower).Tau proteins expressed in the fly retina are detected as two major bands by western blotting with pan-tau antibody . Followiupper and taulower, we used a panel of antibodies capable of distinguishing the phosphorylation status of tau at AD-related sites. Western blotting with TAU-1 antibody, which specifically recognizes tau protein without phosphorylation at several AD-related sites , detected taulower  sites decreases tau binding to microtubules, whereas replacing these phosphorylation sites with unphosphorylatable Ala significantly increases binding [lower  and Thr231 (pThr231)  and Fig . We founIn vivo microtubule-binding assays revealed that, in the GSK3/Sgg knockdown background, co-expression of Aβ42 was still capable of increasing the level of tau in the cytosolic fraction and decreasing the level in the microtubule fraction  suppressed Aβ42-induced augmentation of tau toxicity  and Fig . These mupper  were preferentially reduced in S2A tau.As described above , wild-tytype tau . In addiu  but does not promote tau phosphorylation at SP/TP sites , as the S2A mutation did. Knockdown of PAR-1 significantly reduced the levels of taulower  through phosphorylation at Ser262 and Ser356.Taken together, these results indicate that PAR-1 stabilizes less phosphorylated forms of tau Fig 5A), lower) in the presence of Aβ42. Western blot analyses using the pan-tau antibodies tauC and Tau46 revealed that PAR-1 knockdown reduced the levels of tau Fig 5A), lower . Importantly, blocking this stabilization of tau suppressed tau-mediated neurodegeneration and reduced the level of tau phosphorylated at the AD-associated residue Thr231  at the C-terminal region and no N-terminal insert (N), include a kind gift from Dr. M. B. Feany   and the Western blotting was carried out as described previously . Briefly2 (NEB) and proteinase inhibitor cocktail (Roche), and incubated with λ protein phosphatase (NEB) for 3h at 30°C, then subjected to western blotting as described above.Twenty fly heads for each genotype were homogenized in NEBuffer1  supplemented with 1 mM MnClMicrotubule binding assay was performed using a previously reported . Fifty hPreparation of paraffin sections, hematoxylin and eosin staining, and analysis of neurodegeneration were described previously . To analStatistics was done with the JMP software (SAS) or R (R Development Core Team (2008)) with Student's t, or one-way ANOVA followed by Tukey-Kramer HSD.S1 Figp<0.005, n.s., not significant (p>0.05)). Genotypes are as follows: (control) gmr-GAL4/+, (tau) gmr-GAL4/+;UAS-tau/+, (tau+Aβ42) gmr-GAL4/UAS-Aβ42;UAS-tau/+ and (Aβ42) gmr-GAL4/UAS-Aβ42. Transgene expression was driven by gmr-GAL4.External eyes of flies expressing the gmr-gal4 driver alone (control), human tau (tau), tau and Aβ42 (tau+Aβ42) or Aβ42 (Aβ42). The surface areas of the external eyes are shown as mean ± SE Click here for additional data file.S2 Figp > 0.05 by Student's t-test. Representative blots are shown. Transgene expression was driven by gmr-GAL4. Genotypes are as follows: (tau) gmr-GAL4/+;UAS-tau/+ and (tau+CD8GFP) gmr-GAL4/UAS-CD8GFP;UAS-tau/+.The levels of tau and tubulin in the lysate of fly heads expressing tau alone (tau) or co-expressing tau and non-toxic proteins in the secretory pathway CD8GFP (tau+CD8GFP) before sedimentation (input), in the supernatant (cytosol) and in the pellet containing microtubules (microtubule) were analyzed by western blotting by using anti-tau antibody. The same amount of proteins from each genotype was loaded. Expression of CD8GFP was confirmed by western blotting with anti-GFP antibody (CD8GFP). Mean ± SD, n = 5; (TIF)Click here for additional data file.S3 FigWestern blots of lysate of fly heads expressing tau with or without phosphatase treatment with anti-tau antibody. Following phosphatase treatment, the two tau bands (indicated by arrowheads) merged and were detected as a single faster-migrating band (indicated by asterisk). Transgene expression was driven by gmr-GAL4. The fly genotype is gmr-GAL4/+;UAS-tau/+.(TIF)Click here for additional data file.S4 FigAβ42 was expressed in all neuron and retina with a combination of two GAL4 drivers, the pan-neuronal elav-GAL4 driver and pan-retinal gmr-GAL4 driver. No significant changes in the levels of acetyl tubulin or tyrosinated tubulin were detected in the Aβ42 fly brain. Two independent transgenic fly lines expressing Aβ42 at different expression levels (Aβ42#1 and Aβ42#2) yielded similar results. Genotypes: (control) elav-GAL4/Y;gmr-GAL4/+, (Aβ42#1) elav-GAL4/Y;gmr-GAL4/+;UAS-Aβ42/+ and (Aβ42#2) elav-GAL4/Y;gmr-GAL4/UAS-Aβ42.(TIF)Click here for additional data file.S5 Figp < 0.05, Student's t-test. Tubulin was used as loading control. Expression of UAS-SggRNAi was driven by the pan-retinal gmr-GAL4 driver. Note that Sgg RNAi is only expressed in the retina, while endogenous Sgg is ubiquitously expressed, and protein levels of Sgg were assessed by western blot of whole head lysate. Thus, the observed signal reflects not only Sgg protein in the retina, but also that in other cells in the head in which Sgg expression is not suppressed. Therefore, it is likely that reduction of Sgg protein in the retina is larger than the level shown here. Genotypes: (control) gmr-GAL4/+ and (Sgg RNAi) gmr-GAL4/+;UAS-Sgg RNAi/+. (B) RNAi-mediated knockdown of Sgg reduces tau phosphorylation at SP/TP sites. Western blots of fly heads expressing tau (tau) or that co-expressing tau and Sgg RNAi (tau+SggRNAi) with pan-tau antibody (tau46 and tauC) or antibodies that recognize phosphorylation status of tau at the SP/TP sites . Tubulin was used as loading control. Mean ± SD, n = 5; *, p < 0.05, **, p < 0.01, ***, p < 0.005. Expression of tau and SggRNAi was driven by the pan-retinal gmr-GAL4 driver. Although residual Sgg-mediated phosphorylation of tau may be present, Sgg RNAi caused significant reduction in the levels of pSer202-tau, and pThr231-tau and PHF1 . Representative blots are shown. Genotypes: (tau) gmr-GAL4/+;UAS-tau/+ and (tau+Sgg RNAi) gmr-GAL4/+;UAS-Sgg RNAi/UAS-tau.(A) Reduction in Sgg protein levels by the expression of Sgg RNAi in the retina. Heads lysates were subjected to western blotting with anti-GSK3 antibody. Mean ± SD, n = 5, *, (TIF)Click here for additional data file.S6 Figp > 0.05). Genotypes: (control) gmr-GAL4/+, (Sgg RNAi) gmr-GAL4/+;UAS-SggRNAi/+, (Aβ42+SggRNAi) gmr-GAL4/ UAS-Aβ42; UAS-SggRNAi/+ and (Aβ42) gmr-GAL4/UAS-Aβ42.Heads of flies expressing the gmr-GAL4 driver alone (control), tau (tau), tau and Aβ42 (tau+Aβ42), or Aβ42 (Aβ42). The surface areas of the eyes are shown as mean ± SE Click here for additional data file.S7 Figp<0.005. Representative blots are shown. Genotypes are as follows: (tau) UAS-tau /+;elav-GeneSwitch/+ and (tau+PAR-1RNAi) UAS-tau /+;UAS-PAR1RNAi/elav-GeneSwitch. Transgene expression was induced by feeding newly eclosed flies RU486 for two days.Western blots of fly heads expressing tau (tau) or that co-expressing tau and PAR-1 RNAi (tau+PAR-1RNAi) driven by elav-GeneSwitch with pan-tau antibody (tauC). Tubulin was used as loading control. Mean ± SD, n = 5; ***, (TIF)Click here for additional data file.S8 Figp<0.005. Representative blots are shown. Genotypes: (control) gmr-GAL4/+ and (PAR-1RNAi) gmr-GAL4/+;UAS-PAR-1RNAi/+.Western blots of fly heads expressing gmr-GAL4 driver alone (control) or that expressing PAR-1 RNAi (PAR-1 RNAi) with anti-phospho-Ser21/9 antibody (pSer21/9) or a pan-GSK3 antibody (Sgg). Mean ± SD, n = 5; ***, (TIF)Click here for additional data file.S9 Figp>0.05. Representative blots are shown. Genotypes: (control) elav-GAL4/Y;gmr-GAL4/+ and (Aβ42) elav-GAL4/Y;gmr-GAL4/UAS-Aβ42.Aβ42 was expressed in all neuron and retina with a combination of two GAL4 drivers, the pan-neuronal elav-GAL4 driver and pan-retinal gmr-GAL4 driver. Western blots of fly heads expressing driver alone (control) or that expressing Aβ42 (Aβ42) with anti-phospho-Ser21/9 antibody (pSer21/9), or a pan-GSK3 antibody (Sgg). Mean ± SD, n = 5; (TIF)Click here for additional data file.S10 Fig(A). Western blotting of fly heads expressing tau alone or co-expressing tau and Aβ under the control of the pan-retinal gmr-GAL4 driver. No specific signal was detected with an anti-pSer238-tau antibody. (B) Western blotting of fly heads without expression of tau (control), expressing tau under the control of the pan-neuronal elav promoter (elav-tau (1) and elav-tau (2)), or expressing tau under the control of the gmr-GAL4 driver (gmr>tau). pSer238 was detected in elav-tau (1) and elav-tau (2), but not in gmr>tau. Genotypes: (control) gmr-GAL4/+, (tau) gmr-GAL4/+;UAS-tau/+, (tau+Aβ) gmr-GAL4/UAS-Aβ42;UAS-tau/+, (elav>tau(1)) elav-tau(1)/TM3Ser, (elav>tau(2)) elav-tau(2)/elav-tau(2), and (gmr>tau) gmr-GAL4/UAS-Aβ42;UAS-tau/+.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit, NPHP-4 influences localization and function of a distal ciliary kinesin. Moreover, data suggest human OSM-3 homolog (Kif17) could act as a modifying locus affecting disease penetrance or expressivity in NPHP patients.Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on  C. elegans nphp-4(tm925) mutants. Mutations in ten loci were obtained that severely exacerbated the cilia defects in the nphp-4(tm925) mutants, but importantly, had minimal defects in the absence of the nphp-4 mutation. Here we identified four of these loci, each encoding a cilia protein. Three mutations are in known ciliopathy genes, mks-1, mks-2 and mks-5. The fourth allele is a missense (S316F) mutation in OSM-3, a kinesin required for distal cilia assembly and is the sole kinesin responsible for intraflagellar transport along the cilia distal segment in C. elegans. The osm-3(yhw66) mutation affects a putative phosphorylation site that is important for OSM-3 localization, movement, and function, largely in an nphp-4 dependent manner. These data establish a genetic interaction between osm-3 and nphp-4 that regulates kinesin activity and localization and raises the possibility that mutations in Kif17, the mammalian homolog of osm-3, may influence the phenotypes in human NPHP patients.Nephronophthisis (NPHP) is a genetically heterogeneous ciliopathy that has minimal genotype-phenotype correlation. The cause of this variation is not known, but could result from additional mutations in the patients’ backgrounds capable of modifying the phenotype. To identify candidate NPHP modifying loci, we conducted an enhancer mutagenesis screen using  C. elegquencing . PCR conhttp://www.ncbi.nlm.nih.gov/) or from the Celera Database (http://www.celera.com). Gene sequences were identified using the C. elegans database Wormbase and references therein (http://www.wormbase.org). Sequence alignments were performed using ClustalW .Genome sequence information was obtained from the National Center for Biotechnology Information (C. elegans growth methods [rol-6 [Strains were grown on regular nematode growth media (NGM) using standard  methods  at 20°C  methods  or rol-6s [rol-6  as a marosm-3 from osm-3(yhw66);nphp-4(tm925) DNA and inserting the fragment in cGV7 using Gateway cloning technology. All PCR was performed using AccuTaq-LA DNA Polymerase  according to manufacturer’s instructions. The resulting constructs were sequenced and injected at 5 ng/μl. For each series of outcrosses the resulting F2 offspring obtained from self-fertilization were screened by PCR to identify strains containing required mutations.pcGV6 and pcGV7 Gateway vectors, p328 (XBX-1::TdTomato), and p380 (MKS-5::TdTomato) were described previously , 25. pMH0 population of young adult nphp-4(tm925) mutant worms were grown at room temperature, washed with M9 solution  three times, and placed into a 15-ml conical tube. Worms were concentrated by centrifugation at 2,000 X g and suspended in 2 ml of M9. 2 ml of 60mM EMS (dissolved in M9) solution was added to the 2 ml of worm suspension to reach a final concentration of 30 mM. The vial was rotated for 4 hours at room temperature. Worms were then collected by centrifugation at 2,000 X g and washed three times with M9, rotating between washes. Worms were suspended in 0.5 ml M9 and plated onto NGM plates seeded with OP50 and allowed to recover overnight at 20°C.Mutagenesis was performed as described previously  with mod0) were placed on the edge of an E. coli-seeded 10-cm NGM agar plate, and 8 such plates were made. P0 were allowed to lay F1 generation overnight and the next day were transferred onto new plates. This was repeated two more times with the purpose of F1 generation separation by age. Once F1 worms reached adulthood, 50–60 animals from each plate were transferred onto new plates to produce F2 generation. The F1 worms were transferred two more times with the same goal to produce F2 separated by age. As soon as F2 worms on the earliest set of plates reached adulthood, they were collected by centrifugation and dye-filled. Dye-filling defective animals were isolated by visual inspection and manual picking using a Lieca MZ16FA fluorescent microscope. About 4,000 mutagenized haploid genomes and about 500,000 individual F2 worms were screened.The screen for dye-filling defective animals was performed as follows. The day following the mutagenesis, four mutagenized worms (Phttp://www.wormbase.org).To identify the chromosomal location of the genetic lesions responsible for the Dyf phenotypes, a single nucleotide polymorphism (SNP)-based mapping strategy was applied essentially as described . Brieflynphp-4(tm925) males, and F1 heterozygous males were then crossed to other mutant lines which had mutations on the same chromosome. The F1 progeny from these crosses was assessed for dye-filling defective phenotypes. The lack of dye-filling defective male progeny indicated complementation. If any Dyf males were observed, the two mutant lines were considered allelic. Similar mating schematics were applied to determine whether newly isolated alleles were in previously identified mks genes. For X-linked mutations, F1 hermaphrodite progeny were analyzed.To determine which of the mutant lines were allelic, we intercrossed males and hermaphrodites from different lines that mapped to the same linkage group. The F1 progeny resulting from each cross was tested for their dye-filling phenotypes in male worms to ensure progeny were from a genetic cross. The presence of nonDyf male progeny indicated complementation. If F1 male progeny displayed only Dyf phenotypes, the mutant lines were considered to be allelic. In cases when double mutant males had mating defects, an alternative strategy was taken; double mutant lines were crossed to Worms were anesthetized using 10 mM Levamisole, immobilized on a 10% agar pad coated with polystyrene beads. Fluorescence imaging was performed using a Nikon 2000U inverted microscope  outfitted with a PerkinElmer UltraVIEW ERS 6FE-US spinning disk laser apparatus . Confocal images were captured every 0.2μm in the z-axis with a 100x (NA 1.39) objective and analyzed with Volocity 6.3 software . Images were processed into Figs using Photoshop 7.0 and Illustrator CS5 . Time laps movies of IFT particles moving along the axoneme were acquired for kymographs at a rate between 3-4fps at a single z-plane for 300 frames.C. elegans was performed following protocol previously described [4 for one hour. En bloc staining with 1% uranyl acetate was done for 45 minutes. Samples were dehydrated in increasing levels of EtOH and propyline oxide, infiltrated with Embed812 resin, and then embedded in molds with resin. Molds were incubated at 60°C for 2 days. The samples were serial sectioned with a Leica Ultracut UC6 in 80nm sections. Sections were post-stained in 2% aqueous uranyl acetate and Reynold’s lead citrate for five minutes each. Sections were imaged at 80Kv using a FEI Tecnai T12 electron microscope.Electron microscopy on escribed , 58. BriDye-filling assays were performed using DiI  as described previously with modifications . In brieProcedures for osmotic avoidance assay were performed as described in  with sliDNA libraries were prepared according to Illumina's 'Preparing Samples for Sequencing Genomic DNA' protocol (Part # 1003806 Rev. B March 2008). Samples were sequenced using Illumina's GAIIx machine in the laboratory of Dr. Oliver Hobert (Columbia University) or the Heflin Center for Genomic Sciences (University of Alabama at Birmingham). The data were analyzed using MAQGene programs or the Integrative Genomics Viewer , 61.For osmotic avoidance assays comparisons were performed using the Student’s t-test. If the data was not normally distributed, the Mann-Whitney Ranked Sum was used instead. These tests were performed using SigmaStat3.1 software package (Systat Software IC).Dye-filling experiments were analyzed using a 2x2 chi-square test in R . An FDR postHoc test was completed to account for multiple comparisons. For all tests, p values less than 0.05 were considered significant.Kymograph experiments were analyzed using a two-way ANOVA with a Tukey’s Post-Hoc test. These tests were performed using R version 3.1.2 software package . For all tests, p values less than 0.05 were considered significant.Cilia Length experiments were analyzed using a one-way ANOVA with a Tukey’s Post-Hoc test. These tests were performed using R version 3.1.2 software package . For all tests, p values less than 0.05 were considered significant.Tails of worms were imaged following previously stated protocol. Images were then analyzed using Volocity 6.3 software . Using Volocity a line was drawn along the phasmid cilia starting 1μm before the base of the cilia into the IFT compartment and ending 0.5μm past the tip of the cilia. The line was drawn based of XBX-1::tdTomato fluorescence. Cilia lengths and fluorescence intensity after background fluorescence was subtracted were normalized. Percent total fluorescence of GFP compared to position along the normalized cilia was calculated using excel.(-kt), where A is the maximum recovery of florescence in the cilium as compared to pre-bleach levels and k is the rate constant of entry into the cilium.Worms expressing various forms of OSM-3 were imaged using the protocol above with time-lapse imaging. The phasmid cilia were then bleached (determined by the region at the end of the pre-ciliary compartment to the ciliary tip) leaving the pre-ciliary compartment unbleached. The light path was split using a Dual View (Photometrics) beam splitter to bleach and image both GFP and tdTomato simultaneously. Recovery of fluorescence over 3 minutes was captured at 1–3 frames per second for the first 30 seconds and 0.1 fps afterwards. Background fluorescence was subtracted before analysis. Recovery curves were fit to the one-dimensional diffusion equation F(t) = A*eTails of worms were imaged following previously stated protocol. Images were then analyzed using Volocity 6.3 software . A line drawn from the base of the phasmid cilia to the tip was used to measure the length.S1 Fignphp-4(tm925), and nphp-4(tm925)-dependent synthetic Dyf double mutant lines. Scale, 9μm.Representative confocal images of phasmid (tail) neurons using the XBX-1::TdTomato cilia marker protein in wild-type, (TIF)Click here for additional data file.S2 Figmks-1(yhw146) G/A substitution (denoted by asterisk) is predicted to disrupt splicing between exons 2 and 3 of the mks-1 transcript and cause read-through into intron 2 where a stop codon is immediately located. (B) Sanger sequencing of mks-5(yhw91) cDNA shows that the nt8247 G/A substitution (denoted by asterisk) affects an exon 15 splice donor site. The use of a cryptic splice donor results in an aberrant transcript with a premature translational termination sequence. (C) The altered yhw91 transcript is predicted to introduce 17 unique amino acids onto the C-terminus of the truncated MKS-5 protein.The (TIF)Click here for additional data file.S3 Fignphp-4(tm925), osm-3(yhw66), and nphp-4(tm925);osm-3(yhw66). Numerical data for these experiments can be found in Kymograph images of DYF-11::GFP, OSM-3::GFP, and OSM-3(S316F)::GFP in WT, (TIF)Click here for additional data file.


STAT1, IRF7, MX1, MX2, ISG15, ISG20, IFIT1, IFIT2, IFIT3, IFI27, IFI44L, APOBEC3A, DDX58 (RIG-I), TNFSF10 (TRAIL), and RSAD2 (viperin) were confirmed by real-time quantitative PCR and were consistent with the microarray data. In addition, at the post-translational level, HIV-1 Vpr induced the phosphorylation of STAT1 at tyrosine 701 in human MDMs. These results demonstrate that HIV-1 Vpr leads to the induction of ISGs and expand the current understanding of the function of Vpr and its role in HIV-1 immune pathogenesis.Macrophages act as reservoirs of human immunodeficiency virus type 1 (HIV-1) and play an important role in its transmission to other cells. HIV-1 Vpr is a multi-functional protein involved in HIV-1 replication and pathogenesis; however, its exact role in HIV-1-infected human macrophages remains poorly understood. In this study, we used a microarray approach to explore the effects of HIV-1 Vpr on the transcriptional profile of human monocyte-derived macrophages (MDMs). More than 500 genes, mainly those involved in the innate immune response, the type I interferon pathway, cytokine production, and signal transduction, were differentially regulated (fold change >2.0) after infection with a recombinant adenovirus expressing HIV-1 Vpr protein. The differential expression profiles of select interferon-stimulated genes (ISGs) and genes involved in the innate immune response, including  Antigen-presenting cells (APCs) are critical for both innate and adaptive immunity. Professional APCs such as macrophages play an integral role in the immune pathogenesis of the human immunodeficiency virus type 1 (HIV-1) Vpr, an accessory gene product of HIV-1, is a protein of 96 amino acids and has a predicted molecular weight of 15 kDa that is relatively conserved in HIV-1 and simian immunodeficiency virus (SIV) + T-cells IRF7, and caused phosphorylation of STAT1 at tyrosine 701 in human MDMs. These findings enhance the current understanding of HIV-1 replication and pathogenesis in human macrophages.A striking feature of Vpr is its unique potential to promote viral productivity in monocytes/macrophages and in a small population of CD4To better understand the role of HIV-1 Vpr protein in human MDMs, a recombinant adenovirus expressing ZsGreen1 and FLAG-tagged Vpr, Ad-Vpr, was generated. As a control, a recombinant adenovirus expressing ZsGreen1, Ad-Zs, was used. A schematic diagram of both recombinant adenoviruses is shown in + cells were separated by high-gradient magnetic sorting using MACS beads. The isolated CD14+ cells were differentiated into MDMs for 7 days, and then infected with the Ad-Vpr or Ad-Zs at a MOI of 100. After 48 h, the cells were either observed under a fluorescence microscope or lysed and analyzed for the expression of Vpr and ZsGreen1 protein by Western blotting. Fluorescence microscopy showed that ZsGreen1 was expressed in both Ad-Vpr- and Ad-Zs-infected MDMs compared to mock-infected controls, which remained ZsGreen1-negative , which interrogates more than 47,000 transcripts from 38,500 genes. This approach enabled us to monitor Vpr-induced changes in the global gene profile of the MDMs. Data analysis using GeneSpring GX software showed that Vpr modulated the expression of 557 genes in Donor 1 and 116 genes in Donor 2. Given that the array analyzes more than 47,000 gene transcripts, this is considered a minor change in the global host gene profiles . Heat maThe differentially regulated genes were filtered to determine gene entities common to both donors. Out of 557 genes altered in response to Vpr in Donor 1 and 116 genes in Donor 2, only 66 genes were common to both . Gene onIFI44L (40-fold), CXCL10 (23-fold), MX1 (15-fold), CCL8 (13-fold), IFIT1 (10-fold), TNFSF10 (TRAIL) (8-fold), ISG20 (8-fold), IFIT2 (8-fold), APOBEC3A (7-fold), CXCL11 (7-fold), IFI27 (7-fold), OAS2 (7-fold), IRF7 (6-fold), and ISG15 (5-fold) were the most highly up-regulated genes, whereas PPBP(CXCL7) (96-fold), MARCO (13-fold), CXCL5 (7-fold), MT2A (6-fold), and CCL22 (4-fold) were the most highly down-regulated genes in Donor 1 (IFI44L (12-fold), MX1 (7-fold), APOBEC3A (6-fold), IFIT1 (5-fold), IFIT2 (4.5-fold), IFIT3 (4-fold), ISG15 (3-fold), XAF1 (3-fold), OAS3 (3-fold), CCL8 (3-fold), OAS2 (3-fold), DDX58 (2.5-fold), STAT1 (2-fold), MX2 (2-fold), IRF7 (2-fold) and CCL2 (2-fold) were the most highly up-regulated genes, whereas THBS1 (3-fold), HLA-DQA (3-fold), TLR7 (2.6-fold), CD74 (2.5-fold), CXCL2 (2-fold), CCR2 (2-fold) and CXCL9 (2-fold) were the most highly down-regulated genes in Donor 2  were higher in the microarray data compared to their respective relative expression levels in the qRT-PCR data. On the other hand, in Donor 2 except for IRF7 (4-fold) and MX1 (5-fold) higher expression levels of APOBEC3A (233-fold), ISG20 (132-fold), IFIT2 (97-fold), IFIT1 (51-fold), ISG15 (38-fold), IFI27 (47-fold), IFI44L (36-fold), TNFSF10 (TRAIL) (29-fold), RSAD2 (22-fold), MX2 (17-fold), IFIT3 (16-fold), DDX58 (12-fold) and STAT1 (5-fold) were observed by qRT-PCR compared to their respective microarray data  by qRT-PCR. As shown in APOBEC3A gene transcript failed to express its gene product is not clear; however, differential regulation of gene transcription does not ensure a corresponding change in gene product levels. Taken together, these results clearly indicate that HIV-1 Vpr protein leads to the activation of the type I IFN pathway and the subsequent up-regulation of various ISGs in human MDMs.Finally, Western blotting was performed to examine the effect of HIV-1 Vpr on the protein expression levels of IRF7, STAT1, ISG15, ISG20, APOBEC3A, and TRAIL in MDMs. Cell lysates were prepared from Ad-Vpr, Ad-Zs, or mock-infected MDMs and subjected to Western blotting using specific antibodies. β-actin was used as a loading control. Consistent with the microarray data and the qRT-PCR results, STAT1, ISG15, ISG20, IRF7, and TRAIL were up-regulated in Ad-Vpr-infected macrophages compared to Ad-Zs- or mock-infected controls ; howeverin vitro infection of human MDMs with an adenovirus expressing HIV-1 Vpr protein. Although some previous studies have shown that HIV-1 infection leads to the activation of innate immunity and thus the induction of various ISGs in human MDMs The data presented herein are the first analysis of the changes in gene transcription that occur following MX1, IFI44L, DDX58, RSAD2, and several of the IFITs, which have been shown to play an important role against HIV-1 infection in MDMs MX1, MX2, IFIT1, IFIT2, IFIT3, IFIT27, IFI44L, DDX58, and RSAD2 genes obtained through microarray strongly correlated with the real-time PCR data, it is reasonable to speculate that the expression of these proteins may be up-regulated following HIV-1 infection in human MDMs.In this study, by utilizing an Affymetrix oligonucleotide microarray, we demonstrated that the majority of the genes differentially regulated by Ad-Vpr in both donors were involved in the immune response, indicating the important role played by HIV-1 Vpr protein in human MDMs. A large number of genes from this group is predicted to be activated during the innate immune response  as part STAT1 gene encodes a 91-kDa protein which is activated by both type I and type II IFNs Real-time PCR data and Western blot analysis confirmed the activation of IRF7 by HIV-1 Vpr in human MDMs  and 7. IHIV-1 Vpr protein caused the up-regulation of various ISGs, such as ISG15 and ISG20  and 7, wTRAIL protein is produced after HIV-1 infection in monocytes due to the IFNα/β-mediated activation of the STAT1 signaling cascade CXCL1, CXCL5, CXCL7, CXCL9, CXCL10, and CXCL11  is similar to ISGs induced by Vpr in Ad-Vpr infected MDMs. Our data indicating that Vpr leads to the induction of ISGs and activation of innate immune responses is contrary to some of the previously published reports which showed that Vpr helps HIV-1 to escape the innate immune responses by either counteracting the UNG2, a host cellular intrinsic factor which inhibits HIV-1 replication HIV-1 Vpr is essential for efficient infection of non-dividing cells such as macrophages. It has been shown that HIV-1 Vpr is expressed within infected cells and is packaged into HIV-1 virions. Although, the virion-associated Vpr is able to cause cell cycle arrest of CD4Our data confirmed that HIV-1 Vpr leads to the induction of ISGs in MDMs. However, our findings showed some donor-specific differences in the expression profiles of these ISGs, which might be due to differences in overall susceptibility and the host response to the HIV-1 Vpr infection. These differences and the number of donors used in the study should not be considered a limiting factor because the expression profiles of the selected genes in all donors were independently confirmed by qRT-PCR with reproducible and consistent readouts each time . FurtherIn conclusion, our studies have identified IRF7, STAT1, ISG15, ISG20, and TRAIL as key up-regulated molecules in MDMs harboring HIV-1 Vpr. Based on previous published reports and our present data; we suggest a potential role for these genes in host defense against HIV-1 replication and infection. Future studies to elucidate the mechanisms through which Vpr up-regulates these molecules as well as their roles in HIV-1 pathogenesis will certainly improve our understanding of the replication and pathogenesis of the HIV-1.Human cervical HeLa cells and human embryonic kidney HEK-293 cells were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% heat-inactivated fetal bovine serum  and 100 units/mL penicillin/streptomycin (Sigma). Plasmid transfection was performed using Lipofectamine 2000 (Life Technologies).+ cells were isolated by positive selection with anti-human CD14+ magnetic beads . Purity was greater than 95% (data not shown). Primary MDMs were generated by culturing CD14+ cells in RPMI 1640 medium (Sigma) supplemented with 10% FBS (Cell Culture Bioscience), 5% human AB serum(Sigma), antibiotics, and GlutaMax (Gibco), and containing recombinant human macrophage colony-stimulating factor . After 7 days, cellular differentiation status was confirmed by detection of MDM surface such as CD14 and CD68 (data not shown). All participants provided written informed consent. Ethics approval for this study was granted by the RIKEN Ethics Committees [Certificate No. Wako 21–2 (3)].Human PBMCs were obtained using a standard Ficoll-Paque (Pharmacia) gradient from heparinized blood from healthy individuals. CD14horseradish peroxidase (HRP)-labeled donkey anti-goat or goat anti-mouse secondary antibodies were from Sigma.STAT1 (#9172), phospho-STAT1 , and IRF-7 (#4920) rabbit polyclonal antibodies were from Cell Signaling Technology. The ISG15 mouse monoclonal antibody (MAb) (#AIS0701) was from ATGen. The TRAIL rabbit polyclonal antibody (#54008) was from ANASPEC. The ISG20 rabbit polyclonal antibody (#ARP40392-T100) was from Aviva System Biology. The HIV-1 Vpr mouse MAb #3 was produced by immunization of synthetic peptides N'-CQAPEDQGPQREPYN-C' corresponding to amino acids 3–16 of Vpr. The APOBEC3A goat polyclonal antibody (#NB100-93428) was from Novus Biologicals. ZsGreen1 rabbit polyclonal antibody (#632474) was from Clontech Laboratories. Fluorescein isothiocyanate (FITC)-conjugated MAbs directed against the human surface markers CD14 and CD68 were from Miltenyi Biotec and used at the supplier's recommended concentrations. The β-actin (#1978) MAb and NL43GAAGCTAGCGACTACAAGGATGACGATGACAAAATGGAACAAGCCCCAGAAGA (forward) and GCTCTAGACTAGGATCTACTGGCTCCAT (reverse), and cloned into the pShuttle2 vector at the NheI and XbaI restriction sites. Similarly, the ZsGreen1 gene was PCR-amplified with the FLAG tag incorporated using the primers TAATCTAGAGACTACAAGGATGACGATGACAAAGCCCCTCTCCCTCCCCCCCCCCTAA (forward) and TAGCGGCCGCTCAGGGCAAGGCGGAGCCGGAG (reverse) using the pRetroX-IRES2-ZsGreen1 plasmid (Clontech Laboratories) as a template, and then cloned into the pShuttle vector just downstream of Vpr at the XbaI and NotI restriction sites. The integrity of the generated recombinant plasmids was confirmed by DNA sequencing. Then the entire cassette (flanked by unique I-CeuI and PI-SceI restriction sites) was excised and ligated into Adeno-X viral DNA using the Adeno-X expression system 1, according to the manufacturer' s instructions (Clontech Laboratories).Adenoviruses were constructed using the Adeno-X™ expression system (Clontech Laboratories). Briefly, wild-type (wt) Vpr from HIV-1PacI and transfected into HEK293 cells with Lipofectamine 2000 (Life Technologies). The recombinant adenoviruses were purified using the Adeno-X maxi purification kit (Clontech Laboratories) and titrated using the Adeno-X rapid titer kit (Clontech Laboratories), following the recommendations of the manufacturer. The virus stocks were stored at −80°C for future use.Adeno-X viral DNA containing the FLAG-Vpr or ZsGreen1 was linearized with MDMs were transduced with Ad-Vpr or Ad-Zs at a MOI of 100. The cells were harvested for RNA extraction at 48 h post-transduction. MDMs were washed three times with ice-cold PBS, and total RNA was extracted using the RNeasy mini kit with DNase digestion, according to the manufacturer's instructions (QIAGEN). RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher) and stored at −80°C. For microarray analysis, the quality of the RNA was determined using the Agilent Bioanalyzer (Agilent Technologies).RNA samples were analyzed by microarray using the GeneChip Human Genome U133 2.0 plus array (Affymetrix). Microarray hybridization and fluorescence detection were performed as described in the Affymetrix GeneChip Expression Analysis Technical Manual. The. cel data files generated by the Affymetrix microarray hybridization platform were analyzed using GeneSpring GX ver. 12.0 software (Agilent Technologies). Probe-level analysis was performed using the RMA algorithm. Microarray data have been deposited in NCBI's Gene Expression Omnibus and assigned the GEO Series accession number GSE56591. Fold changes in gene expression, hierarchical clustering, and gene ontology annotations were determined.GAPDH mRNA expression as an internal control.Total RNA was prepared using the RNeasy mini kit as described above. RT-PCR was performed using specific primers and One-Step SYBR Green PCR mix (Takara), according to the manufacturer's manual. qRT-PCR was performed using a Prism 7500 sequence detection system (Applied Biosystems). Samples were run in triplicate and all data were normalized to Mock or virus-infected MDMs were washed with PBS and then lysed with CelLytic™ MT Cell Lysis reagent (Sigma) which was supplemented with a protease inhibitor cocktail (Roche Diagnostics) according to the manufacturer's instructions. Protein concentrations were determined with a BCA protein assay kit (Pierce) using bovine serum albumin as a standard. Proteins were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyl difluoride  membranes. The PVDF membranes were probed with the primary antibodies mentioned above followed by an anti-mouse HRP or anti-goat HRP or anti-rabbit HRP secondary antibody (Sigma), and signals were detected by enhanced chemiluminescence .µg/ml) at 37°C for 20 min and then stained with PI (40 µg/ml). For each sample, at least 7,000 cells were analyzed using a FACS Calibur instrument (Becton-Dickinson) with CELL Quest software (Becton-Dickinson). Ratios of the numbers of cells in the G1 and G2/M phases (G2+M: G1 ratios) were calculated using ModFit LT Software (Verity Software House).HeLa cells were infected with an adenoviral vector expressing Vpr or expressing only ZsGreen1, as a control. At 48 h post-infection, the cells were harvested and fixed with 1% formaldehyde followed by 70% ethanol. Fixed cells were incubated in PBS containing RNase A (50


PIK3CA gene are found in 10–20% of colorectal cancers (CRCs) and are associated with poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic TRAIL death receptor antibodies emerged as promising anti-neoplastic therapeutics, but to date failed to prove their capability in the clinical setting as especially primary tumors exhibit high rates of TRAIL resistance. In our study, we investigated the molecular mechanisms underlying TRAIL resistance in CRC cells with a mutant PIK3CA (PIK3CA-mut) gene. We show that inhibition of the constitutively active phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway only partially overcame TRAIL resistance in PIK3CA-mut-protected HCT116 cells, although synergistic effects of TRAIL plus PI3K, Akt or cyclin-dependent kinase (CDK) inhibitors could be noted. In sharp contrast, TRAIL triggered full-blown cell death induction in HCT116 PIK3CA-mut cells treated with proteasome inhibitors such as bortezomib and MG132. At the molecular level, resistance of HCT116 PIK3CA-mut cells against TRAIL was reflected by impaired caspase-3 activation and we provide evidence for a crucial involvement of the E3-ligase X-linked inhibitor of apoptosis protein (XIAP) therein. Drugs interfering with the activity and/or the expression of XIAP, such as the second mitochondria-derived activator of caspase mimetic BV6 and mithramycin-A, completely restored TRAIL sensitivity in PIK3CA-mut-protected HCT116 cells independent of a functional mitochondrial cell death pathway. Importantly, proteasome inhibitors and XIAP-targeting agents also sensitized other CRC cell lines with mutated PIK3CA for TRAIL-induced cell death. Together, our data suggest that proteasome- or XIAP-targeting drugs offer a novel therapeutic approach to overcome TRAIL resistance in PIK3CA-mutated CRC.Mutations in the oncogenic  PI3K converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate, thereby generating a docking site for pleckstrin homology domain containing proteins such as Akt/PKB. In CRC, approximately 10–20% of tumors exhibit mutations in the p110α catalytic subunit (predominantly H1047R and E545K substitutions in the PIK3CA gene), causing constitutive PI3K/Akt activation2 and worsening clinical outcome.3Colorectal cancer (CRC) is among the three most common malignancies worldwide.4 TRAIL binding to TRAIL receptor 1 (TRAIL-R1) or TRAIL-R2 induces formation of a chain-like death-inducing signaling complex (DISC). This allows stepwise caspase-8 activation and initiates a cascade of proteolytic cleavage events finally activating caspase-3 and triggering the execution phase of apoptosis.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) emerged as a promising anti-cancer agent, capable of selectively inducing cell death in tumor cells.5 tBid activates Bcl2-associated X protein (Bax) and Bcl2-antagonist/killer (Bak), enabling pore-formation in the outer mitochondrial membrane and release of apoptogenic factors such as cytochrome c and second mitochondria-derived activator of caspase (SMAC).6 The pro-apoptotic effect is at least twofold: cytochrome c associates with apoptotic protease-activating factor 1 (Apaf-1), forming a molecular scaffold for caspase-9 activation (‘apoptosome'), which in turn boosts downstream effector caspase activation. Synergistically, SMAC neutralizes cytosolic inhibitors of apoptosis proteins (IAPs), such as cIAP1, cIAP2 and especially XIAP.7In so-called type I cells, initial caspase-8-mediated cleavage of caspase-3 efficiently triggers further autocatalytic caspase-3 processing to the mature heterotetrameric p12-p17 molecule. In type II cells, however, X-linked inhibitor of apoptosis protein (XIAP) inhibits processing of the caspase-3 p19 intermediate to the p17 subunit of the mature enzyme. Death receptor-induced apoptosis in these cells therefore relies on a mitochondria-dependent amplification loop that is triggered by caspase-8-mediated cleavage of the BH3-interacting domain death agonist (Bid) to tBid.8High levels of IAPs or deregulated expression of Bcl2 family proteins are common in human cancers and often confer apoptosis resistance. This hampers efficacy of TRAIL-based therapies and to date, the therapeutic benefit of TRAIL in clinical trials is indeed rather limited.PIK3CA licensed TRAIL and CD95L to induce an amoeboid morphology in CRC cells, which is associated with increased invasiveness in vitro.9 Here, we show that targeting of the aberrantly active PI3K/Akt signaling pathway in TRAIL resistant, PIK3CA-mutated CRC cells only partially restored death receptor-triggered apoptosis induction. We identified impaired caspase-3 maturation by XIAP as the underlying molecular mechanism of TRAIL resistance in HCT116 PIK3CA-mut cells. Inhibition of XIAP or the proteasome efficiently restored TRAIL sensitivity irrespective of mitochondria-dependent death signal amplification. Together, our results indicate that targeting XIAP or the proteasome in CRC with PIK3CA mutations may offer a promising strategy to exploit the therapeutic potential of TRAIL in cancer therapy.We have recently found that mutant PIK3CA in the CRC cell line HCT116 revealed that exclusive expression of a PIK3CA allele harboring an activating H1047R substitution (HCT116 PIK3CA-mut) in exon 20 conferred resistance to TRAIL-induced apoptosis  died in a dose-dependent manner after TRAIL stimulation  differences in TRAIL receptor- or CD95-associated signaling pathways for the following reasons: treatment of HCT116 PIK3CA-wt and the PIK3CA-mut counterparts with the primarily pro-inflammatory ligand TNF exhibited comparable levels of phospho-IκBα, thereby ruling out general defects in the activation of the NF-κB pathway  and deguelin  were observed , but this synergistic effect was lost at higher wortmannin concentrations (CIμM wortmannin5 =1.55). Ly294002, another PI3K inhibitor, also acted synergistically with TRAIL in concentrations up to 20 μM . Again, increased cytotoxic effects by Ly294002 itself were reflected by higher CI-values (CIμM Ly29400240 =1.09) and correlated with observations from the corresponding viability assays .Similar observations were made when challenging HCT116 y assays . A recencovitine  revealedImportantly, although we were able to clearly demonstrate synergistic effects of combinatorial treatments of TRAIL and an Akt, PI3K or CDK inhibitor, the re-sensitization to TRAIL-induced cell death was only partial. As robust cell death induction in transformed cells is crucial for effective TRAIL-based anti-cancer therapy, we concluded that interfering with the PI3K/Akt signaling axis or CDKs is most probably insufficient to fully exploit the therapeutic potential of TRAIL in PIK3CA-mutant CRC cells.14 We therefore investigated whether blockade of the ubiquitin-proteasome system affects TRAIL-induced cell death in HCT116 PIK3CA-mut cells. Indeed, pre-treatment with the proteasome inhibitors bortezomib or MG132 resulted in almost full-blown cell death induction  and MG132 . Importantly, sensitization for TRAIL-induced cell death by proteasome inhibition was not restricted to HCT116 PIK3CA-mut cells and was also evident in the PIK3CA-mutant CRC cell line LS-174T  and Survivin were detectable in our cellular system .To that point, elevations in apoptosis-promoting molecules seemed unlikely to be the molecular basis for bortezomib-induced TRAIL sensitization. Consequently, we analyzed changes in the levels of anti-apoptotic proteins in HCT116 r system . Of noter system . This rePIK3CA-mut cells also reduced cellular XIAP levels t manner . For BV6zVAD-fmk .PIK3CA-mut-protected HCT116 cells to TRAIL-induced cell death  PIK3CA allele exhibited a constitutively activated PI3K/Akt-pathway, high-level resistance against TRAIL-induced cell death10 and increased invasiveness in vitro and in vivo.15 TRAIL resistance was essentially attributed to constitutive PI3K activation and reduced Bax expression.23 We confirmed that mutant PIK3CA shifts TRAIL and Fc-CD95L signaling from cell death induction to pro-survival signaling via robust NF-κB activation. The latter was poor in HCT116 PIK3CA-wt cells  stabilization of the pro-apoptotic proteins Bax29 or tBid16 and (c) increased levels of the pro-apoptotic BH3-only proteins Bik and Bim.30 However, none of these mechanisms was applicable to the bortezomib-induced TRAIL sensitivity in HCT116 PIK3CA-mut cells, as in the presence and absence of bortezomib and/or TRAIL (a) cFLIP levels  downregulation of the anti-apoptotic protein cFLIP with subsequently enhanced activation of caspase-8;P levels  as well P levels  remainedP levels  and (c) P levels  did not PIK3CA-mut cells, the anti-apoptotic protein Mcl-1 was significantly upregulated  domains and is itself a substrate of caspase-8. Caspase-8-generated cleavage fragments of XIAP inhibit further processing of caspase-3 intermediates36 by competitive binding of BIR1 and BIR2 (BIR1-2) to a neo-epitope in the caspase-3 p19 fragment.19 This inhibition can be surmounted by robust caspase-3 activation and cleavage of XIAP in a manner that functionally inactivates the BIR1-2 fragment.36 In TRAIL-resistant melanoma cells, for example, caspase-3-mediated XIAP cleavage acts as a positive feedback loop that sensitizes to TRAIL-induced cell death.37 In line with this, TRAIL-induced caspase-3 activation in bortezomib, BV6 and mith-A pre-treated HCT116 PIK3CA-mut cells further enhanced the initially observed caspase-independent reduction of XIAP of these drugs . These isogenic cell lines exclusively harbor either a wild-type (HCT116 PIK3CA-wt) or a mutated allele  for PIK3CA. Cells were cultured in RPMI 1640 medium  supplemented with 10% fetal calf serum (PAA). Fc-CD95L was produced in HEK293 cells and purified by affinity chromatography targeting an internal Flag epitope. Protein G agarose beads coupled to anti-Flag mAb were purchased from Sigma . Human recombinant TRAIL was purchased from Apronex . Roscovitine, Ly294002 and wortmannin were purchased from Adipogen , deguelin from Cayman chemicals  and mith-A from AppliChem . BV6  was synthesized as described elsewhere.41 zVAD-fmk was obtained from Bachem , MG132 from Merck , bortezomib from US Biological . Antibodies specific for caspase-3, caspase-8, caspase-9, JNK, phospho-JNK, ERK, phospho-ERK, p38, phospho-p38, IκBα, phospho-IκBα, Puma, Bim, Bid, Bak, cFLIP, Bcl-XL, Survivin, Mcl-1 and XIAP were purchased from Cell Signaling . Anti-Bax was from Santa Cruz , anti-β-actin from Sigma, anti-tubulin from Dunn Labortechnik . Anti-RIP1 and anti-PARP were purchased from BD Biosciences .LS-174T and DLD-1 cells were purchased from the German Collection of Microorganisms and Cell Culture . HCT116 4 per well) were seeded in 96-well plates and challenged the next day with the indicated concentrations of TRAIL or Fc-CD95L in triplicates. Cell viability was determined 18 h after stimulation using 3--2,5-diphenyl tetrazolium bromide staining.Cells  glycerol, 0.2 M Tris, pH 8.0) supplemented with phosphatase inhibitor cocktails-I and -II (Sigma). Subsequently, lysates were sonicated and boiled for 5 min at 96 °C. Protein separation for western blot analyses was achieved by SDS-polyacrylamide gel electrophoresis. After transfer to nitrocellulose membranes, nonspecific binding sites were blocked by incubation in Tris-buffered saline containing 0.1% Tween 20 and 5% dry milk. Membranes were incubated with primary antibodies of the specificity of interest. Antigen–antibody complexes were visualized using secondary, horseradish peroxidase-conjugated antibodies  and the ECL western blotting detection system .g). After addition of Laemmli buffer, samples were boiled for 5 min at 96 °C and separated by SDS-polyacrylamide gel electrophoresis.For preparation of Triton X-100 lysates, cells were washed and resuspended in lysis buffer  Triton X-100, 10% (v/v) glycerol, 120 mM NaCl, pH 5) supplemented with complete protease inhibitor cocktail . After incubation for 20 min on ice, lysates were cleared by centrifugation  and 200 pmol of caspase-9-specific or control siRNA were transfected the next day using Lipofectamine 2000  according to the manufacturer's instructions. 48 h post-transfection, knockdown efficacy was determined by western blot analysis.Knockdown of caspase-9 was performed using siRNA oligonucleotides . Briefly, cells were seeded in six-well plates  were seeded in 96-well tissue culture plates and cultured overnight. Medium was changed and the indicated concentrations of TRAIL, Fc-CD95L or medium were added for 6 h in the presence and absence of 100 μM zVAD-fmk. The supernatant was collected and IL-8 was quantified using an ELISA (BD Biosciences). All groups were analyzed as triplicates.For measuring IL-8 secretion, cells .


Signalling through the Hippo (Hpo) pathway involves a kinase cascade, which leads to the phosphorylation and inactivation of the pro-growth transcriptional co-activator Yorkie (Yki). Despite the identification of a large number of pathway members and modulators, our understanding of the molecular events that lead to activation of Hpo and the downstream kinase Warts (Wts) remain incomplete. Recently, targeted degradation of several Hpo pathway components has been demonstrated as a means of regulating pathway activity. In particular, the stability of scaffold protein Salvador (Sav), which is believed to promote Hpo/Wts association, is crucially dependent on its binding partner Hpo. In a cell-based RNAi screen for ubiquitin regulators involved in Sav stability, we identify the HECT domain protein Herc4 (HECT and RLD domain containing E3 ligase) as a Sav E3 ligase. Herc4 expression promotes Sav ubiquitylation and degradation, while Herc4 depletion stabilises Sav. Interestingly, Hpo reduces Sav/Herc4 interaction in a kinase-dependent manner. This suggests the existence of a positive feedback loop, where Hpo stabilises its own positive regulator by antagonising Herc4-mediated degradation of Sav. Drosophila using genetic screens for mutations resulting in tissue overgrowth [Drosophila comprises two kinases, Hpo and Warts (Wts). Activated Hpo phosphorylates the downstream kinase Wts, which leads to the Wts-mediated phosphorylation of the transcriptional co-activator Yorkie (Yki) [myc, bantam and diap1) [Drosophila [The Hippo (Hpo) pathway has emerged as a major regulator of growth and tissue size ,2. It waergrowth –12. The ie (Yki) ,13. Phosie (Yki) ,15. In tie (Yki) ,17, to id diap1) –20. Hencosophila –25.Over the last decade, various genetic screens and proteomic approaches have identified many new components of the pathway . This haSlimb/β-TrCP E3 ligase complex [Slimb/β-TrCP. Moreover, several other components of the Hpo pathway have been reported to be targets of ubiquitin-dependent proteasomal degradation, such as RASSF1A, RASSF5/NORE1, Mob1 and Wts/LATS1 [However, despite the ever-expanding complexity of Hpo upstream signalling, the regulation and function of the core components still remain incompletely understood. In particular, apart from phosphorylation, the role of covalent protein modifications, such as ubiquitylation and SUMOylation has been little explored. One well-characterised instance is the regulation of the Yki orthologs YAP and TAZ by the SCF complex ,30. Sequts/LATS1 –36.Salvador, RASSF, Hippo) domains [sav phenocopies the overgrowth phenotype of hpo mutants [The present study focuses on the tumor suppressor protein Salvador (Sav), which, together with Hpo and Wts, was one of the first Hpo pathway components to be identified ,6. Sav i domains ,6,37. Th domains –40. Howe mutants ,6,41,42, mutants ,41–43. R mutants . Another mutants ,45,46. H mutants ,45. HereDrosophila S2 cells were transfected with Effectene transfection reagent (Qiagen) and grown in Drosophila Schneider’s medium (Life Technologies) containing 10% FBS (Sigma), 50μg/ml penicillin and 50 μg/ml streptomycin. Expression plasmids were generated using Gateway® technology (Life Technologies). Open reading frames (ORF) were PCR amplified from existing plasmids or cDNA  and cloned into Entry vectors (pENTRTM/D-TOPO). Vectors from the Drosophila Gateway Vector Collection (http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.html) were used as Destination vectors. All expression plasmids were sequence-verified. Point mutations were generated using the Quikchange® Multi Site-Directed Mutagenesis Kit (Stratagene).http://www.dkfz.de/signaling2/e-rnai/)RNAi library generation work-plan can be found in the supplementary figures –S4 Figs.6 cells were plated per well and cells left to settle. After 3 h the medium was removed and replaced with 1 ml of serum free Schneider’s medium containing dsRNAs (20 μg). Cells were soaked for 30 min and then 2 ml of full Schneider’s medium was added. Transfections were carried out 24 h after dsRNA treatment.RNAi screening was carried out as outlined in g for 20 min at 4°C. FLAG-tagged proteins were purified using anti-FLAG M2 Affinity Agarose Gel (Sigma), whereas immunoprecipitation of Myc-tagged proteins was carried out using Protein A Sepharose (Sigma). Detection of purified proteins and associated complexes was performed by immunoblot analysis using chemiluminescence (Pierce). Western Blots were probed with mouse anti-FLAG , rabbit anti-FLAG (Sigma), mouse anti-Myc , rabbit anti-Myc (Santa Cruz Biotechnology), rat anti-HA , mouse anti-Tubulin  and mouse anti-GFP (in-house produced), rat anti-Yki [For immunoprecipitation assays, cells were lysed in Lysis Buffer  supplemented with 0.1M NaF, phosphatase inhibitor cocktails 2 and 3 (Sigma) and protease inhibitor cocktail (Roche). Cell extracts were cleared at 16,000 anti-Yki , and rabanti-Yki  and guinFor ubiquitylation assays S2 cells were transfected with Myc-Sav and HA-Ubiquitin and, unless otherwise stated, treated with proteasome inhibitors for 4 h. Cells were harvested and lysed in Lysis buffer (see above). Lysates were immunoprecipitated with an anti-Myc antibody and Protein A Sepharose (Sigma), and subjected to western blot analysis. Ubiquitylation was detected using an antibody against HA-Ubiquitin. Lysis buffer containing 5mM N-Ethylmaleimide (NEM) (Sigma) was used to block deubiquitylating enzyme activity.For proteasome inhibition experiments, cells were treated with 50μM MG132  and 50μM calpain inhibitor I (Ac-LLnL-CHO or LLnL) (Sigma) for 4 h before cell lysis. Ubiquitylation assays were carried out after proteasome inhibitor treatment, unless stated otherwise.g for 10 min at 4°C. 150ug of total lysate was loaded per lane.Heads were cut off from frozen animals and lysed in RIPA buffer  Triton X-100, 0.1% (w/v) SDS, 1% (w/v) Sodium deoxycholate) supplemented with 0.1M NaF, phosphatase inhibitor cocktails 2 and 3 (Sigma) and protease inhibitor cocktail (Roche). Tissue lysates were incubated on ice for 10 min and subsequently cleared by centrifugation at 16,000 155 antibody  was used at 1:20. Secondary anti-rat Alexa 647 antibody (Molecular Probes) was used at 1:200. Fluorescence images were acquired on a Zeiss LSM710 confocal laser scanning microscope (40x objective lens).Wing imaginal discs from third-instar larvae were dissected, fixed for 20 min in 4% formaldehyde, washed three times in PBS supplemented with 0.1% Triton X-100 (PBS-T) and pre-blocked in PBS supplemented with 0.3% Triton X-100 and 10% normal goat serum (NGS) for 1 h. Discs were incubated overnight in the primary antibody diluted in PBS-T containing 10% NGS, followed by three washes with PBS-T, and incubation with a secondary antibody in PBS-T/10% NGS for 2 h at room temperature. After three further washes, discs were mounted in Vectashield mounting medium with DAPI (Vectorlabs). Rat anti-Cihs BAB39389.1 (GenBank), Nedd4 hs AAI44285 (AAI44285), Smurf1 hs EAL23885 (EAL23885), Herc1 hs Q15751.2 (UniProtKB/Swiss-Prot), Herc2 hs O95714.2 (UniProtKB/Swiss-Prot), Herc3 hs Q15034.1 (UniProtKB/Swiss-Prot), Herc4 hs Q5GLZ8.1 (UniProtKB/Swiss-Prot), Herc5 hs Q9UII4.2 (UniProtKB/Swiss-Prot), Herc6 hs Q8IVU3.2 (UniProtKB/Swiss-Prot), Herc4 dm (CG9153) Q5GLZ8.1 (UniProtKB/Swiss-Prot).Multiple sequence alignment and phylogenetic tree analysis was carried out using Clustal Omega (EMBL-EBI). Protein sequence accession numbers were as follows: Itch herc4 gene was performed using the CRISPR/Cas9 system [http://www.flyrnai.org/crispr/. Sense and anti-sense oligos of the selected sgRNA were annealed, phosphorylated and cloned into the expression vector pCFD3 according to the protocol described on http://www.crisprflydesign.org/grna-expression-vectors/. DNA was injected into embryos of y1, M{nos-Cas9.P}ZH-2A w* transgenic flies (Bloomington stock number 54591) by the Fly Facility at the University of Cambridge Department of Genetics. G0 flies were then crossed with the w;;TM3/TM6 balancer strain; subsequently, G1 male flies were crossed with the same balancer strain, followed by genomic DNA screening for frame-shift mutations. We also balanced a wild type progeny of the injected founders to use as a control strain (herc4ctrl).Introduction of a frame-shift mutation in the 9 system . SuitablAdult wings were mounted using Euparal mounting medium (Agar Scientific) and imaged using a Zeiss Axio-plan 2 imaging system, using Optovar optics (1.6x) and a 2.5x objective  connected to a Leica DFC420C camera. Wings were subsequently measured and quantified using ImageJ.w; ubi-GFP::sav/+; hh-GAL4/+, right panels: w; ubi-GFP::sav/+; hh-GAL4/UAS-Herc4.w; UAS-CD8-GFP; rn-GAL4. w; UAS-herc4; rn-GAL4., w; UAS-herc4-C/A; rn-GAL4. w; UAS-CD8-GFP; rn-GAL4, UAS-sav. w; UAS-herc4; rn-GAL4, UAS-sav. w; UAS-herc4-C/A; rn-GAL4, UAS-sav. w; UAS-CD8-GFP; rn-GAL4, UAS-sav-S413A. w; UAS-sav-S413A; rn-GAL4, UAS-herc4. w; UAS-herc4-C/A; rn-GAL4, UAS-sav-S413A.w;; rn-GAL4/ctrl. w;; rn-GAL4, UAS-sav/ctrl. w;; rn-GAL4, UAS-sav-S413A/ctrl. w;; rn-GAL4/herc4C6.3. w;; rn-GAL4, UAS-sav/ herc4C6.3. w;; rn-GAL4, UAS-sav-S413A/ herc4C6.3.Sav is an unstable protein and its stability is regulated by the proteasome system A. This We and others have observed that exogenous Sav is stabilised by Hpo co-expression, while Hpo depletion causes endogenous Sav downregulation C, indicSince Sav ubiquitylation occurs in the C-terminal domain responsible for Hpo binding, Hpo could sterically interfere with ubiquitin ligase binding or ubiquitin transfer. Alternatively, since Hpo is known to phosphorylate Sav ,45,46,51A. Hpo cDrosophila dsRNA sub-library containing known and candidate genes involved in ubiquitylation (378 genes involved in ubiquitin conjugation and ubiquitin binding). The effect of dsRNAs on Sav-Myc expression was analyzed by standard Western-blot analysis. Using this approach we identified the HECT domain protein Herc4 (CG9153) as a Sav E3 ligase (Drosophila Herc4 contains a RLD (RCC1-like domains) domain comprising 7 RCC1-like repeats and a HECT domain [Drosophila there are only 2 small Herc E3 ligases, Herc2 and Herc4. Drosophila Herc4 is marginally more related to human Herc4 than human Herc3  had a reduced effect D. In agin vivo. We generated a GFP-tagged sav transgene, expressed under the control of the ubiquitin promoter (ubi-GFP::sav). Using the GAL4/UAS system, UAS-Herc4 was expressed in the posterior compartment of wing imaginal discs, the larval precursor to the adult wing, using the hh-Gal4 (referred to as hh>herc4) driver. Cubitus interruptus (Ci) staining was used to differentiate the anterior (Ci-positive) and posterior (Ci-negative) compartments of the wing imaginal disc. Herc4 overexpression caused depletion of Sav levels  and therefore more active (SavS413A) [rotund-Gal4 . Overexpression of wild type Sav modestly reduced the size of the wing, while the SavS413A mutant had a stronger effect  had no effect on wing size (in vivo.One of the main effects of Hpo pathway disruption avS413A) . The trar effect A. Expreherc4 mutant using CRISPR technology [herc4, we generated a mutation, herc4C6.3, carrying a one base-pair deletion at codon position C92 (cysteine 92)  A. This eine 92) B. Homozhenotype . We therherc4 affects Sav protein levels in vivo, we analysed Sav protein levels in fly heads by Western blot. herc4C6.3 heads compared to control heads. Together, these results support a role for Herc4 as a Sav E3 ligase.To test whether loss of Drosophila tissue culture cells [The scaffold protein Sav is a core component of the Hpo signalling pathway and its genetic inactivation is sufficient to increase tissue growth in flies and mice ,6,41,43.re cells .Sav levels appear to be tightly regulated by proteasomal degradation, and its association with Hpo/MST promotes both its phosphorylation and stabilisation ,9,45,46,, it seemherc4 depletion reduces Sav ubiquitylation in cell culture  and a single Herc3-6 ortholog . Herc4 mutant mice are viable but males display a spermatozoon maturation defect [In order to shed light on the regulation of Sav stability, we performed a screen for Sav E3 ligases, leading us to identify Herc4 as a negative regulator of Sav . We show culture  and that in vivo . Howeverinal RLD . Mammaliinal RLD . Indeed,n defect . In addin defect . It wouln defect .In this report, we have also shown that the ability of Herc4 to degrade Sav is antagonised by Hpo . FurtherS1 FigFlag-tagged Herc4, Su(dx) and Nedd4 were expressed in S2 cells together with Sav-Myc and Ub-HA. After lysis, Sav was analysed for ubiquitylation.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


Up to ~40 % of Hodgkin and non-Hodgkin lymphomas carry the Epstein-Barr virus (EBV) genome and express type 2 viral latency proteins (T2LPs) EBNA-1, LMP-1, LMP-2 and BARF-1. EBV specific T cells (EBVSTs) can be generated from the blood of EBV+ individuals, but are usually dominated by T cells specific for viral proteins not expressed in type 2 latency lymphomas (T2LPs).To overcome this problem, we used dendritic cells, pulsed with overlapping peptide libraries (pepmixes) spanning T2LPs in the presence of IL-4 and IL-7 to stimulate patient peripheral blood mononuclear cells. Responder T cells were then expanded using pepmix-pulsed, autologous activated T cells and HLA-negative K562 cells expressing co-stimulatory transgenes.5 cells; n=11). High dose IL-15 also increased the frequency of central memory T cells in the final T cell product (36.81±17 vs. 19.09±13 % vs.13.81±22 % (IL-15 Hi vs. IL-15 Lo vs. IL-4) in combination with Il-7).Although T2LP-specific EBVSTs could be generated from healthy-donors, we were unable to consistently generate EBVSTs with significant T2LP specificity from patients. We hypothesized that patient T cells were anergic to antigen expressed within tumors, and since IL-15 has been shown to rescue tolerant or anergic T cells, we replaced IL-4 with IL-15 (in combination with IL-7). This improved antigen specificity up to 10-fold. Further dose optimization showed significant advantages in cytolytic activity, proliferation and antigen specificity with higher dose of IL-15. We achieved higher fold expansion (3 fold mean increase in absolute EBVST numbers) and enhanced specificity for T2LPs .Our results suggest that a high frequency of antigen-specific T cells in the infused product is critical for clinical activity and strategies to further improve specificity will enhance overall tumor responses.


RORA and RORC were down regulated, whereas AhR target gene CYP1A1 was upregulated. IL-22 induction by DEP-PAH was mainly dependent upon AhR whereas IL-22 induction by B[a]P was dependent upon activation of PI3K and JNK. Altogether, these data suggest that DEP-PAH and B[a]P may contribute to increased IL22 production in both healthy and asthmatic subjects through mechanisms involving both AhR -dependent and -independent pathways.Pollution, including polycyclic aromatic hydrocarbons (PAH), may contribute to increased prevalence of asthma. PAH can bind to the Aryl hydrocarbon Receptor (AhR), a transcription factor involved in Th17/Th22 type polarization. These cells produce IL17A and IL-22, which allow neutrophil recruitment, airway smooth muscle proliferation and tissue repair and remodeling. Increased IL-17 and IL-22 productions have been associated with asthma. We hypothesized that PAH might affect, through their effects on AhR, IL-17 and IL-22 production in allergic asthmatics. Activated peripheral blood mononuclear cells (PBMCs) from 16 nonallergic nonasthmatic (NA) and 16 intermittent allergic asthmatic (AA) subjects were incubated with PAH, and IL-17 and IL-22 productions were assessed. At baseline, activated PBMCs from AA exhibited an increased IL-17/IL-22 profile compared with NA subjects. Diesel exhaust particle (DEP)-PAH and Benzo[a]Pyrene (B[a]P) stimulation further increased IL-22 but decreased IL-17A production in both groups. The PAH-induced IL-22 levels in asthmatic patients were significantly higher than in healthy subjects. Among PBMCs, PAH-induced IL-22 expression originated principally from single IL-22- but not from IL-17- expressing CD4 T cells. The Th17 transcription factors  Allergic asthma has strongly increased in the last decades in western countries and is considered mainly as a Th2 mediated disease. There is increasing evidence that environmental pollution contributes to this augmented prevalence . In partIt is now clear that the biological response to many environmental pollutants is a direct consequence of their interactions with the Aryl hydrocarbon Receptor (AhR), a cytosolic transcription factor which binds exogenous ligands such as PAH or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as endogenous ligands derived from tryptophane metabolism such as 6-formylindolo carbazole (FICZ). Recent data suggest that AhR may be a major transcription factor involved in the development of the Th17 and Th22 subsets , and theAlthough AhR has been consistently involved in IL-22 production by T cells ,19, contIn the present study, activated peripheral blood mononuclear cells from allergic asthmatics exhibited an increased Th17/Th22 type profile compared with nonallergic nonasthmatic subjects. Only some of the PAH tested including DEP-PAH- and Benzo[a]pyrene- (B[a]P) exacerbated IL-22 production by stimulated mononuclear cells, whereas IL-17 production was inversely down regulated in asthmatic as well as healthy subjects. The mechanisms of regulation of IL-22 induction were dependent upon AhR, and also involved Mitogen-activated protein kinase (MAPK) and Phosphotidylinositol 3-kinase (PI3K) pathways.Venous blood was collected from 16 healthy nonallergic nonasthmatic (NA) subjects, with negative skin prick tests and no specific IgE to common environmental allergens, and from 16 allergic asthmatic (AA) patients. All these patients had a clinician’s diagnosis of asthma, and exhibited positive skin prick tests and specific IgE (more than 3 KU/L) towards at least one allergen. Skin prick testing included house dust mite, grass and tree pollens, cat and dog danders as well as fungi. Exclusion criteria included upper or lower respiratory tract infection within one month of study, use of anti-inflammatory controller medications within four weeks of study, significant non-asthma pulmonary disease or other medical problems. All subjects were non-smokers. To exclude the potential confounding effects of steroid treatment, only intermittent asthmatic subjects taking only β2 agonists as needed, according to GINA guidelines, were included. Among potential confounding factors for IL-17 and IL-22 expression, we anticipated severity of the disease, recent infections and smoking, however we cannot exclude uncontrolled factors such as encountered environmental pollution or diet. The subject’s characteristics are shown in 6/ml) were stimulated in 48-well flat-bottomed microculture plates coated with the minimal anti-CD3 concentration  and soluble anti-CD28  in complete Iscove's modified Dulbecco's medium , a medium previously shown to favour IL-17 production [As there are some controversies about the effect of AhR ligands on the production of IL-17 which may relate to the use of particular single cell types , we evaluated the effects of PAH in an environment where all these cell types are represented and can interact, in particular with dendritic cells, i.e. peripheral blood mononuclear cells (PBMCs). These cells were prepared from blood collected on heparin as previously described . To actioduction . A mix ooduction ,28, 9–10oduction . For Th1Cytokines were measured by ELISA according to the manufacturer’s recommendations. IL-17A, IL-22, CCL20, IL-13, IL-5, IL-10, TGF-β1, periostin and CCL18 kits were from R&D systems , IL-17F from eBiosciences and IFN-γ from BD Biosciences . The lower detection limit was 15.6 pg/ml for IL-17A, IL-17F and CCL20, 31.2 pg/ml for IL-22, IL-5, IFN-γ, and TGF-β, 93.8 pg/ml for IL-13, 375 pg/ml for periostin and 7.8 pg/ml for CCL18.Quantitative real Time PCR was performed as previously described . Briefly6/ml) were incubated with HAP or DMSO with or without Th17 cytokines. At day 3 IL-2 was added again. At day 6, cells were stimulated by PMA  and Ionomycin  in the presence of Brefeldin A  for 4h. Cell surface and intra cellular staining was performed as previously described [+ (clone RPA-T4), CD8-PE+ (clone HIT8a) for T cells; CD3-FITC- (clone HIT3a) CD56-PE+ (clone B159) for NK cells; iNKT-PE+ (clone 6B11) for iNKT cells; Lin2-FITC-  CD123-PE-+/ (clone 7G3) HLA-DR-V450+ (clone G46.6) for dendritic cells (DCs) (BD Biosciences) and intracellular staining using IL-17A-APC (clone eBio64DEC17), IL-22-PERCP eFluor710 (clone 22URTI) (eBiosciences). Mouse IgG1-APC, IgG1-PERCP eFluor710 isotypes (clone P3.6.2.8.1) were from eBiosciences. Living cells were gated using forward and side scatter properties, and further using livedead fixable aqua dead cell stain kit  and then cell populations were gated using the specific markers indicated above. Data were acquired on a BD LSR Fortessa flow cytometer using the DIVA software (version 6.1.3). Compensations were calculated using BD Compbeads with the automatic compensation programme. Data were expressed as percentage of cytokine positive cells among the considered population after subtraction of the control isotype.IL-17 and IL-22 cell origin was determined by flow cytometry. Stimulated PBMCs  except for CCL18. For CCL18, data were analysed using non-parametric Friedman analysis of variance followed by Dunn’s post hoc test. For the other parameters, within NA/AA comparisons were performed using one way ANOVA followed by Holm-Sidak’s multiple comparison test, and between NA/AA comparisons using unpaired t-test. Statistical analyses were performed using GraphPad Prism 6 software . Values of p<0.05 were regarded as statistically significant. As this was an exploratory study, no adjustment for multiple testing was performed.RORC, AhR target gene CYP1A1, but not RORA were upregulated in AA compared with NA subjects  subjects (n = 10) and allergic asthmatic (AA) patients (n = 12) incubated or not with PAH. Results are expressed as mean ± SEM. *(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigActivated PBMCs from nonallergic (NA) subjects (n = 10) and allergic asthmatic (AA) patients (n = 12) were incubated or not with PAH, in the presence or not of AhR antagonist CH-223191. The dotted line is set on the level of the antagonist-treated control cells. Results are expressed as mean ± SEM.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Quantification of edema and scar maps with cardiac MR images (cMRIs) enables effective Radiofrequency Ablation (RFA) of arrhythmias during the Electrophysiology (EP) procedure . This deAcquiring image gradient is a common step in intensity-based registration methods  (e.g. De® Virtex7-XC7V2000T) was used to investigate the accelerated performance. Figure Figure The performance of the proposed computing architecture demonstrates its high potential for accelerating registration of 3D-gated MRI images to improve visualization of the MRI-guided cardiac therapy.NIH U41-RR019703, R43 HL110427-01, AHA 10SDG261039, EPSRC and Croucher Foundation Fellowship.