SLCO1B3 gene (P<0.05) in the shell gland and oviduct. Predicted transcription factor binding sites in the long terminal repeats (LTR) indicate AhR/Ar, a modulator of oestrogen, as a possible promoter/enhancer leading to reproductive tissue-specific over-expression of the SLCO1B3 gene. Analysis of all jungle fowl species Gallus sp. supports the retroviral insertion to be a post-domestication event, while identical LTR sequences within domestic chickens are in agreement with a recent de novo mutation.Oocyan or blue/green eggshell colour is an autosomal dominant trait found in native chickens (Mapuche fowl) of Chile and in some of their descendants in European and North American modern breeds. We report here the identification of an endogenous avian retroviral (EAV-HP) insertion in oocyan Mapuche fowl and European breeds. Sequencing data reveals 100% retroviral identity between the Mapuche and European insertions. Quantitative real-time PCR analysis of European oocyan chicken indicates over-expression of the  The ancestral avian eggshell is believed to be both white and immaculate Several hypotheses have been proposed to account for the diversity of eggshell pigmentation. These range from the conspicuous, crypsis and recognition de novo origin in South American chicken. Following Castello Oocyan is an autosomal dominant trait in chicken resulting from an accumulation of biliverdin in the eggshell, leading to blue/green shelled eggs et al. SLCO1B3, proposing it to be the causative mutation of the oocyan phenotype in the breed. They reveal the same retroviral insertion and site to be present in another Chinese breed (Lushi), but a different insertion site in North American Araucana believed to be of South American descent. They suggest that these two insertions represent parallel evolution of the oocyan phenotype in the American and Asian continents, but do not provide any evidence of enhanced expression of SLCO1B3 in native fowl from the South American continent.Recently Wang SLCO1B3 in the shell gland and oviduct of European oocyan chickens and that HMOX1, a previous candidate gene, is not over-expressed de novo mutation in domestic chicken. Our results support the parallel, post-domestication, integration of an endogenous retrovirus leading to the oocyan phenotype in South America and Asian chickens vice versa. Moreover, they indicate a likely recent origin for the mutation on the South American continent.Here, we report the sequencing of the 300 kb interval containing the oocyan locus that we previously mapped in Mapuche fowl and European chicken breeds Gga1:67,246,039–67,350,164)  of the chicken reference genome , indicating c26830 to be a possible retroviral insertion rather than a translocation.Target enrichment and sequence capture in 9 chickens  . Variant350,164) . Structu SLCO1B3  [15]. ThG. varius), grey (G. sonneratii), and Sri Lankan (G. lafayettii) jungle fowl, as well as three red jungle fowl subspecies , none of which lays blue eggs, indicating the conserved length of the genomic region amplified and absence of the insertion  and DNA sequences  are found for the South American and European chickens. The EAV-HP sequence of the oocyan Dongxiang chicken is 98% identical to the Mapuche/Araucana, however, the host integration site is different at Gga1:67,324,647  is found in the 96 bp homologous host sequence upstream of the Mapuche/Araucana retroviral integration site, whilst the Dongxiang shared the reference genome ‘A’ allele , Europe , and Asia . This confirms the insertion to be an EAV-HP retrovirus integrated in opposite orientation to the solute carrier ,324,647 . A singl’ allele . No poly’ allele  in any o3′-CCTTCA-5′) and Dongxiang (3′-GAGGAG-5′) chicken. The opposing LTRs within a given sequence are identical for their 313 bp length, although a pair-wise comparison reveals a 1% sequence divergence (3 SNPs) between the Mapuche/Araucana and Dongxiang LTR sequences  is different in the Mapuche/Araucana . The average relative quantitative (RQ) values are presented in t-test. For the qRT-PCR runs, each target gene was analysed in triplicate in each tissue. Significant (P<0.05) over-expression of SLCO1B3 is observed in the shell gland (∼19 fold increase) and oviduct (∼180 fold increase) of oocyan chickens  descendants. Taken together with the independent discovery of the EAV-HP integrations by Wang et al. SLCO1B3 over-expression in oviduct tissue in Dongxiang chickens, the results complement one-another. With the knowledge of distinct genomic insertion sites, the results clearly indicate the independent acquisition of the oocyan phenotype in native Asian and South American chickens.The close genomic proximity of the EAV-HP integration to et al.The origin of chickens in South America is a subject of debate, and genetic links to Chinese chicken have been suggested Historical evidence for the oocyan phenotype indicates that it has been present since at least 500 years ago in the Dongxiang chicken in Asia The EAV-HP integration site of the oocyan Mapuche fowl, the French Araucana, the Moss breed from Spain, and the Araucana and Cream Legbar chickens from the UK are identical and in agreement with historical accounts that the modern European oocyan breeds derive from Mapuche fowl. Similarly the genome integration site of the Mapuche fowl was identical to the North American Araucana Gallus gallus, G. sonneratii, G. lafayettii, and G. varius) are known to lay blue or green eggs G. gallus, and is likely to have been acquired post separation of Gallus from other galliform species due to its absence in the more distant turkey and quail genomes G. gallus. However, the absence of sequence divergence in the opposing LTRs of the EAV-HP insertion within any of the individual chickens sequenced prevents an estimation of the date of the integration other than to suggest that it is recent.None of the jungle fowl species described , has been linked to oestrogen metabolism and oxidative stress SLCO1B3 transcript (GenBank accession no: JN381032) commences 2 bp after the predicted AhR/Ar transcription factor binding site. The results of our analyses therefore suggests that the EAV-HP insertion might enhance the expression of SLCO1B3 in a reproductive tissue-specific manner. This is possibly due to promoter and/or enhancer sequences in the LTRs, such as AhR/Ar – a mediator of oestrogen regulation. Indeed, several studies have identified genes expressed in the chicken oviduct as being under the regulation of oestrogen SLCO1B3, for which biliverdin is a substrate, might increase the accumulation of biliverdin in the shell gland during shell matrix formation, giving rise to blue eggs. However, in absence of full transcriptome data we cannot fully exclude the possibility of other long-range cis-regulatory effects of the EAV-HP insertion, or a possible role for any of the SNPs identified in the oocyan mapping interval on the expression of the phenotype.Avian retroviral LTRs have been extensively studied SLCO1B3, resulting in blue eggs cannot be excluded, calling for further investigation.Our findings address an important question: to what extent are endogenous retroviruses (ERVs) shaping phenotypic diversity in birds? It is not the first reported instance of an ERV insertion affecting a chicken's phenotype. An insertion of avian leukosis virus (ALV) in intron 4 of the tyrosinase gene results in the recessive white plumage of some chicken breeds et al. Taeniopygia guttata, which has more than double the ERV burden compared to chicken The abundance of ERVs identified in three neognaths Passer domesticus, European cuckoo Cuculus canorus, and common guillemot Uria aalge are all laying blue eggs as well as non-blue eggs within a single population. In this context, it is also interesting to remember that brown egg colours of various intensities are found across different bird species including chicken, such a phenotype could be influenced by the presence of multiple retroviral insertions in agreement with the polygenic control of the trait This ERV reservoir together with the capacity of ERVs for horizontal transfer highlights the possibility that ERVs might influence egg colour more generally in birds. For example species like house sparrow Whether the blue egg offers an increased propensity for bi-parental provisioning, confers a luminary-related advantage, anti-microbial properties, or offers some degree of crypsis remains to be seen. At least in the case of the chicken, where it has likely been the subject of artificial selection, the oocyan EAV-HP integration will remain in the genome for as long as man continues to take a shine to blue eggs.All the chickens at the INRA experimental farm were produced, fed and sacrificed according to French regulations in 2007, which did not require the approval by an ethical committee at that time, but the authorization of the facility and the researchers. The farm was, and still is, registered by the Ministry of Agriculture with the license number B37–175–1 for animal experimentation. The experiment was realised under authorization 37–002 delivered to D. Gourichon and authorization 2369 delivered to M. Tixier-Boichard. Animal procedures were approved by the Departmental Direction of Veterinary Services of Indre-et-Loire. Before tissue sampling, animals were sacrificed by cervical dislocation followed by bleeding.Gallus sp.). Details concerning DNA isolation and processing of tissues for RNA isolation are included in the supplementary information  following the manufacturers recommended approach. All samples were recorded as having a calcified egg in the shell gland. The concentration and purity of all RNA samples was determined with a Nano-Drop® ND-1000 UV-Vis Spectrophotometer (Nano-Drop Technologies). Samples were aliquoted into 50 µl volumes at a concentration of 10 µg and stored at −80°C until use. Prior to the qRT-PCR amplification, aliquoted RNA (10 µg in 50 µl reaction volumes) was treated with TURBO Dnase I  to remove any trace genomic DNA arising from the isolation procedure. The High Capacity RNA-to-cDNA Kit (Invitrogen) was used to synthesize XT Custom Target Enrichment Kit (Agilent Technologies Inc). Baits were designed using Agilent's eArray software (https://earray.chem.agilent.com/earray) to capture the region previously mapped for oocyan  in three oocyan  and six non-oocyan chickens . Paired-end (2×100 bp) sequencing was performed using Illumina's GAIIx platform. Reads were mapped using BWA http://picard.sourceforge.net). BAM files from the target-enrichment sequence-capture for the nine chickens sequenced have been uploaded to NCBI (SRA accession no: SRA067224).Target enrichment was performed using Agilent's SureSelectVariant discoveryhttp://www.ensembl.org/info/data/ftp) and BGI (http://chicken.genomics.org.cn/chicken/) SNP databases, and the functional consequences of novel SNPs were summarized using Ensembl's Variant Effect Predictor Gga1:67,051,487–67,364,512). Reads spanning the SV boundaries were assembled into contigs and BLAT against the chicken genome  to resolve the SV break-point to base pair resolution and to identify partial sequences of the insertion. A detailed account of SV analysis is provided in the supplementary information . Data were collected and analysed using the 7500 Fast Real-Time PCR Systems v2.0.6 software (Life Technologies) and the RQ values for each tissue and target were exported to Excel®. An average value was calculated and the relative RQ plots were generated for each target for oocyan, brown and white egg layers respectively. Differences in expression levels for each target between oocyan and non-oocyan chickens were tested for their significance using Student's t-test.All primers used in the study are provided in Gga1 were designed to amplify a 349 bp product in non-oocyan chickens. A PCR amplification band was observed at ∼4.5 kb in oocyan samples extracted from blood or tissue, but was inconsistent in oocyan samples extracted from blood on FTA Classic cards – these samples were later confirmed by multiplex PCR (described below) to contain the retroviral insertion. The failure to consistently amplify the 4.5 kb fragment from the FTA samples might be explained by degraded DNA, or high amounts of inhibitor carry-over following DNA extraction. The c26830 insertion was Sanger sequenced in three oocyan homozygotes, one each of the Dongxiang breed, European Araucana breed and a Mapuche fowl, by primer-walking using the c26830 long-range PCR product as the template. FASTA sequences have been submitted to NCBI GenBank for the Mapuche/Araucana and Dongxiang EAV-HP insertions (GenBank accession no: KC632577 and KC632578).The size of the c26830 retroviral DNA insertion was identified by long-range touch-down PCR. Primers spanning the insertion breakpoint on Gga1 and amplifies a 364 bp product in the absence of the retroviral insertion. In total, 143 samples were screened   is in the LTR sequence of EAV-HP which, with the forward primer, amplifies a 167 bp product (190 bp in Dongxiang) in the presence of the retroviral insertion. Whilst the other reverse primer (Multiplex PCR R1) with the forward primer spans the insertion breakpoint on  breeds) .A BLAST of the complete c26830 sequence confirmed it to be the EAV-HP retrovirus. The heuristic approach for gene prediction Figure S1Long-range PCR spanning the c26830 insertion.(PDF)Click here for additional data file.Figure S2Multiplex PCR reveals a different product size in Dongxiang chicken.(PDF)Click here for additional data file.Table S1Target-enrichment sequence capture mapping summary.(PDF)Click here for additional data file.Table S2Average relative quantitative (RQ) values per sample per tissue following qRT-PCR.(PDF)Click here for additional data file.Table S3Summary of samples used in the study.(PDF)Click here for additional data file.Table S4Primers used in qRT-PCR.(PDF)Click here for additional data file.Table S5Primers used in long-range and multiplex PCRs.(PDF)Click here for additional data file.Methods S1Supplementary information on (a) sampling, (b) structural variant discovery, and (c) PCR cycling profiles.(PDF)Click here for additional data file.Sequence S1Annotated sequence for Mapuche/Araucana oocyan homozygote.(PDF)Click here for additional data file.Sequence S2Annotated sequence for Dongxiang oocyan homozygote.(PDF)Click here for additional data file.Sequence S3Sequence alignment of Dongxiang sequence to GenBank accession no: JF837512.(PDF)Click here for additional data file.


Endothelial function was assessed by the change in FVR following occlusion. Seven subjects returned for a repeat study with hyperglycemia replaced by euglycemia. Preocclusion FVR decreased from euglycemia to hyperglycemia (P = 0.003). Postocclusion fall in FVR during hyperglycemia was less than during euglycemia (P = 0.002). These findings were not reproduced when hyperglycemia was replaced with a second euglycemia. These results demonstrate that acute hyperglycemia causes vasodilation and alters endothelial function in adolescents with type 1 diabetes. In addition they have implications for future studies of endothelial function in type 1 diabetes and provide insight into the etiology of macrovascular and microvascular complications of type 1 diabetes.Alterations of blood flow and endothelial function precede development of complications in type 1 diabetes. The effects of hyperglycemia on vascular function in early type 1 diabetes are poorly understood. To investigate the effect of hyperglycemia on forearm vascular resistance (FVR) and endothelial function in adolescents with type 1 diabetes, FVR was measured before and after 5 minutes of upper arm arterial occlusion using venous occlusion plethysmography in (1) fasted state, (2) euglycemic state (~90 mg/dL; using 40 mU/m Multiple studies have indicated that cardiovascular disease has its origins in childhood and adolescence –3. This Previous studies have indicated that endothelial dysfunction is present in adolescents with type 1 diabetes before the development of overt complications , 11, 12.We measured forearm blood flow and endothelial function in otherwise healthy, pubertal subjects with type 1 diabetes at variable fasting glucose levels, and during euglycemic and hyperglycemic insulin infusion. Secondarily, we assessed other cardiovascular risk factors including inflammatory markers and markers of oxidative stress in all three conditions.2. Mean HgbA1c of 8.3 ± 1.2% and mean duration of diabetes was 4.2 ± 3.9 years. The study was approved by the NCH Institutional Review Board and informed consent was obtained from a parent or legal guardian. Proper assent was obtained from all subjects.Eleven adolescents  with type 1 DM were recruited from the Pediatric Diabetes Clinic of Nationwide Children's Hospital (NCH). Their mean age was 14.5 ± 1.0 years (mean ± SD) and their mean body mass index was 21.5 ± 2.9 kg/mScreening included a history, physical exam, Tanner staging, and fasting laboratory testing. Type 1 DM was defined by American Diabetes Association Criteria plus a fasting C-peptide of less than 0.4 ng/mL, insulin monotherapy since diagnosis, and an absence of a history of oral hypoglycemic agents and acanthosis nigricans on exam. All subjects were nonsmokers by report.All subjects were Tanner stage 2–4 in order to minimize the effects of starting or finishing puberty. In order to limit confounding effects on endothelial function, subjects with BP > 95th percentile, smoking, pregnancy, and uncorrected hypothyroidism, were excluded. Subjects with microalbuminuria, overt nephropathy, or early renal failure  were also excluded.For the main study visit, subjects were admitted to the Clinical Research Center (CRC) at Ohio State University after an overnight fast. Subjects continued their insulin regimen of multiple dose injections (MDI) or continuous subcutaneous insulin infusion (CSII). Subjects on MDI received basal insulin injection the night prior to study and subjects on CSII continued with basal insulin infusion overnight. Subjects were NPO with subsequent omission of morning insulin bolus on the study day. Endothelial function measurements and blood sampling for laboratory analysis of inflammatory, oxidative, and endothelial markers were performed in each subject during three states: (1) fasting, (2) euglycemia, and (3) hyperglycemia.Forearm Blood Flow (FBF) was measured using strain gauge venous occlusion plethysmography, as previously described by Higashi and Yoshizumi , using a2/min to bring the glucose level to a target of 90–95 mg/dL. Insulin rates were increased above 40 mU/m2/mn in 2 subjects because of prolonged hyperglycemia. Blood samples were taken through a second intravenous catheter at five-minute intervals for the immediate determination of plasma glucose using an automated glucose oxidase technique . When the target glucose level was achieved, dextrose was added to maintain euglycemia for 30 minutes with a minimum total of 60 minutes of insulin infusion. In those subjects in whom the insulin level was increased, it was reduced to and maintained at 40 mU/m2/min at the beginning of the euglycemic period. Endothelial function measurement and blood sampling were repeated at the end of 30 min of euglycemia.After the fasting endothelial function measurement was complete, the insulin clamp portion of the study was initiated. A catheter was placed in an antecubital vein for the administration of glucose, insulin, and saline. Insulin infusion was initiated at a rate of 40 mU/mAfter completion of the euglycemic phase the insulin infusion was continued and the dextrose infusion rate was increased to raise the plasma glucose level to a target of 200 mg/dL for 60 minutes following the end of euglycemia. Endothelial function and biochemical markers were again measured after hyperglycemia.As a control for the effects of fluid volume and insulin infusion, seven subjects returned for an additional study in which the hyperglycemic arm of the study was replaced with a second euglycemic phase. Each subject received normal saline to maintain an equal rate of volume infusion as in the previous hyperglycemic clamp. The insulin infusion rate remained unchanged.High-sensitivity C-reactive protein (hsCRP), total plasma antioxidant capacity (TAOC), and a measure of oxidative stress, were measured for each subject at each stage of glycemia. TAOC is a nonspecific assay of antioxidant defense which measures the ability of constituents in plasma to absorb oxidation . Soluble intracellular adhesion molecules (sICAM) were also measured at each stage of glycemia as a marker of endothelial activation. Serum sICAM levels were determined using a commercially available assay .P < 0.05 and tendencies are mentioned at P < 0.1.Repeated measures analysis of variance was used to determine differences in FBF and FVR responses to changes in plasma glucose and upper arm occlusion. Only glucose was used as a repeat factor for TAOC, sICAM, and hsCRP. Systat 11  was used to perform all statistical analysis. Data are expressed as mean ± SE. Differences were considered significant at During the first study clamp, the mean fasting glucose was 166 ± 21 mg/dL, mean glucose during euglycemia was 83 ± 4 mg/dL, and mean glucose at the end of hyperglycemia was 219 ± 7 mg/dL . The meaP < 0.001) and a significant difference in response to upper arm occlusion across the three levels  and further increased from euglycemia to hyperglycemia (P = 0.005). The increased FBF following upper arm occlusion during hyperglycemia was greater than the increase following occlusion during euglycemia (P = 0.013). Repeated measures analysis revealed a tendency for a decrease in the ratio of postocclusion FBF to preocclusion FBF across the three levels (P = 0.070). Although no specific differences were noted between baseline and euglycemia or between euglycemia and hyperglycemia. Repeated measures analysis of variance revealed significant differences in FBF across the three glucose levels  across the three phases of the clamp (P < 0.001) and a significant effect of glucose level on the occlusion-induced change in FVR (P = 0.001). Specifically, preocclusion FVR tended to decrease from fasting to euglycemia (P = 0.073) and significantly decreased from euglycemia to hyperglycemia (P = 0.003). The absolute postocclusion decrease in FVR during euglycemia tended to be less than that at baseline (P = 0.083) and the postocclusion fall in FVR during hyperglycemia was significantly decreased compared to euglycemia (P = 0.003). However, no differences were seen for the pre- to postocclusion percent decrease in FVR between the three stages . The most significant difference was identified between fasting and euglycemia (P = 0.058) concurrent with insulin initiation. No significant changes were identified between the three glycemic states for TAOC or hsCRP.Significant decreases in sICAM values across all three phases were present  but did not increase further during the second euglycemic period. There were no differences in response to upper arm occlusion between the three time periods. The increase in preocclusion FBF from euglycemia to hyperglycemia during the first study tended to be greater than the lack of change from the first to second euglycemic periods (P = 0.082).Repeated measures analysis of variance again revealed significant differences in FBF across the three measurement times  but no differences were noted in regards to diastolic blood pressure or mean arterial pressure . As in the hyperglycemic study preocclusion FVR fell from baseline to euglycemia (P = 0.003) but did not differ between the two euglycemic periods. Similarly, the decrease in FVR following upper arm occlusion was less during first euglycemic period than at baseline (P = 0.004) but did not differ between the two euglycemic periods. In regards to occlusion response, the percent change in FVR during the first euglycemic period tended to be smaller than that at baseline . There were no significant differences found between the studies for postocclusion FBF response. For FVR, the decrease in preocclusion FVR from euglycemia to hyperglycemia was significantly different from the lack of change during continued euglycemia (P = 0.042) and the decrease in postocclusion fall in FVR during hyperglycemia was significantly different from the lack of change during continued euglycemia (P = 0.047).Comparison of the changes between euglycemia and hyperglycemia with the changes between first and second euglycemia periods revealed that the increase in preocclusion FBF from euglycemia to hyperglycemia was significantly different from the lack of change during continued euglycemia (Hyperglycemia causes acute vasodilation in healthy adults . The vasThe mechanism for hyperglycemia-induced vasodilation is not clear. One possible mechanism would be increased vascular volume and baroreflex suppression since systolic blood pressure increased during hyperglycemia. Against this hypothesis is that a similar, although not statistically significant, increase occurred during the same time period of the control, euglycemic saline infusion study without changes in FBF or FVR. Also, against this hypothesis is the previous study which showed that hyperglycemia increases, not decreases, sympathetic nerve activity . Since hIn contrast to the acute vasodilatory effect of hyperglycemia during constant insulin infusion, we also saw vasodilation with correction of hyperglycemia from baseline to euglycemia during both studies. This is likely due to the vasodilator effect of insulin infusion . This deThe direct impact of hyperglycemia-induced vasodilation in patients with type 1 diabetes is not certain. One primary area of interest would be its effect on diabetes complications, nephropathy in particular. Specifically, hyperglycemia-mediated decreased vascular resistance may be responsible for increased renal blood flow and increased glomerular filtration rate seen early in diabetic nephropathy . AdditioThe effect of hyperglycemia on endothelial function in our study is unclear. The total postocclusion fall in FVR was less during hyperglycemia but the percent fall in FVR was not different. The smaller absolute postocclusion vasodilatory response during hyperglycemia is most likely due to the increased preocclusion vasodilation and decreased reserve capacity for additional stress-induced vasodilation or, in other words, a ceiling effect in maximal vasodilation. Kawano et al.  previousMultiple past studies have evaluated endothelial function in adolescents with type 1 diabetes , 11, 12.Limitations to this study are the short duration of hyperglycemia and the study of males and females combined. The lack of effects of hyperglycemia on markers of endothelial damage, inflammation or oxidation, may be secondary to the short duration of hyperglycemia in our study. It is possible that more prolonged hyperglycemia may have induced changes in some of these areas. Although gender differences have been found in endothelial function in adults, no differences were apparent in this study.In conclusion, acute hyperglycemia has profound effects on blood flow, vascular resistance, and endothelial function. These findings have implications for future studies of endothelial function in type 1 diabetes. More importantly, hyperglycemic-induced vasodilation may play a significant role in the development of macrovascular and microvascular complications in patients with type 1 diabetes.A. S. Dye participated in data collection and writing paper. H. Huang performed laboratory measurement. J. A. Bauer supervised laboratory measurements and reviewed edited paper, R. P. Hoffman wrote protocol and obtained research funding, supervised or directly collected data, wrote and edited paper. He is responsible for its content.


The recent finding that inhibition of the glycogen-synthase-kinase 3β (GSK-3β) induces a rapid nuclear export of the AR in androgen-stimulated prostate cancer cells prompted us to analyze the effects of a GSK-3β inhibition in the castration-resistant LNCaP sublines C4-2 and LNCaP-SSR. Both cell lines exhibit high levels of nuclear AR in the absence of androgenic stimuli. Exposure of these cells to the maleimide SB216763, a potent GSK-3β inhibitor, resulted in a rapid nuclear export of the AR even under androgen-deprived conditions. Moreover, the ability of C4-2 and LNCaP-SSR cells to grow in the absence of androgens was diminished after pharmacological inhibition of GSK-3β in vitro. The ability of SB216763 to modulate AR signalling and function in CRPC in vivo was additionally demonstrated in a modified chick chorioallantoic membrane xenograft assay after systemic delivery of SB216763. Our data suggest that inhibition of GSK-3β helps target the AR for export from the nucleus thereby diminishing the effects of mislocated AR in CRPC cells. Therefore, inhibition of GSK-3β could be an interesting new strategy for the treatment of CRPC.In order to generate genomic signals, the androgen receptor (AR) has to be transported into the nucleus upon androgenic stimuli. However, there is evidence from  Transcriptional regulation by nuclear receptors plays a pivotal role in the development and growth of prostate cancer (PC) cells. This is well exemplified by the role of the androgen receptor (AR), a ligand-activated transcription factor belonging to the steroid receptor superfamily. In the absence of androgens, the AR is predominantly located in the cytoplasm stabilized by a multichaperone complex in vitro development of an androgen-independent phenotype is mostly based on the loss of the AR in tumor cells, there is evidence from several clinical studies that the AR is rarely lost in CRPC cells in vivoThe initial androgen dependency of prostate cancer cells is the reason why most PC respond to androgen ablation therapy. Unfortunately, the benefit of androgen ablation is only transitory. After a period of around two years, PC almost invariably progress to a state of the disease termed castration-resistant prostate cancer (CRPC) where tumor cells can grow and survive under castrate levels of circulating androgens. Although the in vitro experiments that in a subset of CRPC cells, the AR acquires the ability to undergo ligand-independent shuttling from the cytoplasm to the nucleus In order to generate genomic signals in hormone-dependent PC cells the AR must be transported into the nucleus upon androgenic stimuli. There is compelling evidence from Various studies have offered insights into the nuclear import of the AR. A bipartite nuclear localization sequence (NLS) has been identified in the DNA binding domain (DBD) and hinge region of the AR in vitro as well as in vivo data presented in this study suggest that induction of nucleocytoplasmic AR export might be a useful strategy to diminish AR signalling, especially in advanced CRPC.The finding that inhibition of GSK-3β induces a rapid nuclear export of the AR in androgen-stimulated PC cells prompted us to analyze the effects of GSK-3β inhibition in castration-resistant LNCaP sublines C4-2 and LNCaP-SSR In order to analyze the effects of GSK-3β inhibitors on AR-activity in CRPC cells we first determined endogenous AR or GSK-3β levels in whole cell extracts of PC cells grown in the absence of DHT. The AR-positive LNCaP sublines C4-2 and LNCaP-SSR, known to proliferate in the absence of androgens, served as models for CRPC Y216), a GSK-3β-activating phosphorylation site, was increased in LNCaP and C4-2 cells  -2 cells . In ordells Y216.Y216 in both cell lines, being most pronounced in the androgen-independent C4-2 cells , we were able to dramatically reduce intracellular GSK-3β levels in C4-2 cells . This doInhibition of GSK-3β has been shown to inhibit proliferation of AR-positive cells grown in presence of physiological levels of androgens in vivo, we used a modified chick chorioallantoic membrane (CAM) model in vitro observations not only C4-2 but also LNCaP expressed high levels of nuclear AR in vivo . Following SB216763 treatment nuclear AR levels in C4-2 cells were reduced by 57% from 63.3 to 27.1% after 48 hours , the activation function of the enzyme, in C4-2 cells Y216 phosphorylation in LNCaP cells following DHT treatment  leads to a rapid export of the AR prompted us to analyze the effects of a long term inhibition of the enzyme in AR-positive CRPC-cells. Silencing of GSK-3β using specific shRNA led to a depletion of nuclear AR in C4-2 cells. Nuclear depletion of AR protein was less pronounced in C4-2 cells grown in the presence of the AR-stabilizing hormone DHT. Although these findings support the assumption that inhibition of GSK-3β triggers the nucleocytoplasmic shuttling of the AR, the data derived from the long term shRNA-experiments must be interpreted carefully. Short term inhibition of GSK-3β activity by small molecules like SB216763 was repeatedly shown to induce a rapid, CRM1-dependent nuclear export of the AR in PC cells in vitro. Response to treatment of PC cells with SB216763 was most pronounced in AR-positive CRPC cells (inhibition of cell growth: C4-2>LNCaP-SSR>LNCaP>PC3). The ability of SB216763 to inhibit cellular proliferation was paralleled by its ability to induce a CRM1-dependent export of the AR in PC cells.The fact that inhibition of GSK-3β affects AR function as well as AR stability prompted us to analyze the effects of a GSK-3β inhibition on the proliferation of PC cells In order to test the efficacy of GSK-3β inhibitors as potential therapeutic agents, we extended our studies to a CAM-xenograft model. To our surprise the AR was predominantly nuclear in LNCaP xenografts as well as in C4-2 xenografts. A possible explanation for this could be the production of androgens by the host organism. Moreover, the AR of LNCaP and its derivatives is carrying a point mutation (T877A) leading to a promiscuous AR that can, at least in part, be activated by various non-androgenic steroids in vitro and in vivo thereby modulating AR signalling.Taken together our study demonstrates that (1) overexpression of the AR in CPRC cells is paralleled by an increase in intracellular GSK-3β, (2) the effects of GSK-3β on AR signalling are not due to a modulation of DHT binding to the AR, (3) nuclear accumulation of the AR in CRPC cells is not an autonomous function of a mutated AR receptor but is mediated by deregulated cellular factors, such as GSK-3β, (4) GSK-3β and CRM1 are part of a putative complex controlling the nuclear localization of the AR in CRPC cells, and (5) inhibition of GSK-3β activity by small molecule inhibitors induces a rapid nuclear export of the AR in CRPC cells The dependence of CRPC on transcriptionally active AR has resurged the interest in developing inhibitors targeting the AR or androgen axis. Next generation hormone therapies recognize the fact that in CRPC the AR can be activated by intrinsic production of tissue androgens as well as by peptide growth factors. Among hormonal agents currently being tested are CYP17 inhibitors like abiraterone, TAK-700 and TOK-001 or the second generation anti-androgen MDV-3100 GSK-3β inhibitor SB216763 -4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione) was provided by Biomol GmbH, Hamburg, Germany. Phosphatase inhibitor cocktail tablets (Phos-Stop), protease inhibitor cocktail tablets (Complete Mini) and dithiothreitol (DTT) were obtained from Roche Diagnostics GmbH, Mannheim, Germany. Leptomycin B was a product of Alexis Biochemicals, Lörrach, Germany. Dihydrotestosterone (DHT), HEPES, ethylene-diamine-tetra-acetic acid (EDTA) and Tween 20 were provided by Sigma-Aldrich GmbH, Taufkirchen, Germany. Bovine serum albumin (BSA) was provided by PAA Laboratories GmbH, Pasching, Austria. Sodium dodecyl sulphate (SDS), Nonidet NP40 (NP40) and Glycine were products of AppliChem GmbH, Mannheim, Germany. All other chemicals, if not specified, were purchased from Sigma-Aldrich GmbH, Taufkirchen, Germany. GSK-3β-directed shRNA (pkD-GSK-3β-v1) or control shRNA (pkD-NegCon-v1) were products of Upstate Biotechnology, Lake Placid, NY.Antibodies for Western blotting: AR rabbit monoclonal antibody  directed against the C-terminal end of the AR was purchased from Biomol, Hamburg, Germany. Polyclonal rabbit antibodies to pGSK-3β (Y216) and Lamin A (H102) as well as mouse monoclonal antibodies to PSA (CHYH2) and GSK-3β (1F7) were provided by Santa Cruz Biotechnology, Heidelberg, Germany. Mouse monoclonal antibody AC-15 to β-actin was purchased from Sigma Aldrich GmbH, Taufkirchen, Germany. Anti pGSK-3β (Ser 9) (D3A4) and anti-mouse and anti-rabbit IgG-horseradish peroxidase linked antibodies were products of Cell Signalling Technology, Frankfurt a.M., Germany.Antibodies for histochemistry: Mouse monoclonal antibodies directed against PSA (ER-PR8) and AR (AR-441) were products of DAKO Diagnostica, Hamburg, Germany. Immunohistochemical detection/staining of primary antibodies was performed using Histostain Plus, Invitrogen, Darmstadt, Germany, according to the manufactures instructions.AR-negative PC3 cells and AR-positive LNCaP cells were provided by the American Type Culture Collection, Manassas, VA, USA. CRPC cell lines C4-2 and LNCaP-SSR were provided by Prof. Sven Reske, Ulm, Germany and Prof. Martin Burchardt, Greifswald, Germany. RPMI-1640, phosphate buffered saline (PBS) and penicillin/streptomycin solution were products of PAA Laboratories, Linz, Austria. Fetal bovine serum (FBS) and steroid-free dextran-charcoal-treated FBS (FBSdcc) were obtained from BioWest, Nuaille, France. Cell culture plastic ware was purchased from Sarstedt, Nürmbrecht, Germany. LNCaP cells and PC3 cells were routinely cultured in RPMI-1640, 1% penicillin/streptomycin (v/v), 10% FBS (v/v) whereas LNCaP-SSR and C4-2 were routinely grown in RPMI-1640 supplemented with 1% penicillin/streptomycin (v/v) and 10% steroid-free dextran charcoal-treated FBS (FBSdcc) (v/v). During experiments, cells were maintained in RPMI-1640 with 2.5% FBSdcc (v/v) and antibiotics in the presence/absence of DHT and the GSK-3β-inhibitor SB216763.For the preparation of whole cell extracts, cells grown in T25 flasks were incubated with 500 µl lysis buffer , pH 7.3) supplemented with phosphatase and protease inhibitors according to the manufactures instructions. Subsequently, cells were scraped thoroughly out of the flask. Cell lysates were centrifuged at 15000 xg for 15 minutes at 4°C in a tabletop centrifuge. Supernatants were collected and protein concentrations were determined using the BCA-Protein Assay . Whole cell extracts were subjected to Western blot analysis.2, 10 mM KCl, 0.5 mM DTT, 0.05% NP40, pH 7.9) and centrifuged 10 minutes at 3000xg to collect cytoplasmic fractions. Residual pellets containing the nuclei were resuspended in 93.5 µl buffer B , pH 7.9) and 6.5 µl of 4.6 M NaCl and centrifuged at 15000xg to remove nuclear debris. Extracts were analysed by Western blotting.Nuclear extracts were prepared as recently described Whole cell extracts or nuclear fractions (30 µg) were subjected to electrophoresis in a 10% SDS-PAGE and electroblotted onto nitrocellulose membranes .AR, PSA and GSK-3β were detected as follows: Non-specific binding sites on the nitrocellulose were blocked by incubation with 5% (w/v) non-fat dry milk  in PBST . Antibodies were applied in PBST supplemented with 1% not-fat dry milk as follows: AR antibody (EP670Y) 1∶2000; PSA antibody (CHYH2) 1∶500; GSK-3β antibody (1F70) 1∶500 and antibodies serving as loading controls for cytoplasm/nucleus, i.e., β-Actin antibody (AC15) 1∶20000 and Lamin A antibody (H102) 1∶1000. Resulting immunocomplexes were detected using horseradish peroxidase-labelled goat anti-rabbit antibody 1∶2000 and goat anti-mouse antibody 1∶2000 (1∶5000 for the detection of AC-15 to β-Actin). Immunoreactive bands were visualized by direct detection with Super Signal West Pico and Dura Chemiluminescence Substrates  using ChemiSmart 500 Vilber Lormat . Immunoreactive bands were quantified by densitometry using ChemiCapt/Bio1D software from Vilber Lormat.Y216 or pGSK-3βS9) was performed using the following modifications: Nitrocellulose membranes were blocked in PBST supplemented with 5% BSA. Subsequently membranes were incubated with a phosphospecific antibody to pGSK-3β (Y216) diluted in PBST with 1%BSA (w/v) at 1∶500. Immunoreactive bands were detected and visualized as described above. After detection of pGSK-3βY216 the phosphospecific antibody was removed by incubating the membrane 2×10 minutes in stripping buffer (1.5% Glycin (w/v), 0,1% SDS (w/v), 1%Tween-20 (v/v), pH 2,2). Following three washing steps (3×10 min) in PBST, the nitrocellulose membrane was blocked in PBST supplemented with 5% BSA and reprobed with the pGSK-3β (S9) antibody (1∶1000). Subsequently, the membrane was stripped once again and reprobed with the GSK-3β antibody (1F7) (1∶500), serving as a loading control. Immunoreactive GSK-3β bands were detected and quantified as described above.Immunodetection of site specific phosphorylation in GSK-3β  C4-2 cells were transiently transfected with shRNA-expression plasmids directed against GSK-3β (pkD-GSK-3β-v1) or with control shRNA (pkD-NegCon-v1) as recently described Cell viability was determined by means of a colorimetric MTT-assay measuring the reduction of tetrazolium salts to formazan derivatives by functional mitochondria. The assay was performed as originally described in vivo, we used a chick chorioallantoic membrane (CAM) assay as an animal substitute model 6 cells were seeded into the ring in 20 µl 50% Matrigel (v/v)  dissolved in serum and antibiotic-free RPMI-1640. Tumor grafts were allowed to grow for 48 hours. In contrast to the classical CAM assay, the GSK-3β inhibitor SB216763 was not applied topically. Instead 50 µl of the GSK-3β inhibitor  were injected into a CAM vein In order to analyze the effects of a GSK-3β inhibition The Mann-Whitney U test was used to evaluate distribution difference across two groups. Statistical significance in this study was set as p≤0.05.Figure S1Phosphorylation of the GSK-3β downstream target β-catenin. AR-positive C4-2 cells were treated with increasing amounts of SB216763 for 24 hours in the presence/absence of DHT. Cell extracts were analyzed by Western blotting as described in (TIF)Click here for additional data file.


Asthma genetics has been extensively studied and many genes have been associated with the development or severity of this disease. In contrast, the genetic basis of allergic rhinitis (AR) has not been evaluated as extensively. It is well known that asthma is closely related with AR since a large proportion of individuals with asthma also present symptoms of AR, and patients with AR have a 5–6 fold increased risk of developing asthma. Thus, the relevance of asthma candidate genes as predisposing factors for AR is worth investigating. The present study was designed to investigate if SNPs in highly replicated asthma genes are associated with the occurrence of AR.A total of 192 SNPs from 21 asthma candidate genes reported to be associated with asthma in 6 or more unrelated studies were genotyped in a Swedish population with 246 AR patients and 431 controls. Genotypes for 429 SNPs from the same set of genes were also extracted from a Singapore Chinese genome-wide dataset which consisted of 456 AR cases and 486 controls. All SNPs were subsequently analyzed for association with AR and their influence on allergic sensitization to common allergens.P-values corresponds well to the expectations in the absence of an effect. However, in the tests of allele effects in the Chinese population the number of significant P-values exceeds the expectations. The strongest signals were found for SNPs in NPSR1 and CTLA4. In these genes, a total of nine SNPs showed P-values <0.001 with corresponding Q-values <0.05. In the NPSR1 gene some P-values were lower than the Bonferroni correction level. Reanalysis after elimination of all patients with asthmatic symptoms excluded asthma as a confounding factor in our results. Weaker indications were found for IL13 and GSTP1 with respect to sensitization to birch pollen in the Swedish population.A limited number of potential associations were observed and the overall pattern of NPSR1 and CTLA4 can be genetic links between AR and asthma and associations of polymorphisms in NPSR1 with AR have not been reported previously.Genetic variation in the majority of the highly replicated asthma genes were not associated to AR in our populations which suggest that asthma and AR could have less in common than previously anticipated. However,  Allergic rhinitis (AR) is a major chronic respiratory disease and the most common allergic disorder with a worldwide prevalence of 10-25% . A largeIt is well known that asthma is closely related to AR since a large proportion of individuals with asthma also present symptoms of AR, and patients with AR have a 5–6 fold increased risk of developing asthma -10. AsthDue to the close relationship between the occurrence of asthma and AR we hypothesize that these two phenotypes have genetic risk factors in common. The present study was thus designed to investigate if SNPs in highly replicated asthma genes as reported in the Ober and Hoffjan study  are assoDermatophagoides pteronyssinus and Blomia tropicalis. A total of 89% of the patients showed a positive SPT for both allergens. AR is thus diagnosed based on the presence of atopic status and typical AR symptoms as defined by the ARIA 2008 guidelines i.e., two or more AR symptoms  persisting for four or more days a week during the past year. In this study, none of the patients suffered from severe asthma and less than 10% had moderate asthma with continuous medication. Control individuals had no history of AR or any other atopic disease and had a negative SPT. More detailed phenotypic characteristics of the Singapore Chinese population have been described previously [The Swedish study population consists of 246 AR patients  and 431 control individuals  and was recruited from southern Sweden in 2003–2009. All subjects are unrelated and of western European origin, with both parents born in Sweden. The diagnosis of birch and/or grass pollen induced AR was based on a positive history of AR for at least two years and a positive skin prick test (SPT) or Phadiatop test with at least class two to birch and/or timothy grass pollen. A total of 59% of the patients showed a positive SPT for both allergens. All patients were classified as having severe symptoms i.e., nasal itching, sneezing, rhinorrhea and nasal congestion during pollen season and they had all been treated with antihistamines and nasal steroids during pollen seasons previous years. Control individuals had no history of AR or any other atopic disease and had a negative SPT or Phadiatop test. Genomic DNA was extracted from blood collected in EDTA using QIAamp DNA Blood Maxi or Mini kits  and DNA concentrations determined by fluorometry using PicoGreen . The Swedish study population has previously been analyzed in several AR studies -17. The IL4, IL13, CD14, MS4A2 (FCERB1), IL4R (IL4RA), ADAM33, GSTM1, IL10, CTLA4, SPINK5, LTC4S, NPSR1 (GPR154), NOD1 (CARD4), SCGB1A1 (CC16), GSTP1, STAT6, NOS1, CCL5, TBXA2R, ADRB2 and TGFB1) were selected from a compilation of 25 genes reported to be associated with asthma phenotypes in six or more unrelated independent studies [HLA-DRB1, HLA-DQB1, TNF, and LTA as they are located at the HLA locus. Since this study investigates asthma genes for their eventual contribution also to the AR phenotype, we use the gene as the replication unit and not the individual SNPs. For genotyping in the Swedish study population, HapMap (release 24) data were used to identify haplotype-tagging SNPs  for each of the 21 genes. Non-synonymous SNPs reported in dbSNP or HapMap with minor allele frequencies >5% was added to this selection. Genotypes were determined using the Sequenom MassARRAY MALDI-TOF system. Assay design was made using the MassARRAY Assay Design ver. 2.0 software  and primers were obtained from Metabion GmbH . A total of 192 SNPs with a total genotyping rate of 98.4% were analyzed for association with AR in 246 patients and 431 controls. Whole genome genotyping in the Singapore Chinese population was performed using the Illumina HumanHap 550 k BeadChip version 3  at the Genome Institute of Singapore as described earlier [LTC4S, NOD1 and GSTM1) had no SNPs in the Illumina Human Hap 550k panel while CCL5 had one SNP but was filtered out after quality control for SNPs. A total of 413 SNPs from 17 unique genes with a total genotyping rate of 98.4% were analyzed for association with AR in 448 patients and 462 controls. Additional samples  from the Singapore Chinese cohort were genotyped for rs324981, using a Taqman-assay. Reactions were carried out according to the manufacturer’s protocol using an ABI PRISM 7900HT and the genotypes determined using SDS 2.4 software .A total of 21 genes  were approximately 0.04 and all other Q-values were >0.1. The lowest P-value (rs1138272) is below the level for a Bonferroni corrected P-value of 0.05, i.e. 0.00022 vs. 0.00026. Although the overall distribution of P-values is close to the expectation under the assumption of absence of any association, the three P-values <0.001 can be considered an indication of an effect that warrants further investigation.A Kruskal-Wallis rank sum test was used to investigate the relationship between genotype and degree of sensitization to birch and timothy grass in AR-patients. A total of 24 SNPs had P-values <0.05 and the corresponding Q-values and ORs , which is in complete linkage disequilibrium with rs324987  of 1.14 for the heterozygous genotype and an increased OR of 1.65 for the homozygous genotype in comparison with the reference genotype. Thus, both analyses support the involvement of NPSR1 in AR.In addition, we investigated the functional P-values corresponding to the loss of power due to a smaller sample size, 2) the nine SNPs with P-values <0.001 detected in the initial analysis of all 448 AR patients, all showed P-values ≤0.01 after elimination of the 144 patients, 3) these SNPs also showed very small changes in their ORs (<0.08), 4) most ORs were highly similar before and after elimination of the 144 patients, the exception being five SNPs in the NOS1 gene that may indicate confounding due to asthma.Since genetic variation of all genes tested for association with AR in the present study has previously been associated with asthma and since patients with AR have an increased incidence of asthma, we investigated asthma as a confounding factor for our results. Since the strongest associations are detected in the Chinese population, we excluded all Chinese patients with any asthmatic symptoms and repeated the association analysis for this population  was evaluated in an independent sample of 921 AR patients and 390 controls from the Singapore Chinese population. The results  further strengthen this hypothesis.In this study, we investigated the SNP associations of well-replicated asthma candidate genes with AR in two independent populations, one Swedish and one Singapore Chinese population. Since there are inherent differences in the genetic architecture between the two populations and this study investigates asthma genes for their eventual contribution also to the AR phenotype, the gene was used as the level of replication and not the individual SNP . A limitNPSR1 gene with asthma even after Bonferroni correction for multiple comparisons (P<0.001). Vergara et al. [NPSR1 (GPR154) gene and found associations with asthma and total IgE. Furthermore, this gene has been replicated in studies of Caucasian [NPSR1 and asthma appear to be strongly supported. Since the present study strongly indicates an association with genetic variation in the NPSR1 gene also in AR, there is an obvious risk of asthma being a cxonfounding factor for our results. This was investigated by comparing the association results before and after elimination of patients with any symptoms of asthma. The results convincingly showed that asthma is no confounding factor for the SNPs in NPSR1 and CTLA4 in the Chinese population.Previous studies have reported significant associations for a large region of 47 kb in the a et al. , investiaucasian -28 and Caucasian , but wasaucasian . Thus, tNPSR1 could be a genetic link between AR and asthma and associations of NPSR1 polymorphisms with AR have not been reported prior to this.The present result indicates that NPSR1 and CTLA4 as potential susceptibility genes for AR. However, these genes need to be replicated in additional populations and further characterized to elucidate their role in AR predisposition and pathogenesis. The majority of the highly replicated asthma genes were not associated with AR in our populations, which suggest that asthma and AR could be less similar at the genetic level than previously anticipated.In summary, we have identified The authors state that they have no financial or non-financial competing interests.AKA, CFT, CH and LOC designed the study. AKA, TS and DN performed the data analysis and AKA, DN, CH and TS wrote the manuscript. All authors critically revised the manuscript and approved the final form of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/14/51/prepub Complete association results for the AR phenotype and allergen sensitization in the Swedish population.Click here for file Complete association results for the AR phenotype and allergen sensitization in the Chinese population.Click here for file Comparison of association test results before (448 patients) and after.Click here for file


Numerous studies with functional magnetic resonance imaging have shown that the fusiform face area (FFA) in the human brain plays a key role in face perception. Recent studies have found that both the featural information of faces  and the configural information of faces  are encoded in the FFA. However, little is known about whether the featural information is encoded independent of or combined with the configural information in the FFA. Here we used multi-voxel pattern analysis to examine holistic representation of faces in the FFA by correlating spatial patterns of activation with behavioral performance in discriminating face parts with face configurations either present or absent. Behaviorally, the absence of face configurations (versus presence) impaired discrimination of face parts, suggesting a holistic representation in the brain. Neurally, spatial patterns of activation in the FFA were more similar among correct than incorrect trials only when face parts were presented in a veridical face configuration. In contrast, spatial patterns of activation in the occipital face area, as well as the object-selective lateral occipital complex, were more similar among correct than incorrect trials regardless of the presence of veridical face configurations. This finding suggests that in the FFA faces are represented not on the basis of individual parts but in terms of the whole that emerges from the parts. Studies with functional magnetic resonance imaging (fMRI) have identified a face-selective region in the fusiform gyrus, termed the fusiform face area (FFA) Extensive behavioral studies have shown that the key difference in the way that faces are processed, compared to non-face objects, is that the featural and configural information are processed together as an integrated whole, termed holistic processing Here we directly tested whether the representation of faces in the FFA is holistic, parts-based, or both. To do this, we examined the effect of configural change on the representation of the featural information in the FFA. Studies of facial structure have drawn two distinctions on configural information: (1) first-order configuration  and (2) second-order configuration  To examine neural correlates of behavioral holistic processing, we used multi-voxel pattern analysis (MVPA) to measure neural information encoded in the FFA. As compared with the traditional measure of the mean magnitude of blood oxygen level-dependent (BOLD) responses pooled across voxels, voxel-by-voxel patterns of activation provide richer information on neural representations at a finer-scale Based on these designs, we predicted that: (1) if the FFA is engaged in holistic representation of faces C, we shThirteen college students  participated in the study. All participants were right-handed and had normal or corrected-to-normal visual acuity. The fMRI protocol was approved by the IRB of Beijing Normal University, Beijing, China. A written consent form was collected before the experiment from each participant.The face stimuli were gray-scale adult Chinese faces with external contour  removed. Three original male faces were used to generate the stimuli and all stimuli were 7×8.3 cm . First, a face template containing eyebrows and a nose was created. Second, for the face set with a veridical face configuration , the eyes and mouth of a face image were randomly selected from those of three original faces, and then placed in a veridical face configuration. The metrical relationship between face parts  of all face exemplars was kept as constant as possible A, top. Each participant finished a single session consisting of (1) two blocked-design functional localizer runs, and (2) ten event-related-design experimental runs. The localizer runs consisted of human frontal-view faces, objects, houses, and scrambled objects. Each localizer run lasted 5 min and 36 sec, and consisted of sixteen 16 sec blocks with five 16 sec fixation periods interleaved. During each block, twenty different exemplars of a given stimulus category were presented for 300 msec in the center of the screen, followed by a blank interval of 500 msec. During the localizer runs, participants pressed a button whenever they saw two identical images in a row .http://surfer.nmr.mgh.harvard.edu/optseq/) so that trials from each condition  were preceded, on average, equally often by trials from each of the other conditions. In addition, there was an 8 sec fixation period at the beginning and at the end of each run. Thus, each experimental run lasted 5 min and 28 sec.During the experimental runs, participants performed a discrimination task on pairs of faces that could differ in face parts. In a trial, a pair of face images was presented sequentially, either both with or without a veridical face configuration A. Each Scanning was conducted on a 3T Siemens Trio scanner  with an eight-channel phase-arrayed coil at BNU Imaging Center for Brain Research, Beijing, China. Twenty-five 4 mm thick (20% skip) near axial slices were collected (in-plane resolution = 3×3 mm), oriented parallel to each subject's temporal cortex and covered the whole brain. T2*-weighted gradient-echo, echo-planar imaging procedures (EPI) were used . In addition, MPRAGE, an inversion prepared gradient echo sequence , was used to acquire 3D structural images.http://froi.sourceforge.net), and in-house Matlab code. After data preprocessing, including motion correction, intensity normalization, and spatial smoothing , voxel time courses for each individual subject were fitted by a general linear model (GLM). Each condition was modeled by a boxcar regressor matching its time course that was then convolved with a gamma function .Functional data were analyzed with the Freesurfer functional analysis stream  p<0.01, uncorrected)  separately for each hemisphere and for each participant .Specificrrected) A.The rerrected) A. Two sFor the experimental runs, instead of pooling BOLD responses across voxels in the ROIs, MVPA was used to measure neural information encoded at a finer-scale. Specifically, we examined whether the spatial pattern of activation in the FFA, as well as the other pre-defined ROIs, was read out for behavioral face perception. That is, we compared the similarity of spatial patterns of activation in an ROI when participants correctly discriminated face pairs  versus when they made incorrect responses . Trials in which participants did not make a response  were excluded from the analysis. For this analysis, the data were first preprocessed without spatial smoothing to maximize the sensitivity to any information present in the spatial pattern of activation. Then, we estimated the magnitude of the BOLD response with a gamma function  to each pair of faces as a compound trial for each voxel of each ROI. Finally, the ten experimental runs were split into five odd runs and five even runs, and the averaged spatial pattern was extracted from each half of the data (odd versus even runs) separately for each ROI, stimulus type , and behavioral response (correct versus incorrect). Within each ROI, we computed the correlation between the averaged spatial patterns on odd and even runs for veridical faces and for scrambled faces respectively; this analysis was performed separately on the data from trials in which the participant performed correctly versus incorrectly in the whole-part task. Before calculating the correlations, mean magnitude of activation across all conditions was subtracted from the magnitude of each condition in each voxel and in each half of the data. In addition to the MVPA, we also calculated the mean magnitude for each stimulus condition and for each response type by pooling BOLD responses across voxels.t(12) = 8.87, p<0.001, Cohen's d = 2.53, veridical: 84.7 %, scrambled: 72.7 %) and slower (t(12) = 5.95, p<0.001, Cohen's d = 1.70) in discriminating face parts when they were placed in a non-face configuration (Scrambled) than in a veridical face configuration   . Thus, fSpatial patterns were extracted from each half of the data (odd versus even runs) separately for each stimulus condition  and for each response type (correct versus incorrect responses). Within an ROI, we calculated correlations between the spatial patterns in odd and even runs from trials in which participants performed correctly versus incorrectly in discriminating face parts either with or without face configurations.F  = 5.09, p = 0.04, partial η2 = 0.30)  <1) and response type   = 1.38, p = 0.26) were not significant. Post-hoc pair-wise t-tests found that when a veridical face configuration was present, spatial patterns of activation in the FFA were more similar among correct than incorrect trials (t(12)  = 3.22, p = 0.007, Cohen's d = 0.92). In contrast, when the configuration was scrambled, there was no effect of correct versus incorrect on the correlations between the spatial patterns (t(12) <1). We also matched the number of correct and incorrect trials between the two stimulus types by randomly selecting a subset of the data for a stimulus type that had more correct (or incorrect) trials. With the matched number of trials, we found a similar result – a significantly higher correlation in the FFA for correct compared with incorrect trials was observed when the face configuration was present (t(12)  = 3.94, p = 0.002, Cohen's d = 1.23), not when the face configuration was absent (t<1) (F<1) nor the difference between the correct and incorrect trials (both ts <1) was found, consistent with previous findings that the holistic representation of faces is found only in the right FFA F<1)  B. The mnt (t<1) . In addiFFA F<1) . This maFFA F<1) , making F  = 21.14, p<0.001, partial η2 = 0.66)  <1), showing that the OFA has no preference for stimuli with veridical face configurations (t(11)  = 1.94, p = 0.08, Cohen's d = 0.56) over stimuli without such configurations (t(11)  = 3.64, p = 0.004, Cohen's d = 1.13). Therefore, the OFA is apparently engaged in the parts-based representation of faces   = 6.13, p = 0.03, partial η2 = 0.36), confirming that the configural information is necessary for the featural information encoded in the FFA to be read out for behavioral face perception. The three-way interaction among cortical region (FFA versus OFA), response type (correct versus incorrect), and stimulus type  did not reach significance   = 2.73, p = 0.13).The interdependence of the featural and configural information in the FFA suggests that faces are represented not on the basis of their individual parts but in terms of the whole that emerges from the parts, consistent with the Holistic model C. Howev = 0.66) C, indicThe functional division of labor between the OFA and FFA suggests that the FFA and the OFA may comprise a hierarchical network for face processing, with the FFA inheriting the parts-based representation of faces in the OFA, and then integrating this information with their spatial relations for the holistic representation of the faces. Here we examined the specificity of the hierarchy of face representation by investigating two regions in the object processing hierarchy, the LO and the pFs, that are located adjacent to the OFA and the FFA, respectively.t(12)  = 3.35, p = 0.006, Cohen's d = 1.02; pFs: t(12)  = 1.86, p = 0.09, Cohen's d = 0.50) or absent (LO: t(12)  = 2.35, p = 0.04, Cohen's d = 0.49; pFs: t(12)  = 3.25, p = 0.007, Cohen's d = 0.80)   = 2.88, p = 0.12), further confirming that faces are represented non-interactively in both the LO and the pFs. Thus, for the ROIs tested, the holistic representation of faces is specifically carried out in the FFA, whereas the parts-based representation of faces is not.The spatial patterns in both the LO and pFs were more similar among correct trials than incorrect trials, regardless of whether face configurations were present (LO:  = 0.80) . ImportaIn this study, we asked whether the featural information of faces is encoded independently of or combined with the configural information of faces in the FFA, by correlating spatial patterns of activation in FFA with a measure of behavioral face perception. In the experiment, participants were instructed to discriminate face parts that were placed either in a veridical or in a scrambled face configuration. We found that the absence of the first-order face configuration impaired discrimination of face parts, showing the importance of overall structure in representing faces. More importantly, only when the face parts were placed in a veridical face configuration was the featural information in the FFA related to behavioral performance. Therefore, face parts were not independently and non-interactively represented in the FFA, suggesting that the FFA is only engaged in the holistic representation of faces.Our study applied several novel methods to assess the nature of the representations in the FFA. First, unlike previous studies where FFA responses to face parts were compared with visually similar stimuli  Our finding dovetails with previous studies showing that the FFA is involved in the holistic representation of faces In contrast to the results we observed in the FFA, the OFA is engaged in the parts-based representation of faces. Spatial patterns of activation in the OFA were more similar among correct than incorrect trials, regardless of configural changes. This result fits with a TMS study where TMS stimulation of the OFA selectively disrupted participants' ability to discriminate face parts, not their ability to discriminate face configurations The dissociation between the holistic representation and parts-based representation of faces in the brain suggests a functional division of labor between the OFA and FFA. One possible explanation from a widely-held “local-to-global” view Figure S1The behavioral relevance of spatial patterns in the FFA with matched number of trials. The numbers of correct and incorrect trials between the two stimulus types were matched by randomly selecting a subset of the data for stimulus type that had more correct (or incorrect) trials. Error bars indicate s.e.m. above and below the mean.(PDF)Click here for additional data file.Figure S2Mean magnitudes of BOLD responses in the FFA. The magnitude was acquired by the traditional GLM analysis. A two-way ANOVA revealed no interaction of stimulus condition  by response type (correct versus incorrect) in the FFA (F <1). Post hoc pair-wise t-tests revealed no significant difference between the correct and incorrect trials in either veridical or scrambled condition (ts <1).(PDF)Click here for additional data file.Figure S3Mean magnitude of BOLD responses in the FFA to stimuli. A) FFA responses to faces with or without veridical face configurations. The FFA showed a strong response to the scrambled faces, which was about 93% of the activation level to the veridical faces. B) FFA responses to faces and objects in the localizer runs. Note that the data shown here were not from an independent data set . Previous studies based on independent data sets show that FFA response to faces is at least two times higher than that to objects .(PDF)Click here for additional data file.


Upon continuous ligand stimulation, β1-AR is internalized and mostly recycled back to the plasma membrane (PM). The recycling endosome (RE) is one of the membranous organelles involved in the protein recycling pathway. To determine whether RE is involved in the internalization of β1-AR upon ligand stimulation, we evaluated the localization of β1-AR after stimulation with a β-agonist, isoproterenol (Iso), in β1-AR-transfected COS-1 cells. After 30 min of Iso treatment and cell surface labeling with the appropriate antibodies, β1-AR was internalized from PM and translocated into the perinuclear region, the same location as the transferrin receptor, an RE marker. We then evaluated whether sorting nexin 27 (SNX27) participated in the β1-AR recycling pathway. When β1-AR and SNX27 were coexpressed, β1-AR coimmunoprecipitated with SNX27. In addition, shRNA-mediated silencing of SNX27 compromised β1-AR recycling and enhanced its delivery into lysosome. Overall, β1-AR on PM was internalized into RE upon Iso stimulation and recycled by RE through binding with SNX27 in COS-1 cells.In cardiomyocytes, β To determine the localization of βSorting nexins (SNXs) are proteins characterized by the presence of the phox-homology (PX) domain, which binds to phosphatidylinositol-3-monophosphate (PI-3P). SNXs are conserved among a wide variety of species; thus far, more than 30 SNXs have been identified in mammals Cullen . Through2-AR is dependent on its C-terminal PDZ-domain binding motif  domain, which functions as a scaffold to organize various proteins. Efficient recycling of β1-AR localizes mainly at PM and the β-agonist isoproterenol (Iso) triggered the internalization of β1-AR from PM and its translocation into RE. Furthermore, we observed that the recycling of β1-AR after Iso-induced internalization is regulated by its binding with SNX27.Using COS-1 cells, we clearly demonstrated that βCOS-1 cells were maintained in DMEM supplemented with 10 % fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin, as described previously , and the cDNA was synthesized using superscript III  according to the manufacturer’s instructions. Using the synthesized cDNA as a template, PCRs were performed using PrimeStar  with the following primers: β1-AR, 5′-CGAGGGATCCCTCGGCATGGGCGCGGGGGCGCTCG-3′ (forward primer), 5′-GGTGCTCGAGCTACACCTTGGACTCCGAGGAGAAGC-3′ (reverse primer) and SNX27, 5′-GAGCAAGCTTGCTCGCAAGATGGCGGAC-3′ (forward primer), 5′-CACCGTCGACGGTGGCCACATCCCTCTGCTG-3′ (reverse primer). Amplified fragments for β1-AR and SNX27 were subcloned into pcDNA3.1Myc.His (Invitrogen) at the BamH1/XhoI or HindIII/SalI site, respectively. The resulting constructs were confirmed by DNA sequencing. β1-AR (HA-tagged) was constructed using PCR-based insertion of the HA-tag sequence into pcDNA3.1Myc.His/β1-AR at the amino-terminal using PrimeStar (Takara-bio). The primers used were as follows: 5′-GTTCCAGATTACGCTGGCGCGGGGGCGCTCGCCCTG-3′ (forward primer) and 5′-ATCGTATGGGTACATGCCGAGGGATCCGAGCTC-3′ (reverse primer).β1-AR (HA-tagged), the DNA encoding β1-AR (HA-tagged) was subcloned into the pLHCX vector  at the HindIII/XhoI site using the following primers: 5′-CGAGAAGCTTCTCGGCATGTACCCATACGATG-3′ (forward primer) and 5′-GTAGATCGATCTACACCTTGGACTCCGAGG-3′ (reverse primer), pLHCX/β1-AR. To make a mutant β1-AR that lacks last four amino acids at the carboxy terminus (∆C4β1-AR), we mutated a pLHCX/β1-AR using a PCR with the following primers: 5′-CGGCCCCGCGGTCCCGAAGAGGAGCATCTAGCTA-3′ (forward primer) and 5′-GTAGATCGATCTACGAGGAGAAGCCCTGGCGCC-3′. A recombinant retrovirus harboring β1-AR (HA-tagged) was prepared using the Retrovirus Packaging kit (Ampho) (Takara-bio) according to the manufacturer’s instructions. The pLHCX/β1-AR (HA-tagged) retrovirus was infected into COS-1 cells, and the infected COS-1 cells were selected by growing in 200 μg/ml hygromycin  for approximately 2 weeks. After evaluating the expression level of β1-AR in the surviving cells, this cell line was used for the experiments. We named the COS-1 cells stably expressing β1-AR (HA-tagged) as “β1-AR cells.”To establish COS-1 cells that stably express β1-AR Ab ; anti-HA Ab and anti-EEA1 Ab . In addition, we used several monoclonal Abs: anti-human TfnR Ab (Invitrogen), anti-GM130 Ab , anti-LAMP1 Ab (Santa Cruz Biotechnology) and anti-Myc Ab .We used the following polyclonal antibodies (Abs) in this study: anti-β1-AR cells were plated on cover glasses in a 24-well plate. After treatment with 50 μg/ml cycloheximide for 4 h, cells were fixed for 10 min, permeabilized for 10 min and blocked in 4 % paraformaldehyde, 0.1 % Triton X-100 and 5 % bovine serum albumin (BSA) for 30 min at room temperature. The primary Ab diluted 1:1,000 with 5 % BSA was added and incubated for 30 min at room temperature. After washing with phosphate-buffered saline (PBS) thrice, the secondary Ab (Alexa546-labeled anti-rabbit Ab for the anti-HA Ab or Alexa488-labeled anti-mouse Ab for the anti-GM130 and anti-TfnR Ab) was added. To track the internalization of β1-AR from the cell surface, Iso and the primary Ab were added simultaneously before fixation.COS-1 cells transfected with the plasmids of interest or βFor confocal microscopy, we used LSM500 . Images of the fixed cells were taken at ambient temperature with a 63 × 1.4 NA Plan Apochromat oil-immersion lens. Excitations were performed using two lasers, one emitting at 488 nm and the other at 543 nm. Emissions were collected using appropriate filters.1-AR on the cell surface, whereas the other was designed to evaluate the degradation of β1-AR on the cell surface.Two types of cell surface biotinylation experiments were performed upon Iso treatment; one was designed to evaluate the reduction in β−6 M Iso. Cells were washed with PBS thrice and lysed with RIPA buffer . Biotinylated proteins were precipitated with avidin-agarose  and eluted with 1× SDS sample buffer . Eluates were subjected to SDS-PAGE and Western blot analysis.Cells were biotinylated preceding Iso treatment. Biotinylated cells were quenched with 0.1 M glycine, washed several times with prewarmed DMEM and then further incubated for 4 h in the presence of 101-AR or β1-AR cells were starved in serum-free DMEM for 2 h and incubated for 30 min with or without 10−6 M Iso. To evaluate the recovery of once-internalized receptors, cells were further incubated for 60 min after the removal of Iso. Cells were then washed with PBS thrice and biotinylated with 0.1 mg/ml sulfo-NHS-biotin (Pierce) in PBS with 1 mM CaCl2 and 0.5 mM MgCl2 for 30 min. After 30 min, cells were quenched with 0.1 M glycine. The preceding steps were mentioned in the previous section.COS-1 cells transiently expressing βFollowing SDS-PAGE, the gel was put into a semidry blotting system  and proteins were transferred onto a PVDF membrane . The membrane was blocked with 5 % skimmed milk in PBS containing 0.1 % Tween 20 , incubated with primary Ab of interest in blocking buffer, washed thrice with PBST and then incubated with the secondary Ab conjugated with horseradish peroxidase. The membrane was visualized with Immobilon Western , and the image was captured with Chemidoc (Bio-Rad).1-AR or ∆C4β1-AR was cotransfected with SNX27 (Myc-tagged) in COS-1 cells. After 2 h of starvation in serum-free DMEM, transfected cells were incubated with or without 10−6 M Iso for 10 min. Cells were then collected, washed thrice with PBS and lysed with lysis buffer  containing a protease inhibitor cocktail . Lysates were incubated with protein G agarose premixed with an anti-Myc Ab for several hours; the protein G agarose was then washed thrice with lysis buffer. Precipitated proteins were eluted in the same volume of 2× SDS sample buffer by boiling for 2 min and subjected to SDS-PAGE, followed by Western blot analysis as mentioned previously.β1-AR cells using Lipofectamine 2000 (Invitrogen). In brief, 2 μg of shSNX27 in 100 μl of Opti-MEM was mixed with 5 μl of Lipofectamine 2000 diluted in 100 μl of Opti-MEM. The mixture was incubated at room temperature for 20 min and then added to cells that were cultured overnight in 1 ml of antibiotic-free medium in 12-well plates. After 48 h, cells were used for the experiments including Western blot analysis, real-time PCR, cell surface biotinylation, coimmunoprecipitation and immunostaining.shRNA against SNX27, targeting the sequence AAGGACCATAGACAGAGATTA (shSNX27), was cloned into the pSIN vector (Takara-bio) according to the manufacturer’s instructions. shSNX27 was transfected into βTotal RNAs were prepared using a TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Real-time RT-PCR analyses were performed using a Thermal Cycler Dice Real Time System Single MRQ (Takara-bio). cDNA was synthesized using a PrimeScript RT reagent kit (Takara-bio), and PCRs were performed using SYPR premix ExTaq II (Takara-bio), according to the manufacturer’s instructions. The mean number of cycles required to reach the threshold of fluorescence detection  was calculated for each sample, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was quantified for normalization of the amount of cDNA in each sample. The specificity of the amplified products was evaluated by the melting curve. The primer sets used to quantitate SNX27 and GAPDH, respectively, were as follows: 5′-ACCACCTGCTTGTGTGTCG-3′ (forward) and 5′-ATCAACGGGGAGCTGTACG-3′ (reverse) and 5′-ATTGCCCTCAACGACCACTT-3′ (forward) and 5′-AGGTCCACCACCCTGTTGCT-3′ (reverse).1-AR. To determine the distribution of β1-AR at steady state in COS-1 cells, we established cells stably expressing β1-AR proteins that were N-terminally tagged with the HA epitope (β1-AR cells). First, the costaining of β1-AR with GM130, a cis-Golgi marker, in β1-AR cells was performed. The results show that β1-AR was expressed not only on the cell surface but also in the Golgi , which is localized predominantly in RE. RE is involved not only in the transit of recycling molecules, such as TfnR or LDL receptor but also in various biological activities including anterograde transport   and SNX27 (Myc-tagged). Cell lysates were coimmunoprecipitated with anti-Myc Ab followed by probing with anti-β1-AR or anti-Myc Ab. As shown in Fig. 1-AR was coimmunoprecipitated with anti-Myc Ab. The interaction was augmented by 10 min of stimulation with Iso, suggesting that β1-AR can physically interact with SNX27. The interaction of β2-AR with SNX27 was reported to be mediated by a PDZ domain-binding motif of β2-AR . Transient transfection of shSNX27 in COS-1 cells diminished the expression of SNX27 by 90 % at the mRNA level (Fig. 1-AR on the cell surface was internalized after 30 min of Iso treatment and recycled back to the cell surface after 60 min of chase following Iso removal. To elucidate the functional roles of SNX27 in β1-AR recycling, we performed the same experiment using shSNX27. In mock-transfected β1-AR cells, β1-AR was diminished after 30 min of Iso treatment, whereas during the 60-min chase time, a considerable amount of β1-AR was recovered on the cell surface compared with the amount of β1-AR on the cell surface at 0 min (Fig. 1-AR after 30 min of Iso treatment was equivalent in β1-AR cells with shSNX27 and mock-transfected β1-AR cells, a lesser amount of β1-AR was recovered after 60 min of chase in shSNX27-transfected cells. These results suggest that shSNX27 inhibited recycling of the internalized β1-AR. To confirm this result, we performed confocal microscopic analysis of cells with or without shSNX27. As shown in Fig. 1-AR on the cell surface had become internalized and was eventually confined to RE corresponding with the localization of TfnR in mock-transfected cells. After 60 min of chase, the internalized β1-AR was recycled back. In contrast, in cells with shSNX27, β1-AR expressed on the cell surface became internalized after 30 min of Iso treatment, most of which was segregated from the TfnR-positive RE. After 60 min of chase, β1-AR was still inside the cells, in which TfnR and β1-AR did not merge.Since β EE Fig. , we nextvel Fig. a. As shomin Fig. b. These 1-AR was examined. Without shSNX27 treatment, most of the internalized β1-AR did not overlap with LAMP1, a lysosome marker, after 30 min of Iso treatment (Fig. 1-AR partly overlapped with the LAMP1-positive organelle, indicating that it was partly in the lysosome, where proteolysis takes place (Fig. Finally, the degradation of β1-AR, which has a class I PDZ-binding motif with an uncharged amino acid at the −5 position, interacts with SNX27 in EE. This is consistent with a previous report showing that the PDZ domain of SNX27 prefers acidic or uncharged residues over basic ones at the −5 position (Balana et al. 1-AR is recycled by binding with SNX27 and degraded when such binding does not occur. In other words, the binding of β1-AR with SNX27 might determine its fate, recycling or degradation. It has been reported that SNX27 regulates the recycling of internalized β2-AR from EE/RE/LE to the PM by its interaction with retromer (Temkin et al. 2-AR. It is possible that the SNX27/retromer complex might be involved in the PDZ-directed recycling of β1-AR as well. We are currently working on the involvement of retromer in β1-AR recycling.We found that β1-AR, but not β2-AR, is highly expressed in heart and regulates heart functions such as contraction and heart rate. Thus, whether β1-AR is recycled or degraded upon sympathetic action is essential to the recovery of sensitivity to catecholamines—particularly upon onset of heart failure, for which administration of adrenergic agents is a common intervention strategy. However, continuous stimulation of β1-AR often leads to desensitization, which is a common problem with this type of therapy. If we could pharmaceutically control the recycling and degradation of β1-AR, this problem could be circumvented. SNX27 could be a therapeutic target for controlling β1-AR or other proteins that have a PDZ-domain binding motif.β1-AR is internalized into RE upon ligand stimulation and then recycled back to PM in COS-1 cells. Furthermore, we present evidence that SNX27 may be crucial in the recycling of β1-AR.In conclusion, we observed that cell surface β


However, the factors that regulate its expression and consequently the signaling pathways in which ZEB1 participates are poorly defined. Because it is induced by estrogen and progesterone and is high in prostate cancer, we investigated whether tcf8, which encodes ZEB1, is regulated by androgen. Data herein demonstrate that tcf8 is induced by dihydrotestosterone (DHT) in the human PC-3/AR prostate cancer cell line and that this induction is mediated by two androgen response elements (AREs). These results demonstrate that ZEB1 is an intermediary in androgen signaling pathways.The zinc finger E-box binding protein 1 (ZEB1) transcription factor belongs to a two-member family of zinc-finger homeodomain proteins involved in physiological and pathological events mostly relating to cell migration and epithelial to mesenchymal transitions (EMTs). ZEB1 (also known as  Although tcf8 was cloned in 1991 [p73 and p63 genes, members of the p53 gene family, and its binding to those genes promotes cell proliferation and prevents differentiation [ZEB1  has been identified as yet. Interestingly, estrogen represses expression of ZEB1 mRNA in LβT2 gonadotrope cells [Despite ZEB1's importance in modulating development, cell proliferation, reproduction, and metabolism, relatively little is known about the regulation of  chicken  although chicken . A secon chicken . The gen chicken . While gβ family  and IGF-β family  regulate oviduct . It is a oviduct , 28, in  oviduct , in mous oviduct . This wape cells , in humape cells , and in pe cells , indicattcf8. This question is particularly relevant as ZEB1 expression is high in aggressive prostate cancer (PCa) cell lines and tissues and as it promotes phenotypic changes consistent with epithelial to mesenchymal transitions (EMT) in those lines [tcf8 through binding or tethering of AR to this gene. Treatment of the PC-3/AR prostate cancer cell line, which overexpresses AR [tcf8 and that both bind AR. This raises the possibility that ZEB1 serves as a direct intermediary in at least some androgen signal transduction pathways.The goal of these studies was to assess whether androgen receptor (AR) directly regulates se lines , 31. Thise lines , 33. Thiesses AR , with diβ-galactosidase assays were performed according to the manufacturer's instructions using the Galacto-Star System , with chemiluminescence measured in a standard luminometer at 1.0 second/tube.The PC-3/AR prostate carcinoma cell line was kindly provided by Dr. K. Burnstein (University of Miami) and was maintained as previously described . The LNCμg aliquots of mRNA were reverse transcribed with MMLV Reverse Transcriptase (Invitrogen) to create cDNA templates for real-time reverse transcriptase PCR (qPCR). For iCycler  using the probes and primers designated in N, N, N′, N′-tetramethylrhodamine (TAMRA). For RPL32, the fluorophore was 6-carboxyfluorescein (6-FAM) and the quencher was TAMRA. Both probes and primer sets were used in each reaction with the following optimized multiplex PCR protocol: 12.5 μL iQ Supermix (BioRad), 2.5 μL 10 mM dNTPs, 2 μL 50 mM MgCl2, 0.5 μL iTaq (BioRad), and 0.5 μL cDNA template. Cycling parameters were as follows: 1 cycle at 95°C for 3 minutes, followed by 60 cycles at 95°C for 10 seconds and 55°C for 1 minute.Total RNA from cultured cell lines was isolated using TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. Two μL iQ SYBR Green Supermix , 2 μL primer mix (10 μM each), 2 μL template (100 ng/μL), and 8.5 μL H2O. This reaction was prepared as a 3x cocktail and distributed in triplicate into a 96-well plate. qPCR was performed using the BioRad iCycler with an annealing temperature of 61°C for both cDNAs.In α-tubulin antibody  followed by horseradish peroxidase-conjugated secondary antibody . Blots were developed with NBT/BCIP solution .Protein extracts were harvested from PC-3/AR cells using the ReadyPrep Protein Extraction Kit (BioRad) according to the manufacturer's protocol. Protein concentrations were determined by Bradford assay (BioRad), and 100 mg of nuclear extract was loaded per well on a 4–20% ReadyGel Tris-HCl (BioRad). Proteins were run at 60 mAmp for 45 min and transferred to a polyvinyldifluoride (PVDF) membrane for blotting with a ZEB1 antibody and a control tcf8 was cloned by PCR from human genomic DNA  using the forward primer 5′-TGGCCTGTGGATACCTTAGC-3′ (−982 to −963) and reverse primer 5′-CGCTTGTGTCTAAATGCTCG-3′ (−9 to −28) into the pBlueTOPO β-galactosidase reporter vector (Invitrogen), and the insert was sequenced. This tcf8 promoter construct was named pBlueZEB974.A 974 bp region of DNA spanning −982 to −9 relative to the translation start site of Site-directed mutagenesis was carried out on pBlueZEB974 using the QuikChange Mutagenesis kit (Stratagene), following the manufacturer's suggested protocol with the oligomers listed in μg of nuclear protein and 80,000 cpm 32P-end-labeled oligomer as indicated per lane. The sequences of the top strands are listed in Nuclear protein extracts were harvested from PC-3/AR cells treated with 5 nM DHT using the ReadyPrep Protein Extraction Kit (BioRad) according to the manufacturer's protocol. Protein concentrations were determined by Bradford Assay (BioRad). GMSAs were performed as described  using 10α-AR . The immunogen was a synthetic peptide corresponding to residues 299–315 of human AR. GMSAs were placed against film for 48 h at −80°C prior to autoradiographic development.Antibody supershift reactions were performed using a 0.1 mg/mL final concentration of tcf8 is induced at the transcriptional level by both estrogen and progesterone, respectively. Because progesterone receptor and AR recognize many of the same binding sites , an androgen that cannot be aromatized to estrogen, was examined. Human PC-3/AR cells were treated with doses of DHT from 1 to 100 nM for 24 hours , and this plasmid was tested in transfection assays with increasing amounts of DHT  are functional and are required for induction . Considerable evidence indicates that there is a reciprocal relationship between ZEB1 and several miRNAs, most notably those of the miR-200 family . In theript and . Interesript and ). As ZEBript and , 40, it tcf8 is regulated at the transcriptional level by androgen, the data herein show that it is a direct target for AR and identify its specific binding sites. This is an important contribution as very few regulatory elements have been characterized in tcf8, although a number have been proposed  includiiew, see . In facttcf8 gene is a direct target for AR, which binds to two AREs in the 1000 bp proximal to the translation start site. The increase in expression of this gene is reflected by an increase in ZEB1 protein. However, expression of tcf8 is silenced in some reproductive cell lines including the LNCaP PCa cell line by mechanisms that remain to be elucidated but that may involve epigenetic silencing or the miR-200 family of microRNAs.The