During the perinatal period, male mice are exposed to higher levels of testosterone (T) than females, which promotes sexual dimorphism in their brain structures and behaviors. In addition to acting via estrogen receptors after being locally converted into estradiol by aromatase, T also acts directly through androgen receptor (AR) in the brain. Therefore, we hypothesized that AR expression in the developing mouse cortex and hippocampus was sexually dimorphic. To test our hypothesis, we measured and determined AR mRNA and protein levels in mouse cortex/hippocampus collected on the day of birth (PN0) and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. We demonstrated that, as age advanced, AR mRNA levels increased in the cortex/hippocampus of both sexes but showed no sex difference. Two AR proteins, the full-length (110 kDa) and a smaller isoform (70 kDa), were detected in the developing mouse cortex/hippocampus with an age-dependent increase in protein levels of both AR isoforms at PN21 and a transient masculine increase in expression of the full-length AR protein on PN7. Thus, we conclude that the postnatal age and sex differences in AR protein expression in combination with the sex differences in circulating T may cause sexual differentiation of the mouse cortex/hippocampus. Sex differences in brain structure and behavior are ubiquitous in many species, including humans and rodents –3. The gIn male mice, the developing testes secrete T with two surges during late gestation  and on the day of birth , respectively –15. The Analysis of AR expression in the developing and adult rodent brain reveals sexual dimorphism in the bed nucleus of the stria terminalis (BNST) and medial preoptic area (mPOA) with higher AR mRNA and protein levels in males than females –28. In mWe have previously observed sex differences in histone modifications in the developing mouse cortex/hippocampus with more H3 acetylation on K9/14 (H3K9/14Ac) in males around E18 and PN0 and more H3 trimethylation on K9 (H3K9Me3) in males around PN0 and PN6 . Sex difBased on the importance of T in brain sexual differentiation, we hypothesized that, during early development, AR expression in the mouse cortex/hippocampus might be sexually dimorphic and therefore responsible for the development and function of neural circuits governing distinct behaviors between the sexes. To test our hypothesis, we determined AR mRNA and protein levels by RT-qPCR and immunblotting, respectively. Our results reveal the presence of AR mRNA and two AR protein isoforms (110 kDa and 70 kDa) in the male and female mouse cortex/hippocampus during early postnatal development. While males have higher AR protein expression than females on PN7 only, the developing mouse cortex/hippocampus showed a robust increase in AR mRNA and protein levels as function of age. The developmental profile of AR expression in the mouse cortex/hippocampus not only implicates AR in the organization and function of neural circuits for higher cognitive function, such as learning and memory, but also supports AR as a direct target through which different levels of circulating androgens can differentially regulate cortical and hippocampal development and function between the sexes. ad libitum. All experimental procedures were approved by CSULB Institutional Animal Care and Use Committee (IACUC) and performed according to Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines.Adult male and female C57BL/6J (000664) mice were purchased from the Jackson Laboratory  and housed in the California State University Long Beach (CSULB) Animal Care Facility. Animals were kept on a 12 : 12 h light : dark cycle (lights on at 0600 h). Food  and water were providedAdult female mice were individually paired with a fertile male to produce the pups used in this study. Male and female pups were sacrificed by rapid decapitation on the day of birth (PN0) and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. These ages were chosen to overlap with the sensitive period for brain masculinization , during which endogenous or exogenous sex steroids have been shown to be effective at inducing male-like brain structure and function as adult  . The bra260), 280 nm (A280), and 320 nm (A320) was detected by a Bio-Rad SmartSpec Plus spectrophotometer . RNA concentration was calculated at A260, and purity was determined by the A260/A280 ratio (between 1.9 and 2.1). The cDNA samples were synthesized from 1 μg extracted RNA in 20-μL reactions using the Bio-Rad iScript cDNA Synthesis Kit. The reverse transcription was performed at 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min in a Bio-Rad MyCycler thermocycler.Using RNeasy Lipid Tissue kits , total RNA was extracted from the mouse cortex/hippocampus block according to the manufacturer's protocol. Individual samples were diluted (1 : 50) in 10 mM Tris (pH = 7.5)  to determine RNA quality and concentration. Absorbance at 260 nm (Ahttp://www.ncbi.nlm.nih.gov/tools/primer-blast/) against RefSeq RNA. The forward and reverse primers match the sequences of exons 7 and 8 of the mouse AR gene, respectively. In addition, the primers for two house-keeping genes, Actb and Rpl13a, were also used as described in our previous study [The PCR primers for AR were the same as those described by Bonthuis et al. . The AR us study . All priμL of cDNA, 1 μL of each of forward and reverse primers (840 nM), and 6 μL of Absolute QPCR SYBR Green Mix  were mixed and amplified on an Agilent Stratagene MX3000P qPCR system with MxPro QPCR software  with an initial 15 min denaturation/polymerase-activation at 95°C, 40 cycles of 15 s at 95°C, 1 min at the optimized annealing temperature, and 30 s at 72°C and a dissociation step with 1 min at 95°C, 30 s at 60°C, and 30 s at 95°C. Cycles of threshold (Ct) were analyzed by linear regression with log base 2 of the dilution as the predictor and Ct of the reaction as the response. All of these primers were suitable for quantification with (1) the R2 of the regression of at least 0.98, (2) Ct values at the dilutions of 1 : 4 (AR and Actb) and 1 : 16 (Rpl13a) below 30, and (3) the dissociation curve with single peaks. The sequences, annealing temperatures used, and sizes of the resulting PCR products were listed in Next, diluted cDNA stocks were prepared with 8 serial dilutions ranging from 1 : 8 to 1 : 1,024. For each qPCR reaction, 4 μL of diluted cDNA (1 : 4 for AR and Actb and 1 : 16 for Rpl13a). Two replicate reactions for each sample were run, and their Ct values averaged before further analysis. For a given sample, unnormalized relative expression values for AR expression were calculated using Pfaffl's method against the PN0 female baseline [ Rpl13a and Actb, to yield normalized relative expression values [ Xist mRNA levels were similarly measured to further confirm the sexes of the pups used.RT-qPCR reactions for gene expression quantification were run under the same conditions as described above, with 4 baseline , and then values . Values n values . In addiMouse brain tissues, including the cortex and hippocampus, were homogenized in cold modified T-PER buffer  with protease inhibitor cocktail (Sigma-Aldrich) and PMSF . Brain tissues were homogenized by drawing and ejecting 15–20 times through sterile 20 G needles, followed by centrifugation at 10,000 g for 5 min at 4°C. The supernatant was separated and stored at −80°C. The lysate protein concentrations of individual samples were determined by bicinchoninic acid (BCA) assays (Pierce Biotechnology) before electrophoresis.n = 6-7 per group) could not be run together on the same gel, immunoblotting was conducted by comparing two different sexes  at each age  or two different ages (PN0 versus PN21) of the same sex . Protein samples of 20 μg each were first separated on 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels or Any kD Mini-PROTEAN TGX precast gels (BIO-Rad) and then transferred to nitrocellulose membranes using the BioRad Trans-Blot Turbo Transfer System. Each membrane was rinsed then blocked in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 10% milk at 4°C overnight. After blocking, blots were rinsed with TBST and then incubated with the primary antibody against AR  for 1 h at room temperature. After rinsing, blots were incubated for 1 h in a horseradish peroxidase- (HRP-) conjugated, anti-rabbit IgG secondary antibody , followed by detection of AR protein bands with SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology) using the ProteinSimple FluorChem E System . The blots were reprobed with the antibody against β-actin  with the anti-mouse IgG secondary antibody (Sigma-Aldrich). The intensities of AR and β-actin on individual membranes were measured by densitometry and analyzed with AlphaView (ProteinSimple). To calculate relative AR protein levels of individual samples, the densities of AR were first normalized with the densities of β-actin, and the ratios of AR and β-actin levels in individual samples were then standardized to the mean level of females at the same developmental stage (sex effect) or the mean level of the PN0 group of the same sex (age effect). Each sample was expressed as the fold-difference from either the female or PN0 value, which were both set at 1-fold.Since the protein samples from eight groups of mice (P < 0.05), a log transformation was applied to the data. After showing normality and homogeneity of variances, the data were analyzed by two-way ANOVA to evaluate the effects of sex, age, and their interaction. When two-way ANOVA showed significant differences, Tukey's post hoc test was used to determine which groups were significantly different. A  P value <0.05 was considered statistically significant.The body weight, tissue weight, and RT-qPCR data from individual groups were first tested for normality of the residuals by the Anderson-Darling test and next tested for equality of variances using Levene's test. If significant deviations were detected  to the full-length (110 kDa) were similarly analyzed with two-way ANOVA to reveal the effects of sex and age and their interactions. A  P value of <0.05 was considered statistically significant.Relative AR protein data were analyzed using Student's P = 0.506) or tissue weight (P = 0.55) was observed (P < 0.001), the minimum occurring on PN0 and the maximum on PN21. Interestingly, while the body weights of these animals continued to increase as age advanced, the maximum brain tissue weight was attained by PN14.The numbers of mice at different ages and sexes used in the current study and their body weights and cortex/hippocampus weights are listed in observed . In contP < 0.001) but not sex (P = 0.058) or their interaction (P = 0.567) on AR mRNA expression. Using Tukey's post hoc test, we further demonstrated that AR mRNA levels in the cortex/hippocampus were significantly higher on PN7  than PN0  and continued to rise on PN14  and PN21   .P = 0.058), there was a trend toward a female bias in AR expression in the mouse cortex/hippocampus, especially on PN14 . In addition, relative Xist mRNA levels were measured in these animals, and this X-linked gene was exclusively expressed in all female pups, not males, regardless of age  were 66% of those in female controls  (P = 0.084); however, this trend was reversed on PN7 when AR protein levels were 1.3 times greater in males  than in females  (P = 0.004). On PN14 and PN21, this sex difference in AR protein levels disappeared. Unlike the full-length AR, there was no sex difference in levels of the smaller AR isoform at any time   .n = 6 versus PN21, 8.45 ± 0.74, n = 7) (P < 0.001) and females  (P < 0.001) (P < 0.001)  , 44.9 ± 4.3% (PN7), 64.7 ± 1.8% (PN14), and 59.5 ± 3.1% (PN21) (< 0.001) .Our study reveals that, as age advanced, levels of AR mRNA and two protein isoforms, 110 kDa (full-length) and 70 kDa, increase in the male and female mouse cortex/hippocampus during the first three weeks after birth Figures  and 4. TGiven sexual dimorphism in AR protein expression in the mouse brain, the explanation could be as simple as regulation in AR protein synthesis (translation) and/or stability (degradation) between the sexes via ligand-receptor interaction. While gonadectomy causes a loss of AR protein, androgen supplement after castration restores AR levels in the adult mouse brain and rat prostate , 43. In In addition, masculine elevation of cortical and hippocampal AR protein might be simply caused by increasing the number of AR-expressing cells located in these brain regions via neurogenesis, migration, and differentiation. In good agreement with masculine expression of cortical and hippocampal AR protein shown in α expression in the developing mouse cortex during first week after birth, the perinatal rise in T might be converted into estradiol locally to activate ERα to similarly drive increased AR protein expression in the male mouse cortex and hippocampus during early postnatal development. Unlike the BNST and mPOA, sexually dimorphic AR protein expression in the developing mouse cortex/hippocampus disappears by PN14  in the developing and adult mouse cortex/hippocampus and hypothalamus Figures . Both ARIn summary, AR mRNA and proteins are present in the developing mouse cortex/hippocampus on the day of birth in both males and females, followed by a robust increase in AR expression at the mRNA and protein levels with age during the early postnatal period. This developmental profile of AR expression indicates that age-dependent regulation of AR transcription, splicing, and/or translation might be involved in programming the structure of the cortex and hippocampus, ultimately contributing to the control of social and cognitive behaviors as well as the underlying neural circuits. Although the sexually differentiated expression of full-length AR protein in the developing mouse cortex/hippocampus is transient, whenever AR protein expression is similar between the sexes, the differences in circulating androgens could still play an important role in modulating AR function in a sex-dependent fashion. Future studies will focus on investigating AR expression specifically in discrete subregions of the cortex and hippocampus.


Vulpes vulpes) for residues of eight ARs and used local parameters (percentage of urban area and livestock density) to test for associations to residue occurrence. 59.8% of samples collected across Germany contained at least one rodenticide, in 20.2% of cases at levels at which biological effects are suspected. Second generation anticoagulants (mainly brodifacoum and bromadiolone) occurred more often than first generation anticoagulants. Local livestock density and the percentage of urban area were good indicators for AR residue occurrence. There was a positive association between pooled ARs and brodifacoum occurrence with livestock density as well as of pooled ARs, brodifacoum and difenacoum occurrence with the percentage of urban area on administrative district level. Pig holding drove associations of livestock density to AR residue occurrence in foxes. Therefore, risk mitigation strategies should focus on areas of high pig density and on highly urbanized areas to minimize non-target risk.Anticoagulant rodenticides (ARs) are commonly used to control rodent infestations for biocidal and plant protection purposes. This can lead to AR exposure of non-target small mammals and their predators, which is known from several regions of the world. However, drivers of exposure variation are usually not known. To identify environmental drivers of AR exposure in non-targets we analyzed 331 liver samples of red foxes ( Vulpes vulpes) population density has been shown in France after bromadiolone applications in open areas against water voles (Arvicola terrestris) [Spermophilus richardsonii) than in control areas [Commensal rodent populations are mainly regulated by anticoagulant rodenticides (ARs)  in plantrestris) ,9. In Caol areas . Such deButeo buteo) [Milvus milvus) [Tyto alba) [Mustela putorius) [Mustela erminea) and weasels  [Liver samples of carcasses are often used to screen for AR poisoning in wildlife because AR active substances accumulate in animal tissue—mainly in the liver , whereaso buteo) ,21, red  milvus) ,19,21 anto alba) ,23 are rto alba) ,25. Wildutorius) , stoats nivalis) ,28.Lynx rufus) [AR residues had been quantified in feces of red foxes  and livex rufus)  and somex rufus) . NeverthRattus norvegicus) are often controlled in sewage systems. Furthermore, Norway rat and house mice (M. musculus) infestations can be found for instance in all sectors of the food industry (including restaurants and supermarkets), living quarters and public parks.Regulation of rodenticide usage varies among countries. In Germany, only difenacoum was authorized in plant protection products during the period in which foxes were collected for the present study, but only for use in and around buildings to protect stored products, whereas eight ARs (see below) were authorized for biocidal use. Therefore, exposure of predators to ARs via field application for plant protection should not have occurred. Three registered substances are first generation ARs  and five are second generation ARs . SGARs have a higher toxicity to vertebrates and persist longer in tissues than FGARs ,34. ThisRed foxes are carnivores with a wide distribution . They inThe aim of our study was to identify factors that influence the exposure of foxes to ARs. We analyzed residue occurrence of ARs in fox liver samples in relation to the intensity of livestock holding and the percentage of urban area. We hypothesized that increased livestock density and increased urban area result in higher AR usage, due to increased rodent manifestations and common AR application resulting in high exposure of foxes to ARs. Furthermore we expected livestock species that are kept in indoor feedlots on a farm to be more associated with increased AR application than species kept outdoors. Information about environmental factors that are associated with FGAR and SGAR exposure in predators can aid optimizing risk assessment and developing risk mitigation strategies.Echinococcus multilocularis) liver samples were frozen for at least one week at -80°C and stored afterwards at -20°C. Samples (N = 303) from districts (N = 14) that provided at least 5 liver samples  were provided by a taxidermist. These foxes were dissected at Julius Kühn Institute. No information was available on age or sex of foxes. For pathogen disinfection (e.g.  samples  were use4 and warfarin-d5) and surrogates  were solved in methanol:water (1:1). The limits of detection with a signal to noise ratio of > 3:1 were between 0.001 μg/g for coumatetralyl, 0.002 μg/g for warfarin, difenacoum, 0.003 μg/g for brodifacoum, bromadiolone and 0.005 μg/g for difethialone, flocoumafen and chlorophacinone. Spectra comparison between sample and references based on Enhanced Product Ion-spectra was done additionally. Recovery rates based on spiking clean turkey  liver  ± standard deviation were for chlorophacinone 83% ±14%, warfarin 118% ±4%, coumatetralyl 100% ±6%, difenacoum 78% ±7%, bromadiolone 77% ±4%, brodifacoum 58% ±6%, flocoumafen 65% ±4% and difethialone 41% ±7% and for the surrogate acenocoumarol 112% ±5%, diphacinone-d4 106% ±9%, phenprocoumon 101% ±1% and coumachlor 91% ±2%. Concentrations are μg AR active substance per g liver wet weight throughout and were extrapolated to 100% based on recovery rates in clean liver samples.The analysis of liver samples is described in detail by Geduhn et al. . ShortlyTo compare the occurrence of FGARs and SGARs in fox liver samples, the percentage of residue occurrence and the median concentration in individuals that carried a residue for each active substance was calculated . Two sidLivestock parameters and the percentage of urban area were obtained from GENESIS-online-database . LivestoThe first aim was to investigate if livestock density and the percentage of urban area within German districts affect the occurrence of residues of pooled ARs and the four most prevalent AR substances found in red foxes . We used binomial linear mixed models to screen data for associations between AR occurrence with livestock density and the percentage of urban area of 14 German districts  as depending variable. The variable with the highest VIF (at least >3) was excluded from the model. VIFs of factors from adapted models were all <3. The final model was selected stepwise by AIC (Akaike information criterion) comparison and resulted in: 1) presence/absence of pooled ARs ~ pig density +  and 2) presence/absence of brodifacoum ~ pig density + .All covariates were standardized before modeling by subtracting the mean and dividing by the standard deviation to reduce associations between parameter estimates. R² values are not provided by lme4. We calculated pseudo r-square values from linear regression of observed vs. predicted values to derive power of the mixed models. All statistical analyses were conducted with R 3.1.3 . Level f198 of 331 liver samples (59.8%) from red foxes contained residues of at least one AR. 128 samples (38.7%) contained more than one active substance, 70 samples (21.1%) contained two active substances, 44 three (13.3%), 6 four (1.8%), 7 five (2.1%) and one sample (0.3%) contained six different AR active substances. Chlorophacinone was detected only once (0.3%) while 151 individuals (45.6%) contained residues of brodifacoum . Median Brodifacoum and bromadiolone residues were found most often and residue concentrations were highest for these active substances . Two indSGARs  residues occurred more often than FGARs   of cattle, pig, sheep and laying hens were tested for associations with pooled AR residues and brodifacoum. Cattle density was highly collinear to other livestock density (verified by the variance inflation factors VIF >3) in both models and was therefore excluded from the analysis. Pig density was positively associated to residue occurrence of pooled ARs and brodifacoum residues . Sheep aOur large-scale study provides clear evidence for AR exposure in red foxes across Germany. Residues of brodifacoum and bromadiolone were most common and at the highest concentration. ARs occurred in foxes from all German districts; therefore, wildlife contamination with ARs is not a local problem. All ARs authorized in the EU/Germany for use in either biocidal products or plant protection products could be found in at least one liver sample. Residues of SGARs were considerably more common than those of FGARs and were detected at higher residue concentrations.Accumulation could explain those differences because SGARs have a remarkably longer persistence in animal tissues than FGARs . AnotherAR usage is common at livestock farms, because often livestock food is easily accessible to commensal rodents and there are plentiful places to hide and rest , resultiPig density was positively associated with AR and brodifacoum residue occurrence whereas sheep density and laying hen density were not associated to pooled AR or brodifacoum occurrence. In Germany management of commensal rodents is mandatory in pig husbandry  but not Microtus species are the predominant rodent taxa [Beside agricultural usage of ARs they are regularly applied in urban areas to control commensal rodents. Commensal rodents occur in a wide range of habitats in urban areas such as sewage systems, waste dumps, in parks and gardens, within the food industry  and in apartment buildings and houses, where waste and food is accessible to rodents ,55. Our ent taxa . Brodifaent taxa  and predent taxa .The high variation of AR occurrence among German districts suggests that also other factors than livestock density and urbanization influence AR distribution in predators. Further research is needed for a better understanding of the pathways of AR exposure in predators.In other studies residues of ARs were found in red foxes, but active substances and concentrations differ greatly. In the UK, France and Spain bromadiolone residues are most commonly found in foxes but liver concentrations vary extremely ,20,52. BNevertheless, residue concentrations may provide rough information on biological effects in non-target animals. Sage et al.  found li50 values of flocoumafen and difenacoum for house mice are between the values of brodifacoum and bromadiolone [In total 20.2% of samples contained a particular SGAR with a concentration higher than 0.2 μg/g. Residues of flocoumafen and difenacoum occurred considerably less often than brodifacoum and bromadiolone and concentrations above 0.2 μg/g occurred in only 6 and 4 samples respectively, a concentration >0.8 μg/g was measured once for flocoumafen. LDadiolone . TherefoResidues of ARs such as brodifacoum and bromadiolone in red foxes are widespread in Germany, which reflects the widespread use of these active substances as biocidal rodenticides in Germany. Our study highlighted that foxes carry residues of SGARs more frequently and in higher concentrations than FGARs. Therefore, risk mitigation strategies should consider especially SGARs when these compounds are used in the biocide sector. The occurrence of AR residues associated positively with livestock density in general and with that of pigs in particular, indicating that ARs do not affect non-target terrestrial predators similarly in all land use types. This is especially the case for the highly persistent SGAR brodifacoum that is commonly used in animal holding. Our results are consistent to the hypothesis that indoor livestock feedlots are a source for AR exposure to non-target predators, in our case mainly from pig (or cattle) production. As a conclusion, risk mitigation strategies are needed for AR application in farmland. In contrast to brodifacoum residues that were associated to livestock density as well as to the percentage of urban area difenacoum residues seemed mainly to originate from urban areas. 7.6% of red foxes in our study may have been biologically affected by bromadiolone, based on the results of Sage et al.  and BernS1 Table(PDF)Click here for additional data file.


Testosterone (T) and related androgens are performance enhancing drugs (PEDs) abused by some athletes to gain competitive advantage. To monitor unauthorized androgen abuse, doping control programs use mass spectrometry (MS) to detect androgens, synthetic anabolic-androgenic steroids (AASs) and their metabolites in an athlete’s urine. AASs of unknown composition will not be detected by these procedures. Since AASs achieve their anabolic effects by activating the Androgen Receptor (AR), cell-based bioassays that measure the effect of a urine sample on AR activity are under investigation as complementary, pan-androgen detection methods. We evaluated an AR BioAssay as a monitor for androgen activity in urine pre-treated with glucuronidase, which releases T from the inactive T-glucuronide that predominates in urine. AR BioAssay activity levels were expressed as ‘T-equivalent’ concentrations by comparison to a T dose response curve. The T-equivalent concentrations of androgens in the urine of hypogonadal participants supplemented with T  were quantitatively identical to the T measurements conducted by MS at the UCLA Olympic Analytical Laboratory (0.96 ± 0.22). All 17 AASs studied were active in the AR BioAssay; other steroids were inactive. 12 metabolites of 10 commonly abused AASs, which are used for MS monitoring of AAS doping because of their prolonged presence in urine, had reduced or no AR BioAssay activity. Thus, the AR BioAssay can accurately and inexpensively monitor T, but its ability to monitor urinary AASs will be limited to a period immediately following doping in which the active AASs remain intact. The abuse of unapproved PEDs remains a barrier to fair athletic competition –4. For eThe abuse of PEDs, particularly androgens, is common also amongst non-elite athletes and by those in the general population seeking to improve body image , 8. AnnuMost methods of androgen monitoring rely on detecting, by MS, the chemical signature of T and known AAS within the urine of the athlete –17. SincFor pharmacologic research purposes, androgenic activity has long been assessed by the long-term androgenic and anabolic alterations in tissues of animal models injected with purified compounds . HoweverHere, the efficacy of an AR BioAssay for detecting T and AAS abuse was examined in detail. AR BioAssay accuracy was evaluated by comparison of androgen measurement against MS measurement of T in urine samples and by detailed characterization of the AR BioAssay’s ability to detect AASs and the long-lived AAS metabolites most commonly monitored in urine by anti-doping laboratories. We discuss the types of PED abuse that are most amenable to detection by an AR BioAssay.The AR BioAssay used here quantifies the androgen-mediated movement of a fluorescent protein (FP)-tagged human AR from the cytoplasm to the cell nucleus and was previously developed for high throughput quantification of AR response to androgenic drugs , 29. ThaThe AR BioAssay is conducted by plating reporter cells into a 384-well dish and adding known amounts of the androgen testosterone  or a uriThe accuracy of AR BioAssay measurement first was examined with urine samples obtained from T-treated males, in whom almost all androgens are T. In those participants, comparison of the T-equivalent concentration of androgens measured by the AR BioAssay should be the same as the T concentrations measured on the same urine samples by the UCLA Olympic Analytical Laboratory.Androgen levels in 21 urine samples provided by 6 T-supplemented males were determined by incubating 0.5 μl of urine with the AR BioAssay cell line grown in 40 μl total volume cell culture media; up to 2.0 μl urine/40 μl was tolerated by the cells. Since >98% of urinary T is excreted into urine as an inactive conjugate with glucuronic acid , 34, theNuclear YFP fluorescence was measured following overnight incubation with the AR BioAssay cell line. Nuclear translocation was complete within 5 hours . Thus, t2 = 0.98, slope = 1.04, y-intercept = 2.4). For those samples above the concentration considered to be measureable by the AR BioAssay , the findings indicate that the bulk of glucuronidase-released, active androgens naturally present in the urine is T.For PED testing, urinary T measurements are compared to the levels of epitestosterone (epiT), a stereoisomer of T. epiT-glucuronide and T-glucuronide are present at approximately equal concentrations in the urine of most individuals. Individuals receiving exogenous T experience an increase in the T-to-epiT ratio, which forms an initial assessment for doping with T , 36, alt-6M or lower are shown as dark and light blue bars , indicating that those compounds are not antagonists competitively inhibiting the AR and therefore are not being recognized by the AR BioAssay.For the detection of PEDs beyond T, the AR BioAssay must respond to natural or synthetic androgenic compounds, and be non-responsive to chemically related, non-androgenic steroids. To examine the specificity of the AR BioAssay for androgens, a broad variety of steroids, androgens, AASs and their metabolites were characterized in dose response studies. The structures of the compounds investigated, their sources and the number of independent studies averaged in the data presented are provided in lue bars . CompounAll other natural steroids and steroid intermediates examined . All 16 -6M compound (dark and light blue bars) and for a limited number of poorly active compounds in which the maximum could be approximated (gray bars). Many of the AASs had a higher potency (more negative logEC50) than did the natural androgens T and DHT. Those with lowest efficacy (light blue bars) also tended to be those with lowest potency .Beyond defining the efficacy of different compounds in the AR BioAssay , the dosIt may be possible to detect AAS abuse as an elevation in AR BioAssay activity in relationship to the T measured in the same urine sample. However, in the body most AASs are directly degraded prior to excretion. Thus, unlike the urinary T-glucuronide from which the intact T can be rescued by glucuronidase treatment, AR BioAssay efficacy for AAS detection rests with the extent to which AAS metabolites remain active in the urine. MS detection of the long-lived AAS byproducts constitutes the current method of detecting PED abuse .Very little is known about the AR activity of the most prevalent AAS metabolites commonly surveyed by accredited anti-doping laboratories. We therefore examined the potency and efficacy of 12 known, long-lasting AAS metabolites generated from 10 common AASs . The effThe concentrations of androgens, AASs or their metabolites by the AR BioAssay that would have to be present in urine in order to be detected  was calcAn AR BioAssay, based upon the direct measurement of the nuclear levels of fluorescent protein-tagged AR, was extensively characterized against a wide variety of androgenic and non-androgenic compounds Figs –6. The AThe quantitative accuracy of the AR BioAssay for urine samples was validated in males undergoing T supplementation therapy Figs  and 3. IThe ability to track changes in androgen levels following T supplementation  suggestsThe motivation for using the AR BioAssay in PED testing is its theoretical ability to detect previously unknown urinary AASs –20, 32. The best method to assess by which an AR BioAssay detects unknown, functionally active AAS metabolites in urine samples is to conduct extensive studies on humans doped or not with each of the known AASs. A prior report of a prolonged elevation in urinary androgen levels detected by a yeast-based AR BioAssay two weeks after administration of methyl-testosterone to a volunteer suggested that some AASs may be detectable by an AR BioAssay . Indeed,Overall, the data reported here indicates that the urinary metabolites of known AASs will be more reliably detected by existing MS procedures than by AR BioAssay methods, which implies that there is no substantive benefit for using the AR BioAssay to detect known AASs. Enthusiasm for applying the urinary AR BioAssay to detect AASs currently unknown to the anti-doping authorities, which was our primary motivation for the studies, also was dampened by our findings that most of the known, long-lasting AAS metabolites are functionally inactive . Thus, tHeLa cells stably co-expressing an mCherry-NLS-mCherry nuclear marker (linked to a blasticidin-resistance expression cassette) and a CFP-AR-YFP reporter  were previously described , 45, 46.The reporter cells were maintained in DMEM-H21 cell culture media  Cell Culture Facility) supplemented with 5% fetal bovine serum , 2 mM glutamine (UCSF Cell Culture Facility), 700 μg/ml G418 and 10 μg/ml blasticidin. Cell lines were maintained in culture for less than 15 passages before new vials were thawed and propagated.Prior to the start of an AR BioAssay measurement, the plated cells were androgen-depleted by washing three times with phosphate buffered saline then maintained for 20 or more hours in ‘androgen-free’ media consisting of a 50:50 mixture of phenol-red-free DME-H21/Ham’s F-12 media  supplemented with 5% fetal calf serum charcoal/dextran-stripped of steroids  and 0.4 mM glutamine. Cells then were seeded at 1500 cells per 30 μl of androgen-free media in each well of a 384-well optical imaging plate .-7M final concentration from a 10-3M T stock in ethanol or b, the same volume the ethanol (0.08% in 140 ml). For each mixture, 10 μl was added to the 30 μl of cells/media in quadruplicate wells for final concentrations per well of a, 0.5 μl urine with 10-7M T/0.01% ethanol or b, 0.5 μl urine with 0.01% ethanol. The purpose for collecting parallel wells treated with urine and a high concentration of T is described under the section ‘Correcting Urine Amplification of YFP Fluorescence’.One day after seeding, each well was treated with a urine sample diluted in media and treated with glucuronidase as follows: 30 μl of urine was incubated at 37°C for 60 minutes in 300 μl total volume with 270 μl of phenol-red-free DME-H21/Ham’s F-12 media containing 100U of recombinant β-glucuronidase . Following incubation, two aliquots of 140 μl each of the mixture were then diluted 2-fold with 140 μl of phenol-red-free DME-H21/Ham’s F-12 media into which had been diluted either with a, T to a 8x10Helix pomatia also were examined as a source of glucuronidase but showed some batch variations that had modest toxicity on the reporter cells at the highest levels.No AR BioAssay activity was detected when urine samples were sham-incubated without glucuronidase (not shown) consistent with the majority of T being glucuronidated in urine. Pilot studies with differing levels of glucuronidase (not shown) were conducted to ensure that the amount of enzyme used was sufficient to fully release active T from the T-glucuronide with up to 2 μl urine/well. There was no visual impact on cell health with the amounts of urine and glucuronidase used in this study. Glucuronidase-containing crude extracts from the snail One day following the addition of urine, green fluorescence from the YFP-tagged AR and red fluorescence from the mCherry-linked nuclear marker were collected on an IXMicro High Throughput Microscope  using filter sets previously described  and a 10The amounts of background-subtracted mCherry and YFP fluorescence intensities within the mCherry-defined nuclear areas were averaged from all nuclei in each of two fields collected for each well. Those measurements were transferred, together with other quality control information (see below) into Microsoft Excel . The average YFP fluorescence intensity within the nuclei was compared for the two fields within each well. If they varied more than 30% from each other, these wells were flagged as inaccurate. When encountered, which was seldom, visual inspection indicated that one of the fields either had some non-cellular fluorescent object present or was grossly out of focus. Discarding those fields helps to quality control the data for improved accuracy. Also examined were other quality controls expected to remain constant from well to well, including the average size of the cell nuclei and the average mCherry intensity within the cell nuclei.Alternative AR quantification methods to the measurement of the average fluorescence intensity of YFP-tagged AR in the cell nucleus also were evaluated. Those alternatives included the total amount of background-subtracted YFP-AR fluorescence in the nucleus , the percent of background-subtracted AR fluorescence in the nucleus over the total background-subtracted AR fluorescence in the cell (nucleus and cytoplasm), and the ratio of the average fluorescence intensities in the nucleus relative to the cytoplasm. The presence of the mCherry-tagged nuclear marker enabled precise identification of the nuclear boundaries whereas, in the absence of a cytoplasmic marker, the boundaries of the cell were less rigorously identified particularly when cell density was high obfuscating the boundaries between cells. As a result, measurements limited to quantification of AR levels in the nucleus were found to be most precise and had the added benefit of being amenable to high cellular density  and compatible with rapid collection of only two fluorescent channels (nuclear marker and YFP-tagged AR). We therefore conducted our quantification using the most robust and rapid measurement of YFP-tagged AR fluorescence intensity in the cell nucleus.- concentration and/or pH [Control studies which compared T dose response curves without and with urine showed that most urine samples directly affected the intensity of the YFP reporter . The origin of this proportional amplification in YFP fluorescence intensity is unknown, although YFP fluorescence is known to be environmentally sensitive, particularly to Clnd/or pH , 51. Unl-7M T, which is sufficient to saturate the response of the AR BioAssay to T , including the written consent form signed by study participants, were reviewed and approved by the UCSF Committee on Human Research Institutional Review Board. Urine samples were provided by consented subjects undergoing evaluation or T-supplementation therapy for hypogonadism at the UCSF Endocrine Clinic. Subjects receiving T-supplementation by injection were preferred for tracking the rise and decline in urinary T levels following T-injection . Samples−6 to 10-13M final concentration in the 40 μl BioAssay volume. Each dose point was added to triplicate wells, each of which had two fields collected per well. Dual field collections acted as a control for measurement accuracy, as described in the “Quantification’ section. For the standard T curve, a greater density of dilution points between 10−7 to 10-10M was collected Click here for additional data file.S2 FigAR BioAssay is not affected by incubation with 5 μM of the four indicated anti-androgens whereas co-incubation with 5 μM of those anti-androgens with 5 nM DHT diminishes the strong AR BioAssay response to DHT.(PDF)Click here for additional data file.S3 FigA, Crystal structure of human AR (left panel) bound with DHT. AR amino acids T877 and L704  flank carbon-17 in in the ‘D-ring’ of T and DHT. Structure is displayed using Cn3D from the publicly deposited coordinates of Zhou et al. [B, Fluorescence microscopy images depicting a selective response of the AR BioAssay (left panels) and the epiT AR BioAssay (right panels) to T and epiT, respectively. To design an epiT-binding AR, the hydrogen bond between T and AR amino acid T877 was disrupted by changing T877 to V, L or I. Those AR mutants were unresponsive to T and to epiT (not shown). To enable epiT binding, L704 on the 17α side of the D-ring was replaced with OH-containing amino acids (T or S) to generate a hydrogen bond to epiT. Only the L704T/T877V double mutant created a sensor that responded to epiT but poorly to T. The epiT AR BioAssay also included a V715M mutation that further sensitized the epiT AR BioAssay to epiT (not shown), possibly by creating a more snug interaction with the steroid ring. C, Quantification of nuclear YFP (AR) fluorescence levels in response to different doses of T or epiT. The normal specificity of AR for T over epiT (left panel) was reversed for the epiT AR BioAssay (right panel). The epiT AR BioAssay did not respond to 10-6M of the steroids and steroid metabolites listed in u et al. . The C-1(PDF)Click here for additional data file.S1 Source DataAR BioAssay measurement of androgen concentrations and mass spectrometry measurements of T and epiT concentrations for each of the 39 urine samples examined in this study.(PDF)Click here for additional data file.S2 Source DataDose response curves were conducted for each of the 51 compounds examined in examine in triplicate wells (two fields per well) in 45 384-well plates prepared independently on 22 separate days. Data shown represent the outcome of curve fitting  for each of the 22 studies. Empty fields in the figure represent days in which those particular compounds were not examined. The mean EC50 for each compound is shown above the first set of data for each compound .(PDF)Click here for additional data file.S3 Source DataThe difference between the top and bottom of the dose response curves (see data in (PDF)Click here for additional data file.


Evidence from cachectic cancer patients and animal models of cancer cachexia supports the involvement of Forkhead box O (FoxO) transcription factors in driving cancer-induced skeletal muscle wasting. However, the genome-wide gene networks and associated biological processes regulated by FoxO during cancer cachexia are unknown. We hypothesize that FoxO is a central upstream regulator of diverse gene networks in skeletal muscle during cancer that may act coordinately to promote the wasting phenotype.To inhibit endogenous FoxO DNA-binding, we transduced limb and diaphragm muscles of mice with AAV9 containing the cDNA for a dominant negative (d.n.) FoxO protein (or GFP control). The d.n.FoxO construct consists of only the FoxO3a DNA-binding domain that is highly homologous to that of FoxO1 and FoxO4, and which outcompetes and blocks endogenous FoxO DNA binding. Mice were subsequently inoculated with Colon-26 (C26) cells and muscles harvested 26 days later.Stat3, Fos, and Cebpb. FoxO was also necessary for the cancer-induced downregulation of several gene transcripts that were enriched for extracellular matrix and sarcomere protein-encoding genes. We validated these findings in limb muscles and the diaphragm through qRT-PCR, and further demonstrate that FoxO1 and/or FoxO3a are sufficient to increase Stat3, Fos, Cebpb, and the C/EBPβ target gene, Ubr2. Analysis of the Cebpb proximal promoter revealed two bona fide FoxO binding elements, which we further establish are necessary for Cebpb promoter activation in response to IL-6, a predominant cytokine in the C26 cancer model.Blocking FoxO prevented C26-induced muscle fiber atrophy of both locomotor muscles and the diaphragm and significantly spared force deficits. This sparing of muscle size and function was associated with the differential regulation of 543 transcripts  which changed in response to C26. Bioinformatics analysis of upregulated gene transcripts that required FoxO revealed enrichment of the proteasome, AP-1 and IL-6 pathways, and included several atrophy-related transcription factors, including These findings provide new evidence that FoxO-dependent transcription is a central node controlling diverse gene networks in skeletal muscle during cancer cachexia, and identifies novel candidate genes and networks for further investigation as causative factors in cancer-induced wasting.The online version of this article (doi:10.1186/1471-2407-14-997) contains supplementary material, which is available to authorized users. Cachexia is a devastating condition that affects up to 80% of patients with cancer, particularly those with cancers of the lung and upper GI tract . The conSkeletal muscle wasting during cancer displays marked similarities to other atrophy-inducing conditions, in that the loss of muscle mass is characterized by increased protein degradation and decreased protein synthesis , 5. HoweSkeletal muscle expresses three FoxO family members, including FoxO1, FoxO3 and FoxO4, with both FoxO1 and FoxO3a significantly upregulated in cachectic muscles from LLC and C26 tumor-bearing mice , 16. MorThe purpose of the current study was to determine the requirement of FoxO for locomotor muscle and diaphragm muscle wasting and weakness in response to Colon-26 (C26) adenocarcinoma, and provide the first genome-wide analysis of the genes and biological networks targeted by FoxO in response to C26 tumor burden. We found that FoxO is necessary for C26-induced muscle wasting of both locomotor muscles and the diaphragm, and that this was associated with its regulation of genes involved in not only proteolysis, but additional atrophy-related transcriptional pathways, including the IL-6 and AP-1 pathways. In addition we also identified FoxO as a novel regulator of gene repression during cancer cachexia, with the most enriched gene networks related to the structure and functional integrity of the extracellular matrix and muscle sarcomere. The data presented in this study thus highlights novel candidate genes and biological networks that are regulated downstream of FoxO that may be further explored as causative factors in cancer-induced muscle wasting.ad libitum. The University of Florida Institutional Animal Care and Use Committee approved all animal procedures.Male CD2F1 mice weighing ~20 g were purchased from Charles River Laboratories  and used for all animal experiments. Mice were maintained in a temperature and humidity-controlled facility with a 12-h light/dark cycle and water and standard diet were provided The d.n.FoxO construct used to inhibit FoxO-dependent transcription encodes for amino acids 141–266 of human FoxO3a which encompasses only the FoxO3a DNA binding domain. The amino acid sequence of the d.n.FoxO protein shares 100% sequence identity with mouse FoxO3a (aa140-265), 85% sequence identity with mouse FoxO1 and 75% sequence identity with mouse FoxO4  were injected in 25 μl along the tibia into the TA and EDL muscles, as previously described by others [11 vector genomes/mouse in 400 μl of sterile lactate Ringer’s solution. This minimally invasive technique causes high and widespread transduction of the diaphragm [Mice were acutely anesthetized with isoflurane gas  delivered via a nose cone. For AAV delivery to the TA and EDL, a small incision was made on the lateral side of the lower leg and the TA muscle exposed. Each vector was diluted in lactated Ringer solution such that 1 × 10y others . For tariaphragm , 23.Expression plasmids for the FoxO1 triple phosphorylation mutant  and the FoxO3a triple phosphorylation mutant , have been described previously  and were5 C26 cells (or 1× PBS as control) subcutaneously into each flank on the same day as AAV delivery. Muscles were harvested when the largest tumor diameter reached 1.5 cm (~26 days post-inoculation) when mice had lost ~15% of tumor-free body weight.Murine C26 cells were obtained from the National Cancer Institute Tumor Repository  and cultured as described previously . Cancer Transduction efficiency of AAV9 was determined in 10 μm cross-sections via direct visualization of GFP fluorescence using a Leica DM5000B microscope  prior to and/or following fixation with 4% paraformaldehyde and labeling of muscle fiber borders with Alexa Fluor-conjugated wheat germ agglutinin (Invitrogen) for 1 hr. Leica application suite, version 3.5.0 software was used to trace and measure fiber CSA as described previously .The solutions and methods used for measurements of muscle isometric function in EDL muscles were described in detail previously , 28.Mouse C2C12 skeletal myoblasts were purchased from American Type Culture Collection , and were cultured and transfected with plasmids as described previously by our lab . MyotubeRNA was extracted from TA and diaphragm muscles using TRIzol as previously described . Isolate20, a min.fold.change ≥ 2 and a min delta ≥ 1.2. The last two variation filters were set to eliminate genes that showed no expression change among the 15 samples but to include genes showing changes at low expression values. In the Pre-ProcessDataSet module, min.fold.change is defined as the fold change of the 2nd highest expression value among the 15 samples compared to the 2nd lowest value among the 15 samples, whereas min delta is defined as the difference between the 2nd highest expression value and the 2nd lowest value among the 15 samples [.cel files and expression values were deposited into MIAME compliant NCBI Gene Expression Omnibus [For microarray analysis, 16 total RNA samples from two conditions (control and tumor bearing) transduced with either AAV9-ev or AAV9-d.n.FoxO  were sent to the Boston University Medical Center Microarray Core Facility for amplification, labeling, and hybridization on the mouse Affymetrix Gene 1.0 ST array . This microarray is designed to measure the expression of 28,132 well-annotated genes. A total of sixteen array images were acquired by GeneChip Scanner 3000 TG and the image (expression) quality was assessed by the Affymetrix Expression Console . The Expression File Creator module of the GenePattern platform was used to generate gene expression signal values . Brainar Omnibus  with accDifferential gene expression analyses were subsequently performed using the Comparative Marker Selection module in GenePattern , which cUpregulated or downregulated FoxO target genes in response to the C26 tumor were analyzed separately for their associated functional annotations using the DAVID Bioinformatics database . EnricheP ≤ 0.05.Methods used for statistical analysis of the microarray data are described in the results section. All other data were analyzed using ANOVA followed by Bonferroni post hoc comparisons  and significance was set at Wasting of locomotor muscles is an important component of whole body weakness and fatigue in cancer patients. In addition, the diaphragm muscle also undergoes significant wasting and is believed to play a key role in respiratory complications and mortality in cancer patients. Despite this, studies aimed at understanding the mechanisms of muscle wasting during cancer have largely focused on locomotor muscles. Thus, in the current study we focused on the role of the FoxO factors in cancer-induced wasting of both locomotor muscles and the diaphragm in response to Colon-26 tumor. To inhibit endogenous FoxO1, FoxO3a and FoxO4 DNA binding-dependent transcription we transduced muscles with recombinant AAV9-d.n.FoxO (or AAV9-ev as the respective control) both of which also express GFP as a non-fusion protein to visualize transduction efficiency. Importantly, the d.n.FoxO sequence consists of only that which codes for the FoxO3a DNA binding domain, and shares 85% sequence identity with the respective DNA binding domain of FoxO1 and 75% sequence identity with that of FoxO4  for force measurements is due to two main reasons: 1) the relatively small size of the EDL allows for efficient diffusion of oxygen and nutrients necessary for force measurements (which is not possible in the TA), and; 2) the EDL contains tendons on both sides which allows for both specific and maximal absolute force measurements . We found that EDL muscles from C26 mice transduced with AAV9-ev showed a 40% decrease in maximum absolute force and an 11% decrease in specific force when compared to EDL muscles of control mice transduced with AAV9-ev, both of which were statistically significant . Subsequent comparison of these genes between C26 mice injected with either AAV9-ev or AAV9-d.n.FoxO showed that 544 genes were differentially expressed in the presence of AAV9-d.n.FoxO (q ≤ 0.01and fold change ≥1.5). Out of these genes, 1 gene (Ip6k3) was significantly changed by AAV9-d.n.FoxO during control conditions (q < 0.01) and was thus eliminated as a downstream target of FoxO in response to the C26 tumor. Out of the remaining 543 genes regulated via a FoxO-dependent manner, 296 genes were upregulated in skeletal muscle due to the C26 tumor  transcription factors, the proteasome complex, transcriptional regulation and apoptosis. Ranked in order of significance, the most highly enriched annotation term from each of the top 10 non-redundant annotation clusters identified via DAVID are shown in Figure Cebpb), and factors which heterodimerize within the AP-1 transcription factor complex, including v-fos FBJ murine osteosarcoma viral oncogene homolog (Fos), Fosl2, Fosb and Jun B proto-oncogene (Junb). Based on these findings we postulated that transcription factor binding motifs for C/EBPβ and AP-1, in addition to FoxO, would be enriched in the promoter regions of genes identified as indirect or direct targets of FoxO. Thus, in a separate analysis we used Broad’s Gene Set Enrichment Analysis tool to analyze the -2 kb to 2 kb cis-regulatory regions surrounding the transcriptional start site of upregulated FoxO target genes to identify the most commonly shared conserved transcription factor consensus motifs. This tool uses overlap comparison of user-provided gene lists and gene sets defined in the TRANSFAC (version 7.4) database as those which share a cis-regulatory motif conserved across the human, mouse, rat and dog genomes. Due to the conservation of these motifs across species, these sites are more likely to reflect putative gene regulatory elements. As expected, a TTGTTT consensus motif, which is annotated to FoxO4 (q = 3.76E-13), and is part of the core FoxO consensus motif also regulated by FoxO1 and FoxO3a, was identified as the second most commonly shared motif .Among the FoxO-regulated genes annotated to the canonical IL-6 and/or AP-1 pathways are several bZIP transcription factors including CCAAT/enhancer-binding protein beta  and the 19S regulator  which confers substrate specificity to the 26S complex. Several additional genes which function as E3 ligases were also identified as downstream targets of FoxO, including F-box/WD repeat-containing protein 11 (Fbx11), Socs3, kelch-like ECH-associated protein 1 (Keap1), splA/ryanodine receptor domain and SOCS box containing 1 (Spsb1) and F-box protein 31 (Fbxo31)  and MuRF1 (Trim63), although slightly repressed by d.n.FoxO, did not pass the statistical criteria set for the identification of FoxO-dependent targets in limb muscles of cachectic mice , Homer1 and Myozenin 3 , all of which play critical roles in muscle fiber integrity and function [In addition to the 296 FoxO target genes upregulated in skeletal muscle of C26 tumor-bearing mice, FoxO was also necessary for the C26-induced downregulation of 247 genes. Analysis of these genes using the Functional Clustering Tool within DAVID identified the Extracellular Matrix, Leucine-Rich Repeats, the Z-disc and Glycosylation among the most highly enriched annotations , FOXO4, myocyte-specific enhancer factor 2A (MEF2A), serum response factor (SRF), and myogenic differentiation 1 (MyoD) . In contrast, the levels of Cypher/Ldb3 were not statistically different between the AAV9-ev C26 vs. AAV9-d.n.FoxO C26 groups.To validate a subset of the transcripts identified via microarray as downstream targets of FoxO during C26, we performed qRT-PCR using cDNA generated from the same RNA samples used in the microarrays. As shown in Figures Stat3 and the ubiquitin E3 ligases atrogin-1/MAFbx, MuRF1, Ubr2 and Fbxo30, the latter of which was recently identified as a FoxO target gene necessary for denervation-induced atrophy [Socs3, Ubqln1, Psma2, Fbxo31, Fbxo30, and Ubr2, all of which were significantly repressed in diaphragm muscles of C26 mice transduced with AAV9-d.n.FoxO. We also found, similar to the TA, numerous transcription factor transcripts to be upregulated in the diaphragm in response to C26, including the b-ZIP transcription factors, Fos, Bach2 and Cebpb, as well as Stat3 and Bcl3  and MuRF1 (Trim63) in the diaphragm were significantly repressed by d.n.FoxO. This finding is in contrast to the TA, where their repression by d.n.FoxO did not reach statistical significance. This discrepancy could be related to the increased sensitivity of qRT-PCR in comparison to microarray. We therefore validated the effect of d.n.FoxO on the C26-induced increase in atrogin-1 and MuRF1 in TA muscles via qRT-PCR, and found a greater magnitude of repression than indicated on the microarray , both of which reached statistical significance . However, since the magnitude of repression by d.n.FoxO was still considerably greater for both atrogin-1 and MuRF1 in the diaphragm than in the TA, these data suggest that additional transcription factors perhaps more active in limb muscle also play a role in atrogin-1 and MuRF1 transcription during cancer cachexia, which has been reported previously [As shown in Table  atrophy . As show3 Figure B, each oO Figure C. In addeviously .Figure 4Cebpb, Fos and Stat3 as downstream targets (direct or indirect) of the FoxO factors during cancer cachexia is significant, as each of these transcription factors have been identified to regulate the muscle atrophy program. More specifically, Cebpb is necessary for muscle wasting during LLC-induced cancer cachexia [Stat3 for muscle wasting during C26 cancer cachexia [Fos for denervation-induced muscle loss [Cebpb, Fos and Stat3 in skeletal muscle. We also determined whether FoxO1 and FoxO3a were sufficient to increase the expression of Socs3 and Ubr2, which are bona fide targets of STAT3 and C/EBPβ, respectively. We therefore injected and electroporated into skeletal muscle expression plasmids encoding “constitutively active” FoxO1 (FoxO1 TM) or FoxO3a (FoxO3a TM) which are mutated at the three Akt phosphorylation sites and harvested muscles 4 days later for qRT-PCR analysis. As shown in Figure Cebpb (3.8-fold) and the C/EBPβ target gene, Ubr2 (3.9-fold). On the other hand, FoxO3a TM significantly increased the mRNA levels of Stat3 (1.4-fold), Fos (4.3-fold), Cebpb (3.1-fold) and Ubr2 (3.8-fold), but not Socs3. Since both FoxO1 and FoxO3a were sufficient to increase Cebpb mRNA to similar magnitudes, we analyzed the Cebpb proximal promoter and identified two potential Forkhead boxO binding elements (FBEs) within 300 base pairs upstream of the transcription start site. A literature search revealed that FoxO1 binds to these sites within the Cebpb promoter to regulate Cebpb transcription in adipocytes in response to TNFα [Cebpb promoter are also necessary for increased Cebpb transcription in response to IL-6, a predominant cytokine in the C26 model of cancer cachexia. To test this we transfected C2C12 skeletal muscle cells with luciferase reporter plasmids driven by either a wild type fragment of the Cebpb proximal promoter (−516 to −1) or a mutated fragment (mFBE1/2) which contains base pair substitutions at both FBEs within the Cebpb promoter which prevent FoxO DNA binding [Cebpb promoter reporter activity, which required the intact FBEs.The identification of cachexia , Stat3 fcachexia , and Foscle loss . Moreovecle loss . To furt to TNFα . We ther binding . FollowiMuscle wasting during cancer is a unique and multifactorial process which likely involves the differential regulation of multiple signaling pathways and downstream targets that together promote the wasting phenotype. However, the specific muscle proteins which mediate these intracellular responses to tumor burden are only beginning to be defined. In the current study we extend recent findings that the FoxO transcription factors are necessary for locomotor muscle fiber atrophy during Lewis Lung carcinoma  and S-18Cebpb and Fos. The identification of Cebpb as a downstream target of FoxO in skeletal muscle during cancer is of particular interest, since the protein expression of C/EBPβ is increased in muscles of tumor bearing mice, and mice lacking C/EBPβ are resistant to LLC cancer-induced muscle wasting [Cebpb mRNA in skeletal muscle. Although we cannot ascertain, based on our findings, whether FoxO1 or FoxO3a directly induce Cebpb gene transcription during cancer cachexia, there are two putative FBEs located within the Cebpb proximal promoter that FoxO1 has previously been documented to bind [Cebpb promoter activation in skeletal muscle in response to IL-6, a predominant cytokine in the C26 model of cancer cachexia. Given the requirement of C/EBPβ for cancer-induced muscle wasting, it seems likely that FoxO-dependent upregulation of Cebpb plays a role in the muscle wasting phenotype induced by FoxO during cancer.Several transcription factors were identified through microarray analysis as downstream targets of FoxO in response to tumor-burden, including the atrophy-related bZIP transcription factors,  wasting . Moreove to bind . Here weFos was the third most highly upregulated FoxO target gene in response to C26 (16-fold), which is in alignment with a previous microarray study showing upregulation of Fos in muscles from both moderately and severely cachectic C26 tumor-bearing mice [Fos is also upregulated in skeletal muscle following denervation and knockdown of Fos prevents the associated muscle atrophy, thus highlighting its role in the atrophy program [Fos could play a role in the enrichment of this pathway. Notably, in addition to Fos we also found that FoxO was necessary for the C26-induced increase in other early response genes related to the AP-1 pathway, including immediate early response 5 (Ier5), and early growth response 1 (Egr-1). Importantly, both Egr-1 and Ier5 were recently identified as cachexia-associated genes upregulated in muscles of pancreatic cancer patients together with Foxo1[The significance of the immediate early gene and oncogenic transcription factor, FOS, as a FoxO target in skeletal muscle during cancer cachexia is currently unknown. However, ing mice . Importa program . Despite program . Since tith Foxo1. Erg-1 wUbqln1) which encodes for an ubiquitin-like (UBL) protein which physically interacts with both proteasomes and ubiquitin ligases and is involved in protein degradation [Socs3, Fbxw11, Keap1, Spsb1 and Fbxo31. While little is known about the substrates and functions of these E3 ligases in skeletal muscle, Fbxw11  plays a role in targeting IκBα for ubiquitin-dependent degradation [Bnip3, Cathepsin-L and Gabarapl1), as well as Fbxo30/MUSA1, which was recently reported as a novel FoxO target gene which encodes an ubiquitin E3 ligase that is required for denervation-induced muscle loss [Many of the upregulated genes identified as downstream targets of FoxO in muscles of C26 tumor-bearing mice are involved in protein degradation, and are coordinately upregulated during multiple wasting conditions –50. Inclradation . In addiradation . This firadation , 9, 10. radation , 40, 53,cle loss . Thus, b(Id1) and 3 (Id3), which act as transcriptional repressors of myogenic basic helix-loop-helix (bHLH) proteins such as MyoD [Unexpectedly, nearly half of the genes regulated by FoxO during cancer were downregulated in response to the C26 tumor. While FoxO could mediate gene repression through direct binding to gene promoters, as has been documented previously –56, FoxO as MyoD , were boA significant number of the downregulated FoxO targets during cancer encode for proteins that localize to the ECM and the Z-disc of the muscle sarcomere. Despite the relatively long half-life of typical structural proteins, prolonged downregulation of these transcripts over several weeks or even months during the progression of cancer should logically be sufficient to decrease protein expression. Provided this holds true for even a small subset of the downregulated targets, the impact of their downregulation could be highly significant to the progression of cancer-induced wasting and weakness. Indeed, many of the target genes downregulated via FoxO play critical roles in maintaining muscle integrity, fiber size and contractile function. Just one example, the extracellular matrix protein, Tenascin C (Tnc), is increased in response to muscle damage, exerts anabolic and proliferative effects on interstitial and myogenic cells, and intriguingly, its genetic ablation is sufficient to decrease muscle mass, cause selective atrophy of type II muscle fibers, and slow muscle contractile properties . Since tMmp8) and Mmp9 in muscles of cachectic tumor-bearing mice Additional file 1:List of FoxO target genes downregulated in skeletal muscle during C26 cancer cachexia.(PDF 189 KB)Additional file 2:Tables S1, S2 and S3. Enriched transcription factor binding motifs in promoter regions of FoxO target genes upregulated (Table S1) and downregulated (Table S3) during C26 cancer cachexia. Table S2. Expression data for additional gene transcripts of interest. (PDF 90 KB)Additional file 3:


Small Heat Shock Proteins (sHSPs) are molecular chaperones that transiently interact with other proteins, thereby assisting with quality control of proper protein folding and/or degradation. They are also recruited to protect cells from a variety of stresses in response to extreme heat, heavy metals, and oxidative-reductive stress. Although ten human sHSPs have been identified, their likely diverse biological functions remain an enigma in health and disease, and much less is known about non-redundant roles in selective cells and tissues. Herein, we set out to comprehensively characterize the cardiac-restricted Heat Shock Protein B-2 (HspB2), which exhibited ischemic cardioprotection in transgenic overexpressing mice including reduced infarct size and maintenance of ATP levels. Global yeast two-hybrid analysis using HspB2 (bait) and a human cardiac library (prey) coupled with co-immunoprecipitation studies for mitochondrial target validation revealed the first HspB2 “cardiac interactome” to contain many myofibril and mitochondrial-binding partners consistent with the overexpression phenotype. This interactome has been submitted to the Biological General Repository for Interaction Datasets (BioGRID). A related sHSP chaperone HspB5 had only partially overlapping binding partners, supporting specificity of the interactome as well as non-redundant roles reported for these sHSPs. Evidence that the cardiac yeast two-hybrid HspB2 interactome targets resident mitochondrial client proteins is consistent with the role of HspB2 in maintaining ATP levels and suggests new chaperone-dependent functions for metabolic homeostasis. One of the HspB2 targets, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), has reported roles in HspB2 associated phenotypes including cardiac ATP production, mitochondrial function, and apoptosis, and was validated as a potential client protein of HspB2 through chaperone assays. From the clientele and phenotypes identified herein, it is tempting to speculate that small molecule activators of HspB2 might be deployed to mitigate mitochondrial related diseases such as cardiomyopathy and neurodegenerative disease. Studies of molecular chaperones and their regulatory pathways are among the most challenging and illuminating ones for understanding the hierarchical integration among genotype-phenotype relationships, biological systems and cellular networks –3. AmongHSPB5 knockout but inadvertently eliminated both genes resulting in double knockout (DKO) mice . Tr. TrHSPB2st Century Biochemicals using the following peptide sequence: CPATAEYEFANPSRLGEQ-amide. In addition, anti-HspB3 antibodies were obtained from StressMarq Biosciences Inc. and HspB3 levels were determined on a separate western blot.Mouse hearts were removed and rinsed with ice cold PBS as previously described from our laboratory . The tisth intercostal space and a 7–0 suture, attached to a balloon, was applied around left descending coronary artery [Age-matched male mice 10–12 week-old) selected for the study were bred according to approved guidelines of Institutional Animal Care and Use Committee (IACUC) at the University of Utah. Animals were anesthetized with a ketamine (100mg/kg) and xylazine (0.5mg/kg) cocktail administered intraperitoneally. Following tracheal intubation of the trachea, the mouse was placed on a small rodent ventilator at a respiratory rate of 120 breaths per m and 0.25 ml tidal volume. To maintain temperature control, animals were placed on a water-heated surgical bed  with temperature setting at 37°C. Under sterile conditions, the heart was exposed through the 4 week-oldTwenty-four hours (h) after reperfusion start, the mice were euthanized, the hearts were excised and cannulated with a 20-gauge needle through the aorta, and then the hearts were perfused with Krebs-Henseleit solution . The LADMouse blood samples were set for clotting before centrifugation for 15 m at 800g. The serum was collected, diluted in series from 1:10 to 1:1000, and then loaded into a 96-well sample plate from the High Sensitive Mouse Cardiac Troponin-I kit . Troponin I level was measured at 450 nm by following the manufacturers recommendations.2PO4. The mitochondria were equilibrated for 5 m at room temperature and 250 uM CaCl2 was loaded to induce swelling. Data was read at 540 nm.Mitochondria were isolated from mouse hearts as previously described  and dilu31P NMR data collection, mice were heparizined  and anesthetized . Excised hearts were retrogradely perfused (60 mm Hg) with modified Krebs–Henseleit buffer  equilibrated with 95% O2/5% CO2, at 37°C, and containing 0.4 mM palmitate/fatty acid free albumin complex (3:1 molar ratio) and 10 mM glucose [For isolated heart studies and  glucose . A water31P NMR spectra. 31P NMR data provided detection of high-energy phosphate content, including phosphocreatine (PCr) and ATP throughout an I/R protocol. Following collection of baseline, pre-ischemic baseline NMR data, perfused hearts were subjected to 16 m of global ischemia at 37°C, followed by 32 m of reperfusion. Relative ATP content in each heart during the pre-ischemic, ischemic, and reperfusion periods was determined from the signal intensity of the β-phosphate resonance signal at -16 ppm. Time-averaged spectra were collected at 243 MHz from the intact beating hearts in 4 m time blocks using 64 scans with a 45° flip angle and 2 s interpulse interval, using previously described data collection and processing/analysis schemes for high-energy phosphate levels and intracellular pH [The isolated perfused hearts were placed inside a 10 mm broadband NMR radiofrequency probe within a 14.1 T NMR magnet  for collection of sequential, lular pH –17.HSPB2 plasmid (pJG591). The bait plasmid (pJG591) was constructed by standard cloning techniques in which HSPB2 was amplified by PCR from a human template using primers GGCGAATTCTCGGGCCGCTCAGTGCCAC and GGCGGATCCTCAGGGCTCAACTATGGCTGCC and cloned into the EcoR1/BamHI sites of pGBKT7 (Clontech) for fusion to the Gal4 DNA binding domain at the N-terminus; the C-terminus is native. Over 22 million yeasts were mated, from which 10,000 colonies arose on SD-Trp-Leu plus Aureobasidin (Clontech) selection plates. Colonies were then patched to alternate Y2H selection plates (SD-Trp-Leu-His-Ade) for phenotype validation through alternate transcriptional reporters (HIS and ADE). The plasmid inserts of the library were identified for over 1,000 colonies arising on the SD-Trp-Leu-His-Ade plates by colony PCR (primer agatggtgcacgatgcacag and ctattcgatgatgaagataccccacc) followed by sequencing and NCBI BLASTN [HSPB2-bait plasmid or empty bait plasmid, selected on SD-Trp-Leu and streaked to SD-Trp-Leu-His-Ade to test for an HSPB2-dependent interaction. For HspB2 specificity testing, HSPB5 was PCR amplified from a human template and cloned into the EcoRI/SalI sites of pGBKT7 (Clontech) using primers GGCGAATTCGACATCGCCATC CACCACCCC and GGCGTCGACCTATTTCTTGGGGGCTGCGGTGAC. The HSPB5 bait plasmid was then co-transformed with a library plasmid into yeast, selected on SD-Trp-Leu and streaked to SD-Trp-Leu-His-Ade to test for an HSPB5-dependent interaction.Yeast harboring a human cardiac cDNA Y2H library (Clontech) were mated by the standard Matchmaker mating protocol (Clontech) to yeast harboring a full-length human I BLASTN  analysisHSPB2 (HSPB2wt), transgenic cardiac HSPB2 (HSPB2cTg), or an HSPB2 cardiac knockout . For eaSPB2cKO, . The IP mentha, Reactome-Fls, Reactome, IntAct and MINT databases (partners reported for either human or mouse proteins) and mapping them into a single merged network. Gene Ontology analysis for the resulting networks was done using the Cytoscape ClueGO plugin [http://omim.org/).Cytoscape version 3.2.1  was usedO plugin . AssociaGAPDH and HSPB2 were cloned into pet15b (Novagen) for expression and purification. GAPDH was amplified from Origene plasmids SC118869 (accession number NM_002046) using the following primers, GGCCATATGGGGAAGGTGAAGGTCGGAGTC and GGCGGATCCTTACTCCTTGGAGGCCATGTGGGC, and cloned into pet15b at the NdeI/BamHI sites. HSPB2 was PCR amplified from pJG591 (using primers GGCCATATGTCGGGCCGCTCAGTGCC and GGCGGATCCTCAGGGCTCAACTATGGCTGCC) and cloned into pet15b at the NdeI/BamHI sites. Expression vectors were transformed into BL21 (DE3) E. coli (Novagen). Overnight cultures of transformants were diluted 1:500 into LB-AMP and grown for 3 h at 37°C. Expression was induced by 0.5 mM ITPG for 5 h at 37°C. Pelleted bacteria were then resuspended in 20 mL XWA lysis buffer . Cells were lysed using the Microfluidics M-110P homogenizer and debris pelleted at 15,000 rpm for 60 m. The supernatants were incubated with 250 μL of Ni-NTA agarose (Qiagen) for 2–3 h, beads were washed twice with 15–25 mL of XWA lysis buffer and then transferred to a column to be washed with 50 mL of lysis buffer containing 25 mM imidazole. Protein was eluted three times per sample with 0.3 mL of lysis buffer containing 250 mM imidazole and only 100 mM NaCl. The control, porcine citrate synthase was purchased from Sigma-Aldrich. Proteins were dialyzed into 40 mM HEPES-KOH buffer (pH 7.5) prior to in vitro chaperone assays. In vitro chaperone assays were conducted as previously described [escribed , and pelIn vivo GAPDH chaperone activity assays were performed by transfecting plasmids bearing human HspB2 fused to myc peptide or the empty plasmid control (pCMV-myc) into C2C12 cells in a 6-well plate using 6.5 μL lipofectamine 2000 (Life Technologies) and manufacturers protocol. After 48 h at 37°C, cells were shifted to 45°C for 30 m and then placed back at 37°C for three h prior to lysis. Cells were resuspended in 300 μL lysis buffer (20 mM HEPES (pH 7.8), 10 mM KCl, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40 and 10% glycerol), homogenized in a Dounce homogenizer ten times for five seconds on ice, and debris pelleted at 13,000 rpm for 5 m. The pellet was resuspended in 100 μL of 2X SDS buffer  bromophenol blue, 100 mM fresh dithiothreitol, 2.5% (w/v) SDS, 25% glycerol) while 6X SDS buffer was added to the supernatant for a final concentration of 1X. Samples were boiled 2 m, analyzed by 12% SDS-PAGE and transferred to nitrocellulose paper. Proteins were visualized using anti-GAPDH antibody, anti-beta-Tubulin antibody, and anti-Myc antibody . Protein bands were analyzed using ImageJ software [software  and pellt-test used for statistical analysis using two-tailed distribution and two-sample equal/unequal variance for all experiments with the exception of the NMR experiments, which were analyzed by ANOVA with Student-Newman Keuls post test [p<0.05. The HspB2 interactome described herein has been deposited in the Biological General Repository for Interaction Datasets  where iThis study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah (Permit Number: 10–05006). All surgery was performed under ketamine and xylazine cocktail anesthesia administered intraperitoneally, and all efforts were made to minimize suffering.HSPB2 overexpressor mice (HSPB2cTg) were generated using human HSPB2 under the control of α-myosin heavy chain (α-MyHC) promoter as schematically shown in (P = 0.035) and HspB8 was non-statistically significantly elevated (P = 0.064) in the detergent insoluble fraction, respectively (HSPB2 OE in intact mouse hearts.Cardiac-specific ectively . In contHSPB2 KO and/or HSPB2/HSPB5 DKO in I/R studies [HSPB2 (HSPB2cTg) were compared with cardiac HSPB2 KO mice (HSPB2cKO) and their littermate controls . Mouse hearts of HSPB2cTg (n = 11), HSPB2cKO (n = 9) and their corresponding controls underwent 45 m of ischemia followed by 24 h of reperfusion in situ. The size of infarction in each mouse heart was analyzed as described in Materials and Methods. An infarction area of 35.4 ± 5.6% was significantly lower in HSPB2cTg mouse hearts compared with the infarction area of 48.7 ± 10.0% for the HSPB2NTg controls . We previously reported a deficit in the recovery of phosphocreatine (PCr) and ATP levels in the ring I/R . The abi2O → ADP + Pi + H+) that occurred during ischemia among all groups is consistent with the similar reduction in intracellular pH among the groups, from a preischemic baseline of 7.1 to the final pH range observed at the end of ischemia of 6.2–6.1. The peak at 3.5 ppm is signal from the intracellular inorganic phosphate (Pi), which shifts to lower values due to the increasing concentration of diprotonated Pi versus mono-protonated Pi as the pH becomes increasingly acidic during ischemia. The peak at 5 ppm is a combined signal from both extracellular pH and residual buffer surrounding and in the isolated perfused heart, which also contains inorganic phosphate and reflects neutral pH.As shown in HSPB2 alone via cardioprotection during ischemia and sustained mitochondrial function upon reperfusion.In contrast to the similarities observed during ischemia, ATP content and recovery was augmented in HSPB2cTg hearts upon early reperfusion in comparison to all other groups . These binding partners were further grouped by their frequencies, depending on if they were retrieved only once  as prey and human HSPB2-dependence. Briefly, prey plasmids encoding 90 of the putative HspB2 binding partners (49 from the multi-hit list and 41 from the single-hit list) were purified from yeast and reintroduced into naïve yeast harboring either the empty bait plasmid or the HSPB2 bait plasmid. A remarkable 81.6% of the 49 multi-hit library plasmids tested showed a positive Y2H result that was dependent on HSPB2 , 10.2% were false positives , and 8.2% were true negatives . In contrast, we found that only 51.2% of the 41 single hit plasmids harbored HspB2 dependent-binding partners, 12.2% were false positives, and another 36.6% were true negatives. The multi-hit binding partners with an 82% rate for validation were then sequenced to determine frame, and are used for all subsequent studies and analysis due to its reliability.The advantage of high sensitivity in the Y2H method comes at the expense of specificity  , 33, 34.in vivo support for non-redundant clientele among sHSPs, consistent with expression and phenotypic analyses that have revealed unique functions for HspB5 and HspB2 chaperones [In order to test the specificity of the HspB2 Y2H interactome, the interactome was compared to the recently published HspB1 (Hsp27) interactome in which 228 binding partners were identified by Katsogiannou et al. from large-scale Y2H screens of testis and Hela cancer cell libraries . Of the aperones .The 84 high confidence binding partners identified by Y2H were further analyzed for their cellular localization and molecular functions. The majority of the binding partners have been reported to primarily localize to the mitochondria 19%), the cytoskeleton (24%) and the cytoplasm (19%) . The fun%, the cyThe identification of multiple mitochondrial binding partners poses a novel hypothesis that HspB2, a resident cytosolic molecular chaperone, might also have distinct mitochondrial clientele. Such clientele would be consistent with the transient association with the outer mitochondrial membrane reported by Nakagawa et al. from tissue culture studies . To testA total of 68 proteins  were uniWhen combined, the 84 high confidence targets from the Y2H screen and 68 from co-IP studies identify 149 unique proteins as putative HspB2 clientele. Three proteins were identified by both methods . The low level of overlap between the two independent screens is due to the difference in using whole-cell verses mitochondrial extract and is also consistent with previous reports on differences due to the diverse strengths of each approach . Of noteHspB2 is one of only a few sHSP to date (including HspB9 and HspB10) without identified disease-associated human alleles. Therefore, the combined high confidence Y2H and co-IP interactome  was analyzed for diseases associated with the putative HspB2 binding partners. There were 35 Y2H and 14 co-IP HspB2 binding partners associated with disease, and of these, 28 (57%) were associated with myopathies and neurodegenerative diseases such as Alzheimer’s disease (AD) . These rAlthough the high confidence Y2H and co-IP HspB2 interactome revealed proteins with a wide range of biological functions, it was apparent that many of these proteins overlap in their biological pathways. In order to identify key pathways influenced by HspB2, the combined Y2H and co-IP interactome (149 proteins) was analyzed using protein-protein interaction and functional annotation datasets in Cytoscape . DetaileGyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a central protein in the network (indicated in orange) with many connections to the major protein cluster with myofibril functions. GAPDH catalyzes the sixth step of glycolysis and is a key metabolic switch for partitioning glucose to TCA for respiration , and hasGAPDH was chosen to validate by further characterization because it was a central node in the bioinformatic analysis of the HspB2 interactome, it is a soluble protein (facilitating its characterization as chaperone target), and it has been associated with all of the HspB2-related cardiac phenotypes including maintaining ATP levels , Table 1in vivo and in vitro chaperone assays . No significant differences in soluble GAPDH were observed when compared with β-Tubulin and GAPDH was not observed in the pellet of homogenized mouse cardiac tissue  of the GAPDH where ~50% of GAPDH aggregated without HSPB2  where it reduced I/R stress-induced infarct size . In suppcKO mice , with ATin vivo binding partners, whereas only four prior HspB2 clientele binding partners were reported to date . Interate  have alThe 84 HspB2 binding partners identified by Y2H are supported by the fact that the HspB2 bait interacted with less than 0.05% of the cardiac cDNA library and was specific for HspB2 when compared to other sHSPs. The HspB2 interactome was compared to the recently published HspB1 (Hsp27) Y2H interactome of 228 binding partners , and onlIn order to identify key pathways of HspB2 function from among the binding partners, a systems biology approach was used to map previously known protein-protein interactions, protein localization patterns, and related diseases. Such approaches have proven fruitful in identifying key pathways involved in protein function as well as human disease . FunctioThe abundance of putative mitochondrial clientele posit HspB2 as a key factor in maintaining cardiac mitochondrial function, particularly when combined with the increased ability to maintain ATP seen in the HSPB2cTg mice . SeveralThe mitochondrial HspB2 clientele obtained by Y2H were supported by 68 proteins retrieved from co-IP with cardiac, mitochondrial HspB2 , with thin vivo significance of the interactome and analysis, GAPDH was studied in more detail due to its identification as a central hub in the protein-protein interaction network  plaques [Although many other sHSPs (including HspB5) have been found to play a role in various diseases including neurodegenerative and muscular disease, disease-associated alleles of HspB2 have not been identified. The systems biology analysis herein, however, posits HspB2 as a player in neurodegenerative, mitochondrial, and muscular disease . In supp plaques . In addi plaques . This is plaques . Both ne plaques , 69, 70. plaques , 72, mitS1 Fig2 was loaded to induce swelling. Data was read at 540nm.Mitochondria were isolated from HSPB2 overexpressors (HSPB2cTg) or control hearts (HSPB2NTg), equilibrated for 5 m at room temperature and 250uM CaCl(TIF)Click here for additional data file.S1 TableProteins retrieved a single time from a Y2H screen for HspB2 binding partners are given along with the following information: protein name, UniProt  protein (PDF)Click here for additional data file.


Although prostate cancer (PCa) initially responds to standard luteinizing hormone releasing hormone agonist–based androgen-deprivation therapy (ADT), most advanced, lethal PCa eventually becomes castration-resistant PCa (CRPC) and metastasizes to bone . The useThe heterogeneous nature of PCa and the difficulty in obtaining sufficient clinical samples of CRPC for genomics analysis have impeded understanding of this disease. However, recent genomics studies have identified potentially actionable signaling pathways that contribute to CRPC. In addition to mutations in genes or pathways, including PI3K-Akt and Wnt/β-catenin, mutations were found in numerous proteins that interact with AR in CRPC samples . These ret al demonstrated that ADT or treatment with ENZ increased the phosphorylation of Akt in PTEN-deleted LNCaP cells and LAPC4 cells, whereas the combination of ENZ and BEZ235, a PI3K inhibitor, caused marked regression of PCa in animal models [Although the fact that mutations cause persistent AR signaling has long been understood, suppression of AR signaling through pharmacologic inhibition was only recently shown to lead to derepression of specific oncogenic signaling pathways in PCa. Carver et al, who showed that androgen-liganded AR reduced AR target gene expression through ARBS2, a transcriptional enhancer [The concept that AR inhibition may derepress particular AR-suppressed genes was extended by the recent findings of Cai enhancer . The autenhancer . Importaenhancer . Overallet al demonstrated that glucocorticoid receptor (GR) expression was upregulated in LNCaP/AR xenografts that are resistant to ENZ, as well as in CRPC bone metastases from patients that had a relatively poor response to ENZ compared with those from patients who had a more favorable response [et al [Recently, Arora response . Additioresponse . These re [et al  with reget al recently found that ENZ increased expression of the proto-oncogene c-MYB, a transcription factor that has been shown to be upregulated in multiple malignancies [Li gnancies . The resgnancies . c-Myb egnancies . These rgnancies . On the gnancies . Thus, tOverall, the results of these recent studies (i) demonstrate the persistence and importance of AR-regulated gene expression in CRPC; (ii) support a model of anti-AR agent–mediated gene derepression as an adaptive resistance mechanism; (iii) suggest that this adaptive response is linked to further progression of a therapy-resistant cell population; and (iv) suggest that overlapping AR and GR, and AR and c-Myb, target genes are strongly selected for drug resistance and should be considered for further study as potential therapy targets for CRPC Figure .