DKK1 and DKK3, leading to enhanced expression of c‐MYC, CYCLIN D1 and SURVIVIN. Through chromatin immunoprecipitation (ChIP) studies, we found that PRMT5 binds to the promoter region of WNT antagonists, DKK1 and DKK3, and induces symmetric methylation of H3R8 and H4R3 histones. Our findings also show that PRMT5 inhibition using a specific small molecule inhibitor, compound 5 (CMP5), reduces PRMT5 recruitment as well as methylation of H3R8 and H4R3 histones in the promoter regions of DKK1 and DKK3, which consequently results in reduced expression CYCLIN D1 and SURVIVIN. Furthermore, CMP5 treatment either alone or in combination with 5‐Azacytidine and Trichostatin A restored expression of DKK1 and DKK3 in TNBCs. PRMT5 inhibition also altered the growth characteristics of breast cancer cells and induced their death. Collectively, these results show that PRMT5 controls breast cancer cell growth through epigenetic silencing of WNT/β‐CATENIN pathway antagonists, DKK1 and DKK3, resulting in up‐regulation of WNT/β‐CATENIN proliferative signalling.Protein arginine methyltransferase 5 (PRMT5) activity is dysregulated in many aggressive cancers and its enhanced levels are associated with increased tumour growth and survival. However, the role of PRMT5 in breast cancer remains underexplored. In this study, we show that PRMT5 is overexpressed in breast cancer cell lines, and that it promotes WNT/β‐CATENIN proliferative signalling through epigenetic silencing of pathway antagonists,  PRMT5 binds to the FOXP1 promoter and induces symmetrical methylation of histone H3R2, which in turn promotes recruitment of the WDR5 subunit of the SET1/MLL methyltransferase complex that is known to methylate H3K4me3, resulting in elevated expression of FOXP1. These results indicate that PRMT5 plays an important role in maintaining breast cancer stemness.The role played by PRMT5 in breast carcinogenesis remains underexplored. A prior study by Scoumanne et al. (2009) demonstrated that PRMT5 regulates proliferation of MCF7 cells, and that its knockdown inhibits their proliferation by inducing G1 cell‐cycle arrest, indicating that PRMT5 is a key regulator of cell‐cycle progression.c‐MYC, CYCLIN D1 and SURVIVIN.APC, DKK3, SFRP1 and SFRP2 has been reported in many breast cancer samples.Several reports have shown that dysregulation of WNT signalling is tightly linked to carcinogenesis.AXIN2 and WIF1.DKK1 and DKK3. ChIP assay confirmed binding of PRMT5 to the promoter region of WNT antagonists, DKK1 and DKK3, and hypermethylation of promoter H3R8 and H4R3. Moreover, PRMT5 inhibition either alone or in combination with Trichostatin A (TSA) and 5‐Azacytidine (5‐Aza) led to transcriptional derepression of DKK1 and DKK3, and decreased expression of WNT/β‐CATENIN target genes, CYCLIN D1 and SURVIVIN. These changes were also accompanied by reduced proliferation, migration, and invasion and enhanced cell death of breast cancer cells.We have recently shown that PRMT5 activates WNT/β‐CATENIN signalling pathway in three different types of non‐Hodgkin’s lymphoma cell lines, mouse primary lymphoma cell lines and clinical samples through epigenetic silencing of 22.1Human breast cancer cell lines HCC1937 and BT549 cells were cultured in RPMI 1640 medium  supplemented with 10% foetal bovine serum (FBS). BT549 cells were supplemented with insulin (0.023 U/mL). MCF7 cells were cultured in DMEM medium  supplemented with 10% FBS. Human mammary epithelial cells (HMECs) were grown in mammary epithelial cell basal medium (American type culture collection) supplemented with rH‐insulin (5 μg/mL), L‐Glutamine (6 mmol/L), Epinephrine (1 μmol/L), Apo‐transferrin (5 μg/mL), rH‐TGF‐α (5 ng/mL), pituitary extract (0.4%) and hydrocortisone (100 ng/mL).2.2TM). Briefly, TNBC cells from a T75 flask were removed using 750 μL of TRIzol reagent, transferred to an eppendorf tube and incubated at room temperature for 5 minutes. Next, 300 μL of chloroform was added and samples were mixed vigorously and incubated at room temperature for 10 minutes. Samples were then centrifuged at 4°C for 15 minutes before the upper layer containing RNA was transferred to a new eppendorf tube containing 500 μL of isopropanol. After incubation at room temperature for 10 minutes, the reactions were centrifuged at 4°C for 15 minutes. The RNA pellet was rinsed with 75% ethanol, air‐dried before resuspension in 20 μL of nuclease‐free water. Next, mRNA was converted into cDNA using High‐Capacity cDNA Reverse Transcription Kit as per manufacturer’s instructions (Applied BiosystemsTM). Briefly, 2 μg of total RNA was reverse transcribed in a 20‐μL reaction mixture containing 2.5 μmol/L random primers, 100 mmol/L dNTP Mix and Taqman reverse transcription reagents. To measure the mRNA levels of WNT target genes and antagonists, real‐time PCR was carried out using TaqManTM Universal PCR Master Mix in a 20‐μL reaction as described previously.PRMT5 , CYCLIN D1 , c‐MYC , SURVIVIN , AXIN1 , AXIN2 , WIF1 , DKK1 , DKK2 , DKK3 , DKK4 , SFRP2 , SFRP3 , SFRP4 , SFRP5 , and β‐ACTIN . β‐ACTIN was used as internal control to normalize expression of tested genes.Total RNA was extracted using TRIzol reagent (Invitrogen2.3Whole‐cell extracts were prepared in radioimmune precipitation assay (RIPA) buffer . The extracts were subjected to Western blot analysis as described previously.2.42, 3 mmol/L MgCl2) supplemented with protease inhibitors, Aprotinin (10 μg/mL), PMSF (100 mmol/L), Pepstatin (2.25 μg/mL), and Leupeptin (10 μg/mL), and fragmented using Q55 sonicator (Qsonica). Sonicated chromatin was further digested by micrococcal nuclease (MNase) (0.6 Units) treatment at 37°C for 20 minutes. MNase‐treated chromatin was analysed by agarose gel electrophoresis to ensure that DNA fragment sizes did not exceed 500 bp. To evaluate PRMT5 recruitment as well as PRMT5‐induced H3R8 and H4R3 symmetric methylation marks, chromatin was immunoprecipitated overnight at 4°C using either pre‐immune or immune antibodies against PRMT5, H3(Me2)R8 and H4(Me2)R3 in the presence of protein A‐Sepharose beads, which were pre‐blocked with sheared salmon sperm DNA (0.2 mg/mL) and BSA (0.5 mg/mL). The retained complexes were washed successively with mixed micelle buffer , buffer 250 , and wash buffer . Next, chromatin was eluted with 200 μL of elution buffer  at 65°C for 10 minutes, and cross‐links were reversed by incubating the samples at 65°C overnight. Chromatin was then incubated with proteinase K (5 μg/mL), yeast tRNA (0.5 μg/mL) and TE [pH 7.6] at 37°C for 2 hours before 50 μL of 4 M LiCl was added, and DNA was purified by phenol and chloroform extraction. Purified DNA was resuspended in 30 μL of Tris‐EDTA (pH 8.0) and 3 μL of RNase A (10 mg/mL). To amplify promoter sequences of PRMT5 target genes, 6 μL of eluted DNA was used in a 20‐μL real‐time PCR reaction mixture containing the following primer pairs and specific Roche Applied Science universal probes .Chromatin immunoprecipitation was carried out as described previously.2.54 cells resuspended in 500 μL serum‐free medium with or without PRMT5 inhibitor were added into the upper well of Boyden chamber. The lower chamber was filled with 1.3 mL of culture medium containing serum for 24 or 48 hours. After incubation, cells that migrated to the lower surface of the upper chamber were fixed by treating them first with 4% formaldehyde, followed by absolute methanol, and then cells were stained with 1% Crystal violet. Images from 4 random fields were captured using an inverted microscope, and cells were counted using ImageJ software.Boyden migration assay was carried out using a 24‐well plate with polycarbonate transwell inserts of pore size 8.0 μm . Boyden invasion assay was conducted using the BD Bio‐Coat Matrigel system. Approximately 3 × 102.64 cells were seeded into a 24‐well plate, and 24 hours later, cells were treated with or without PRMT5 inhibitor for different time intervals. Next, cells were collected, resuspended in 20‐μL solution containing equal volume of culture medium and Trypan blue dye, and counted in order to determine the number of viable cells.Approximately 2 × 102.74 cells were seeded in a 6‐well plate prior to treatment with or without inhibitor for 24 or 48 hours. Next, cells were harvested, washed and resuspended in 1× Annexin binding buffer containing Annexin V‐FITC and PI, and then incubated for 15 minutes in the dark at room temperature. Stained cells were analysed with BD LSRFortessa flow cytometer using FACS Diva software (Becton Dickinson).Approximately 6 × 102.82O2) and colour reagent B (Tetramethylbenzidine) was added before incubating at room temperature in the dark for 20 minutes. After incubation, 50 μL of stop solution (2N H2SO4) was added, and the optical density of each well was read at 450 nm .Levels of secreted antagonists of the WNT/β‐CATENIN pathway were detected by ELISA technique using DuoSet ELISA kit (R&D system). Essentially 100 μL of capture antibody was added to 96‐well culture plate and incubated at room temperature overnight. Next, the antibody solution was removed, and wells were washed twice with 400 μL of wash buffer (0.005% Tween‐20 in filter‐sterilized 1× PBS). Wells were blocked by incubating with 300 μL of blocking buffer (1% BSA in filter‐sterilized 1× PBS) for 1 hour at room temperature. Blocked wells were washed twice with wash buffer before adding 100 μL of either specific standard protein or sample solution. Wells were sealed with adhesive plastic strips, incubated at room temperature for 2 hours before washing twice with wash buffer. Next, 100 μL of Streptavidin‐HRP A was added to each well and incubated in the dark for 20 minutes at room temperature. Wells were then washed, and 100 μL of Substrate solution containing 1:1 mixture of colour reagent A  in MCF7, 9.9‐fold (P < 10−3) in HCC1937 and 3.7‐fold (P < 10−3) in BT549 cells compared with normal human mammary epithelial cells (HMECs)  Figure . In acco) Figure . These rAXIN2 and WIF1.CYCLIN D1, c‐MYC and SURVIVIN increases.CYCLIN D1 was increased by 2.5‐fold (P < 10−3) in MCF7, and 2.4‐fold (P < 10−3) in HCC1937 compared with control HMECs  in MCF7, twofold (P < 10−3) in HCC1937 cells and 1.8‐fold (P < 10−3) in BT549 cells  in all three tested cell lines. Similarly, SURVIVIN mRNA was enhanced by 12.9‐ to 49.3‐fold (P < 10−3) in MCF7, HCC1937 and BT549 cell lines compared with normal HMECs , while AXIN2 mRNA levels were augmented by 25.9‐ to 27.3‐fold, (P < 10−3) in MCF7, HCC1937 and BT549 cells compared with normal HMECs. Similarly, WIF1 also showed increased mRNA levels in MCF7 cells , HCC1937  and BT549 cells  compared to normal HMECs  and BT549 cells   and 21.6‐fold (P < 10−3), respectively. In contrast, DKK2 mRNA levels were elevated by 6.9‐fold (P < 10−3) in BT549. DKK3 mRNA expression was undetectable in MCF7 and HCC1937 cells and was reduced by 38.6‐fold (P < 10−3) in BT549 cells. Expression of DKK4 mRNA was unaffected in MCF7 and BT549 cells compared with normal HMECs and was up‐regulated by 2.4‐fold (P < 10−3) in HCC1937 cells  Figure . DKK2 mRs Figure .SFRP2 mRNA was unaffected in MCF7 and HCC1937 cells compared with control HMECs . Two other members of the SFRP family, SFRP3 and SFRP5, showed substantial increase  in their mRNA levels in breast cancer cells compared with normal HMECs. Expression of SFRP4 mRNA in MCF7 cells did not differ from normal HMECs; however, its level was elevated in HCC1937  and reduced in BT549 cells  , we found that s Figure . This wa) Figure .DKK1 and DKK3 showed significant reduction in their mRNA levels in both TNBCs, HCC1937 and BT549, we evaluated their protein levels in both normal and transformed breast cells. In accord with our RT‐PCR results, Western blot analysis showed that both DKK1 and DKK3 proteins were suppressed in HCC1937 and BT549 compared with normal HMECs  experiments were carried out. Cross‐linked chromatin from MCF7, HCC1937 and BT549 cells was immunoprecipitated with either pre‐immune, anti‐PRMT5, anti‐H3(Me2)R8 or anti‐H4(Me2)R3 antibodies, which were previously characterized and shown to be highly specific.transcriptional repression of pathway antagonistsIn light of our recent work, which showed that PRMT5 epigenetically represses transcription of WNT/β‐CATENIN antagonists 2)R8 and H4(Me2)R3 epigenetic marks at the DKK1 promoter in MCF7 cells  increase in PRMT5 binding to the promoter region of DKK3, which was accompanied by enhanced symmetric methylation of H3R8  and H4R3  in MCF7 cells  and elevated methylation of H3R8  and H4R3  at the DKK1 promoter  were observed in the promoter region of DKK3 , whereas PRMT5 binding to the promoter region of DKK3 was increased by 2.5‐fold (P = .038) in BT549 cells R8  and H4(Me2)R3  were detected in the promoter region of DKK1. Furthermore, symmetric methylation of H3R8  and H4R3  was also detected in the promoter region of DKK3  at lower concentrations of CMP5 (3 μmol/L to 9 μmol/L) and was significantly up‐regulated by 24‐fold (P < 10−3) at 12 μmol/L in BT549 cells  in the presence of 12 μmol/L of CMP5 in BT549 cells , respectively .DKK1 and DKK3 promoters in HCC1937 cells. To address this question, we treated HCC1937 cells with the DNA methyltransferase inhibitor, 5‐Azacytidine (5‐Aza). HCC1937 cells were incubated with increasing amounts of 5‐Aza for either 24, 48 or 72 hours, and viability was determined by Trypan blue dye exclusion assay, which showed that even a higher concentration of 5‐Aza (24 μmol/L) does not have significant cytotoxicity on HCC1937 cells (data not shown). Next, we examined the effect of 5‐Aza (24 μmol/L) alone or in combination with suboptimal amount of CMP5 (9 μmol/L), on transcriptional derepression of DKK1 and DKK3 in HCC1937 cells. Treatment of HCC1937 cells with 24 μmol/L of 5‐Azacytidine for 48 hours did not impact transcription of DKK1  and DKK3 . Furthermore, sequential incubation of HCC1937 cells with 24 μmol/L of 5‐Aza first for 24 hours, followed by addition of 9 μmol/L CMP5 for another 24 hours also did not induce significant transcriptional derepression of DKK1  and DKK3   Figure .DKK1 and DKK3, we treated HCC1937 cells with Trichostatin A (TSA). Real‐time RT‐PCR analysis showed that treatment with either 100 nmol/L or 300 nmol/L of TSA for 24 hours had no effect on DKK1 and DKK3 transcription in HCC1937 cells. Similarly, combinatorial treatment with 9 µmol/L of CMP5 and either 100 or 300 nmol/L of TSA for 24 hours also failed to induce transcriptional activation of WNT antagonists, DKK1 and DKK3 in these cells (data not shown).Deacetylation of histone lysine residues by histone deacetylase enzymes (HDAC) has been shown to be essential for transcriptional silencing of genes.DKK1 and DKK3 in HCC1937 cells. We incubated HCC1937 cells with a combination of suboptimal doses of TSA (100 nmol/L) and 5‐Aza (10 μmol/L) for 24 hours  and 2.5‐fold (P < 10−3), respectively , suggesting that combinational treatment with all three inhibitors was not able to induce further expression of DKK1 and DKK3 in these cells.Previous studies have shown that simultaneous inhibition of histone deacetylase enzyme and DNA methyltransferase enzyme results in restoration of epigenetically silenced genes.s Figure . Real‐tiy Figure . As histDKK1 and DKK3 expression was not affected  and those of DKK3 were elevated by 3.9‐fold (P < 10−3) , whereas DKK3 mRNA was derepressed by 4.5‐fold (P < 10−3)  and threefold (P < 10−3), respectively  Figure . Further) Figure . Similary Figure . Collect3.4DKK1 and DKK3, we wished to evaluate expression of WNT/β‐CATENIN target genes, c‐MYC, CYCLIN D1 and SURVIVIN. HCC1937 treatment with 12 μmol/L of CMP5 for 24 hours triggered reduction in mRNA levels of CYCLIN D1 and SURVIVIN by 1.8‐fold (P < 10−3) and 2.8‐fold (P < 10−3), respectively  and SURVIVIN mRNAs , after 48 hours incubation with 12 μmol/L of CMP5  upon treatment of HCC1937 cells with 9 μmol/L and 12 μmol/L of CMP5 for 24 hours  and 3.7‐fold (p < 10−3) treatment with 9 μmol/L and 12 μmol/L of CMP5 for 48 hours, respectively  and 2.3‐fold (P < 10−3) in the promoter regions of DKK1 and DKK3, respectively, in control DMSO‐treated BT549 cells. Consistent with these results, symmetric methylation of H3R8 was enriched by 9.6‐ (P < 10−3) and 7.1‐fold (P < 10−3) in the promoter of DKK1 and DKK3, respectively. Similarly, methylation of H4R3 histones in the promoter region of DKK1 and DKK3 was increased by 2.6‐ to 6.3‐fold (P < 10−3). When BT549 cells were treated with CMP5, there was a 3.4‐fold (P < 10−3) decrease in PRMT5, which was accompanied by a 2.6‐fold (P < 10−3) and 1.5‐fold (P = .072) reduced methylation of H3R8 and H4R3, respectively, in the DKK1 promoter , and H3R8 and H4R3 methylation by twofold, (P < 10−3) and 6.6‐fold (P < 10−3), respectively, in the DKK3 promoter region  and 5.2‐fold (P < 10−3), respectively. Consistent with this result, symmetric methylation of H3R8 was enhanced by 7.06‐ to 10.2‐fold (P < 10−3) on both promoters, respectively. Similarly, symmetric methylation of H4R3 was increased by 2.8‐ and 2.5‐fold (P < 10−3) on DKK1 and DKK3 promoters, respectively. Treatment of HCC1937 cells with CMP5 reduced enrichment of PRMT5 by 2.3‐fold (P < 10−3) and decreased methylation of H3R8 and H4R3 by 2.8‐fold (P < 10−3) and 2.1‐fold (P = .007) at the DKK1 promoter, respectively  and symmetric methylation of H3R8 by 2.6‐fold (P < 10‐3) at the DKK3 promoter in HCC1937 cells. However, there was no reduction in symmetric methylation of H4R3 at the DKK3 promoter upon treatment of HCC1937 cells with CMP5 , 39% (P < 10−3) and 61.2% (P < 10−3) after 24, 48 and 72 hours, respectively, compared to control DMSO‐treated cells. Treatment with 16 μmol/L of CMP5 showed significant decrease in viability by 18% (P < 10−3), 46.9% (P < 10−3) and 69.1% (P < 10−3) after 24, 48 and 72 hours, respectively. Based on these results, we found that CMP5 had a LC50 value of 15.46 µmol/L at 48 hours for MCF7 cells , 72.2% (P < 10−3) and 88.5% (P < 10−3), respectively. HCC1937 cell viability was substantially reduced by 82% (P < 10−3) after 24 hours treatment with 16 μmol/L of CMP5, and all cells were dead  after 48 and 72 hours treatment. Data analysis showed that CMP5 had a LC50 of 11.7 μmol/L after 24 hours treatment for HCC1937 cells , 38% (P < 10−3) and 55.6% (P < 10−3) after 24, 48 and 72 hours post‐treatment, respectively. In addition, treatment with 16 μmol/L of CMP5 reduced viability of BT549 cells by 65.5% (P < 10−3) and 82% (P < 10−3) after 24 and 48 hours, respectively. Treatment for 72 hours with 16 μmol/L of CMP5 resulted in complete loss of cell viability. When we plotted the results, we found that CMP5 had a LC50 of 10.35 µmol/L at 48 hours for BT549 cells  for either 24, 48 or 72 hours   Figure . To asse9 Figure .P < 10−3) and 57.1% (P < 10−3) cell death, respectively  and 60.3% (P < 10−3) cell death  that is overexpressed in many cancers, results in transcriptional derepression of DKK1 in colorectal cancer cells.DKK1 in MCF7 cells.Epigenetic silencing of WNT/β‐CATENIN antagonists is frequently observed in breast cancer, and reduced expression of DKK1 and DKK3, we inhibited its activity and monitored expression of both WNT/β‐CATENIN antagonists. We found that PRMT5 inhibition brings about DKK1 and DKK3 transcriptional derepression, which correlates with reduced expression of WNT/β‐CATENIN target genes CYCLIN D1 and SURVIVIN in TNBCs  showed that when expressed in the absence of proliferation signals, c‐MYC induces cell death.et al. (2008) showed that distinct c‐MYC expression levels regulate proliferation and apoptosis.To evaluate the contribution of PRMT5 to transcriptional regulation of  Figures . HoweverDKK1 and DKK3 in HCC1937 and BT549 cell lines, and that its inhibition only restores expression of both WNT/β‐CATENIN antagonists in BT549 cells. CMP5 treatment did not induce transcriptional derepression of DKK1 and DKK3 in HCC1937 cells, indicating that there are other epigenetic modifications involved in their regulation . However, combinatorial treatment of HCC1937 cells with CMP5, 5‐Aza and TSA resulted in increased transcriptional derepression of DKK1 and DKK3, indicating that histone lysine deacetylation, histone arginine methylation and DNA methylation work in concert to epigenetically silence DKK1 and DKK3  studies, it was confirmed that PRMT5 binds to the promoter region of WNT antagonists, DKK1 and DKK3, and induces symmetric methylation of H3R8 and H4R3 histones. Furthermore, PRMT5 inhibition using a specific small molecule inhibitor, compound 5 (CMP5), reduced PRMT5 recruitment as well as methylation of H3R8 and H4R3 histones in the promoter regions of DKK1 and DKK3, which consequently results in reduced expression CYCLIN D1 and SURVIVIN. In addition, CMP5 treatment either alone or in combination with 5‐Azacytidine and Trichostatin A restored expression of DKK1 and DKK3 in TNBCs. PRMT5 inhibition also altered the growth characteristics of TNBC cells including proliferation, migration and invasion, and induced their death.Protein arginine methyltransferase 5 (PRMT5) activity is dysregulated in many aggressive cancers and its enhanced levels are associated with increased tumour growth and survival. The current study was focused on investigating the role of PRMT5 in promoting breast tumorigenesis. The results of the study showed that PRMT5 is overexpressed in breast cancer cell lines of varying aggressiveness, and that it promotes their growth by activating WNT/β‐CATENIN proliferative signalling through epigenetic silencing of pathway antagonists, The authors declare that they have no conflict of interest with the contents of this article.Harshita shailesh: Formal analysis (lead); Methodology (lead); Software (lead); Validation (supporting); Writing‐original draft (lead); Writing‐review & editing (supporting). Siveen Kodappully Sivaraman: Formal analysis (supporting); Investigation (supporting); Resources (supporting). Saïd Sif : Conceptualization (lead); Data curation (lead); Formal analysis (lead); Funding acquisition (lead); Investigation (lead); Project administration (lead); Resources (lead); Supervision (lead); Writing‐review & editing (lead).


Obesity is a common disease over the world and is tightly associated with diabetes mellitus, cardiovascular and cancer disease. Although our previous study showed that the synthetic vanadium-protein (V-P) complex had a better effect on antioxidant and antidiabetic, the relative molecular mechanisms are still entirely unknown. Hence, we investigated the effect of the synthetic V-P complex on adipocyte differentiation (adipogenesis) using human preadipocytes to clarify its molecular mechanisms of action. The primary human preadipocytes were cultured with and without V-P complex during adipocyte differentiation. The cell proliferation, lipid accumulation, and the protein expression of transcription factors and related enzymes were determined for the differentiated human preadipocytes. In this study, the 20 μg/mL of V-P complex reduced the lipid and triglyceride (TG) content by 74.47 and 57.39% (p < 0.05), respectively, and down-regulated the protein expressions of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element-binding protein 1 (SREBP-1) and fatty acid synthase (FAS). Additionally, the V-P complex significantly up-regulated the protein levels of total β-catenin (t-β-catenin), nuclear β-catenin (n-β-catenin), phosphorylated adenosine monophosphate-activated protein kinase alpha (p-AMPKα) and liver kinase B1 (p-LKB1). These showed that the inhibitory effect of V-P complex on human adipogenesis was mediated by activating Wnt/β-catenin and LKB1/AMPK-dependent signaling pathway. Therefore, the synthetic V-P complex could be considered as a candidate for prevention and treatment of obesity. Obesity is not only a disease between adult males and females but also a worldwide epidemic , 2. It iIt has been demonstrated that many pathways such as Wnt/β-catenin, and LKB1/AMPK signaling pathway involved in the regulation of adipocyte differentiation through the regulation of transcription factors PPARγ and C/EBPα 10, 12]. In the Wnt/β-catenin signaling, Wnt can affect the adipocyte differentiation based on the β-catenin-dependent  and β-catenin-independent  mechanisms, in which β-catenin plays a central role in inhibiting adipogenesis . LKB1 an, 12. In in vivo and in intro. Inorganic vanadium compounds including ammonium metavanadate (AMV) and sodium orthovanadate showed insulin-like action in female wistar rats -2Htetrazolium disodium salt] (EZ-cytox assay kit) was purchased from DoGenBio Co., Ltd , 4% Paraformaldehyde (PFA) in PBS was purchased from Thermo Fisher Scientific . Preadipocyte medium (PM) and adipocyte differentiation medium (DM) were purchased from Zenbio . Vandal sulfate (VS) was purchased from Daejung Chemical & Materials Co. Ltd .The V-P complex was synthesized according to the method of Zhang and Kim . Briefly2. Human preadipocytes were grown in PM at 37 oC in a humidified 5% CO2 atmosphere . Upon reaching confluence, the culture media were replaced with DM and considered as Day “0”. Subsequently, the culture media were changed every two days until the cells were harvested. Human preadipocytes were treated with V-P complex during differentiation from day 0 until the cells were harvested for analysis.Cryopreserved primary human preadipocytes (Item # SP-F-SL) were purchased from Zenbio , which derived from subcutaneous adipose tissues of five female donors with an average age of 47.2 years and average body mass index of 28.1 kg/m4 cells/ mL) were seeded into 96-well plates and treated at day 0 with different concentrations of V-P complex . After culturing for 5, 10, 14 days, 10 μL of WST was added to each well and then the cells were incubated at 37 oC for 4 h. The absorbance at 450 nm was measured using a microplate reader . The viability (%) was calculated by the following equation:Cell viability was assayed with the EZ-cytox assay kit according to the manufacturer’s instruction. Human preadipocytes  in 96-well plates during differentiation. Cellular TG content was measured with the triglyceride assay kit according to the manufacturer’s instruction. The TG content was calculated by the following equation:oC overnight, followed by incubating with the secondary antibody (1:2000) (horseradish-peroxidase-conjugated anti-rabbit antibody) at room temperature for 1 h. The immune reactive bands were visualized by ECL reagents . Imaging and densitometry were analyzed using a ChemiDoc XRS+ System with Image Lab Software .Total proteins were extracted from the human preadipocytes treated with V-P complex at day 14. The differentiated human preadipocytes were washed with cold PBS and lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitor . Protein samples (25 μg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked at room temperature for 1 h in 5% non-fat skim milk, and then incubated with the primary antibodies (diluted 1:2000) in tris-buffered saline with Tween-20 (TBST) at 4 All experiments were performed in triplicate, and the data were presented as mean values ± SD. A one-way analysis of variance (ANOVA) and Duncan’s test with a significant threshold set at 0.05 (p < 0.05) were used to evaluate the differences using SPASS software . All figures were made using GraphPad Prism version 7.0 for Mac .The differentiation of preadipocytes leads to an increase in adipocyte number which is closely associated with obesity . The groin vitro  ([oda = O(CH2COO-)2]) and [VO(IDA)phen]·2H2O were synthesized and characterized [2·-) and organic radicals (ABTS·+ and DPPH·) [Actually, vanadium compound has attracted much attention in the past decade due to it is considered as one of the promising antioxidant and antidiabetic agents , 45, 46.d DPPH·) . The newd DPPH·) . Additiod DPPH·) . The stuIt has been reported that the vanabin1 (12.5 kDa) and vanabin2 (15 kDa) formed by recombinant protein can bind 10 and 20 vanadium ions, respectively . The difIn the present study, the effect of synthetic V-P complex on adipocyte differentiation in primary human preadipocytes was investigated. The data demonstrated that the V-P complex reduced lipid accumulation and TG content in a dose-dependent manner in differentiated human preadipocytes. It was observed that V-P complex was effective in suppressing adipogenesis by down-regulating the protein expression of adipogenic transcription factors PPARγ, C/EBPα, SREBP-1 as well as by down-regulating the protein expression of adipogenesis-related gene FAS. Moreover, V-P complex also inhibited adipocyte differentiation in primary human preadipocytes through activating Wnt/β-catenin and LKB1/AMPK signaling pathways. Additionally, our findings indicated that synthetic V-P complex could be used as the nutraceutical in management of diabetes or obesity.S1 Fig(PDF)Click here for additional data file.


Aberrant activation of a Wnt/β-catenin pathway results in nuclear accumulation of β-catenin in colon cancer. Inhibiting β-catenin is one strategy for treating colon cancer. Here, we identified Z-ajoene, a sulfur containing compound isolated from crushed garlic, as an inhibitor of colon cancer cell growth. Z-Ajoene repressed β-catenin response transcriptional activity, intracellular β-catenin levels, and its representative target protein levels (c-Myc and cyclin D1) in SW480 colon cancer cells. To clarify the regulatory mechanism of decreased β-catenin levels, we examined the effect of Z-ajoene on β-catenin phosphorylation, which is involved in β-catenin degradation. Z-Ajoene promoted the phosphorylation of β-catenin at Ser45 in a casein kinase 1α (CK1α)-dependent manner, which is an essential step in β-catenin degradation in the cytosol. These findings indicate that Z-ajoene from garlic may be a potential chemotherapeutic agent by modulating CK1α activity and the Wnt/β-catenin signaling pathway. Colon cancer is a common cancer associated with high incidence and high mortality . About 7Allium sativum L., Amaryllidaceae) is one of the most important spices from ancient times and has been intensively studied for therapeutic properties, such as antithrombotic [E- and Z-isomers  is found in crushed garlic and has a broad variety of biological activities. Previously, we reported the anti-inflammatory [Garlic  is responsible for the phosphorylation of β-catenin at Ser45 . TherefoWe confirmed the inhibitory effect of Z-ajoene on the Wnt/β-catenin signaling pathway using a Wnt3a-CM-activated HEK293 reporter cell system. Z-Ajoene repressed intracellular levels of β-catenin without changing the transcriptional expression of β-catenin. In addition, Z-ajoene suppressed TOPFlash reporter activity in LiCl (GSK3β inhibitor) activated HEK293 reporter cells, which means that inhibitory activity of Z-ajoene on Wnt/β-catenin signaling pathway is not involved in GSK3β activity. Consistent with the effects of Z-ajoene in SW480 colon cancer cells, phosphorylated β-catenin was increased by Z-ajoene. A CK1 inhibitor reversed the effect of Z-ajoene treatment in HEK293 reporter cells by increasing β-catenin/TCF activity. These results suggest that Z-ajoene works as an inhibitor of the Wnt/β-catenin signaling pathway by the amplification of CK1α-dependent β-catenin phosphorylation .Taken together, the results indicate that Z-ajoene inhibited the Wnt/β-catenin signaling pathway by downregulating intracellular β-catenin levels through the promotion of CK1α-dependent phosphorylation of β-catenin, resulting in the suppression of SW480 colon cancer cell growth.Z-Ajoene was isolated and the structure was elucidated as described previously .SW480 cells, human colorectal adenocarcinoma cells with truncated APC, was obtained from the Korea Cell Line Bank  and cultured in DMEM with 10% fetal bovine serum (FBS). HEK293 reporter cells  and control cells  were kindly provided by Sangtaek Oh . Wnt3a-secreting L cells (L-Wnt3a cells) were obtained from the American Type Culture Collection . The TOPFlash reporter plasmid was transfected into SW480 cells using Lipofectamine PLUS reagent  according to the manufacturer’s instructions. After 24 h transfections, cells were treated with Z-ajoene for 15 h. The luciferase activity of the SW480 cell lysates was measured by a luminometer .Wnt3a-CM was prepared according to the manufacturer’s instruction. Wnt3a-secreting L cells were cultured in DMEM with 10% fetal bovine serum (FBS) for four days. The medium was harvested and followed by sterilization using a 0.22-μm filter. Fresh medium was added to the cells and cultured for another three days. The collected medium was combined with the previous medium.The HEK293 reporter cells (with TOPFlash reporter plasmid) or control cells (with FOPFlash reporter plasmid) were inoculated at 1000 cells/well and grown for 24 h. After 15 h incubation with Z-ajoene and/or Wnt3a-CM, luciferase activity in the cell lysates was measured by a luciferase assay system .3 cells/well) were treated with Z-ajoene (various concentrations) for 24, 48, and 72 h, and the cell viability was measured in triplicate using a 3--2,5-diphenyl tetrazolium bromide (MTT) method. Cells . The cell lysates were then centrifuged at 10,000× justment . Protein6 cells/dish) were treated for 15 h with/without Z-ajoene. Total RNA was isolated using RNA-isolation Trizol reagent  and then reverse transcribed into cDNA using reverse transcriptase and random hexamer. The cDNA was mixed with buffer, dNTP, Taq DNA polymerase  and primers. The sense and antisense primers for β-catenin were 5′-GGGATGTTCACAACCGAATTGTTATC-3′ and 5′-ACCAGAGTGAAAAGAACGATAGCTAGGA-3′, respectively. The sense and antisense primers for β-actin were 5′-TGTGATGGTGGGAATGGGTCAG-3′ and 5′-TTTGATGTCACGCACGATTTCC-3′, respectively. The amplified DNA was separated on 1.5 % agarose gels and stained with ethidium bromide.Cells (1 × 10p-value of < 0.05 was considered to indicate a significant difference.The data were expressed as the mean ± S.D. of three experiments, and statistical analysis was performed by the Student’s t-test. A


Arabidopsis thaliana (AtGOX1 and AtGOX2). Phosphorylation sites at T4, T158, S212 and T265 were selected and studied using Arabidopsis and maize recombinant glycolate oxidase (GOX) proteins mutated to produce either phospho-dead or phospho-mimetic enzymes in order to compare their kinetic parameters. Phospho-mimetic mutations  led to a severe inhibition of GOX activity without altering the KM glycolate. In two cases (T4D and T158D), this was associated with the loss of the cofactor, flavin mononucleotide. Phospho-dead versions exhibited different modifications according to the phospho-site and/or the GOX mutated. Indeed, all T4V and T265A enzymes had kinetic parameters similar to wild-type GOX and all T158V proteins showed low activities while S212A and S212D mutations had no effect on AtGOX1 activity and AtGOX2/ZmGO1 activities were 50% reduced. Taken together, our results suggest that GOX phosphorylation has the potential to modulate GOX activity.In photosynthetic organisms, the photorespiratory cycle is an essential pathway leading to the recycling of 2-phosphoglycolate, produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase, to 3-phosphoglycerate. Although photorespiration is a widely studied process, its regulation remains poorly understood. In this context, phosphoproteomics studies have detected six phosphorylation sites associated with photorespiratory glycolate oxidases from  This has led to efforts to minimize the negative effects of photorespiration to improve plant yield by producing plants containing a chloroplastic bypass to metabolize photorespiratory glycolate . According to transcriptomic analyses, At3g14415 (AtGOX2) and At3g14420 (AtGOX1) are highly expressed in leaves and they represent the major GOX isoforms in Arabidopsis thaliana [AtGOX3 is mainly expressed in senescing leaves and roots, where it has been proposed to also function as a lactate oxidase and thus play a role in lactate metabolism [AtHAOX1 and AtHAOX2 are expressed in seeds and encode proteins preferring medium- and long-chain hydroxyl acids as substrates [GOX gene do not exhibit a photorespiratory growth phenotype although they were all more sensitive to Pseudomonas syringae and to ozone [2 (3000 ppm) was observed however in an Arabidopsis artificial miRNA GOX line (amiRgox1/2) with both AtGOX1 and AtGOX2 knocked-down and only 5%–10% of wild-type Arabidopsis leaf GOX activity [amiRgox1/2 plants from high CO2 to ambient air led to a 700-fold accumulation of glycolate and a reduced carbon allocation to sugars, organic acids and amino acids that induced an early senescence of older leaves [AtGOX1 and AtGOX2 showed a redundant photorespiratory function, AtGOX1 appeared to have a more predominant role in photorespiration since it attenuated the phenotype of a cat2 mutant [2 assimilation rate [Photorespiration begins with the fixation of Oes being  is normaes being . In Arabthaliana . AtGOX3 to ozone ,10. A phactivity . The tra2 mutant . The impion rate ,13,14,152O2 production by GOX [AtGOX1 and AtGOX2, several phosphorylation sites  have been reported . Indeedn by GOX  as was An by GOX . The glyn by GOX . Arabidon by GOX ,23. Protn by GOX . Concernrted see .AtGOX1 and AtGOX2 enzymatic activities and kinetic properties were analysed using purified recombinant wild-type, phospho-dead and phosphorylation-mimetic GOX proteins. This was also carried out to test the equivalent residues  of maize photorespiratory GOX (ZmGO1) [In this study, the consequences of T4, T158, S212 and T265 phosphorylation on AtGOX1 and AtGOX2 from published phosphoproteomics studies and the PhosPhAt 4.0 database (http://phosphat.mpimp-golm.mpg.de/)  see . In thish AtGOX1 .AtGOX1, AtGOX2 and AtGOX3 proteins while T4 and T158 were replaced by a valine in AtHAOX1 and AtHOAX2 , whereas T4 was conserved only in the plant GOX proteins while T158 was conserved in all GOX proteins, human HAOX2, Aerococcus viridians LOX and Lactococcus lactis LOX  [AtGOX1 and AtGOX2 were chosen for this study that corresponded to T5, T159, S213, T266 of ZmGO1  were replaced by an alanine while T4/5 and T158/159 were changed to a valine to also mimic the sequence of AtHOAX1 and AtHOAX2 (and Homo sapiens HAOX2 for the T4 position)  and kcat  values  the kcat of the three recombinant GOX enzymes without significantly altering the Km glycolate  but a 4–5 fold decreased kcat compared to their wild-type GOX counterparts  were inactive   so as to) values . The T4/lycolate . Similar and 95% . AtGOX1Tterparts . When thinactive . Perhapslycolate .AtHOAX1 and AtHAOX2 proteins preferentially use long-chain hydroxy-acids as substrates although AtHOAX1 can also quite efficiently use both lactate and glycolate [WT proteins were compared and as previously reported, the Km L-lactate was higher than the Km glycolate [AtGOX1 and AtGOX2 while only a 4-fold difference was seen for ZmGO1, and surprisingly, the calculated kcat values for the lactate oxidase reaction were not significantly different when compared to the glycolate oxidase activities , while the Km 2-hydroxyoctanoate for ZmGO1 remained unchanged  using glycolate as a substrate, although ZmGO1 appeared to be less selective.lycolate . Since tlycolate . First, lycolate  with an tivities ; Table 3M and kcat (T158/159V) and activity (T158/159D) using either L-lactate or 2-hydroxyoctanoate when compared to glycolate  while the KM 2-hydroxyoctanoate of AtGOX2T4D and ZmGO1T5D was increased by 3.5-fold and 3-fold, respectively, when compared to their corresponding GOXWT proteins  phospho-site mutations on the calculated kinetic parameters of the lactase oxidase and 2-hydroxyoctanoate oxidase reactions was compared. It was found that T158V/D and T159V/D mutations led to similar effects on Klycolate . For thelycolate . As seenwas used . In thesproteins . When thobserved . Therefo280nm/450nm), it was possible to determine the presence of FMN in the purified GOX proteins used to measure enzymatic activities. An example of absorption spectra of WT and T158/159 mutated GOX proteins highlights the typical spectra associated with FMN that was missing in the GOXT158/159D forms thereby indicating an absence of cofactor  and loop6  together with a loop situated between residues 28–33 (27–32 for AtGOX) formed a lid preventing the loss of FMN [NbGOX, with α-helix4 (next to loop4) forming H-bonds with E3, T5 and N6  allowing a cooperative mechanism in FMN binding between GOX subunits of the same tetramer [Arabidopsis thaliana GOX1 and GOX2 as well as ZmGO1 based on the structure of Spinacia oleracea GOX (PDB 1AL7) indicated that T4 formed H-bonds with R163, E165 and K169 of α-helix4 of a neighbouring GOX subunit.Phospho-mimetic GOX proteins exhibited different degrees of inhibition of their glycolate oxidase activity, except in one case, AtGOX1activity . This inactivity , Table 4proteins . These ms of FMN   the resulting protein had only 3.5% of GOXWT activity and a kcat of only 0.74 s−1 instead of 20 s−1; however, FMN content was not affected [When T4 was replaced by an aspartate in these models, only an H-bond with D163 of α-helix4 was present. Moreover, T158 is located close to a residue implicated in FMN-binding (T155) ,32, the affected . Therefocat of AtGOX1/2T265D and ZmGO1T266D compared to the wild-type recombinant protein but only changed the KM glycolate of AtGOX1T265D  which is part of the “lid” structure involved in FMN loss in the pH sensor model [T265/T266D inhibited glycolate oxidase activity by bringing about a conformational change that interfered with catalysis via the displacement of H254.The mutation of T265/266 to aspartate decreased the glycolate-dependent kOX1T265D . As ment protein  even thoor model . Again, AtGOX1 versus AtGOX2 and AtGOX1/2 versus ZmGO1) did not always respond in a similar manner thus suggesting subtle differences between them even though they have a conserved photorespiratory role in planta. Both mutations at S212/S213 led to a similar decrease in the glycolate-dependent kcat of AtGOX2 and ZmGO1 while the kcat of AtGOX1 remained unaltered .yl-acids , and HsHly shown , AtGOX1 lycolate . Howeverlycolate . Intereslycolate . Thus, eeaction   . In suchs tested , even whactivity . Thus, Tactivity ,35,36, aeatments . SnRK2.2eatments  and we heatments . Thus, Geedlings .2/O2, and stresses like heat, drought, salt and high light as well as pathogen attack. To simplify the detection of GOX phosphopeptides by mass spectroscopy, GOX should be specifically immunoprecipitated from soluble protein extracts at different times during the stress treatments or during the day/night cycle. In this way, it should be possible to identify conditions where GOX phosphorylation is modulated.Therefore, phosphoproteomics studies have had very limited success in providing insights into the eventual role of GOX phosphorylation. Since this post translational modification is a rapid and reversible response to environmental stimuli, experiments should be conducted to identify when GOX is differentially phosphorylated. With respect to GOX photorespiratory function, different conditions expected to modulate photorespiratory flux should be examined such as day/night cycle, variable COEscherichia coli as a recombinant phospho-mimetic protein and therefore we do not know whether this led to a non-physiological alteration of GOX structure (and activity). Since the modification was constitutive and present as soon as the protein was synthesized, it does not reflect a reversible phosphorylation of a protein. For instance, is the absence of FMN in GOXT4/5D and GOXT158/159D due to structural changes induced by the aspartate that inhibits FMN entry into the apo-protein to form the halo-protein, and therefore would phosphorylation lead to changes that favour FMN removal from the halo-protein? Furthermore, phospho-mimetic mutations do not always lead to the modifications in protein function brought about by an actual phosphorylation. Thus, to assess the real role of GOX phosphorylation, identification of the protein kinases  involved would be crucial. Based on the SUBA database (http://suba.plantenergy.uwa.edu.au/), 33 protein kinases are predicted to be addressed to the peroxisome. Using machine learning methods to identify proteins carrying plant peroxisomal PST1 targeting signals, 11 protein kinases were identified as being addressed to the peroxisome [AtGOX1/2 kinases could be to retrieve knock-out mutant lines for predicted and/or verified peroxisomal protein kinases and compare AtGOX1/2 phosphorylation status by mass spectroscopy after AtGOX1/2 immunoprecipitation from soluble proteins extracted from wild-type and mutant plants treated to previously identified conditions that induce AtGOX1/2 phosphorylation.Of course, our in vitro data using recombinant phospho-mimetic GOX proteins only give an indication of what GOX phosphorylation might actually be doing with respect to enzyme activity. Indeed, GOX was produced in roxisome . More reroxisome . A stratZmGO1 did appear to have a relaxed substrate specificity when compared to C3-plant AtGOX1 and AtGOX2.In conclusion, photorespiratory GOX has the potential to be regulated by protein phosphorylation at several distinct sites. In general, phospho-mimetic recombinant GOX proteins exhibited reduced activities and this could be explained by predicted changes in structural interactions affecting key residues involved in FMN binding and catalysis. Phospho-mimetic GOX did not show any alteration in substrate specificity although C4-plant AtGOX1, pET28a-AtGOX2 and pET28a-ZmGO1 expression plasmids [® II XL site-directed mutagenesis kit , according to the manufacturer’s instructions. This strategy generated T4V, T4D, T158V, T158D, S212A, S212D, T265A, T265D (AtGOX1 and AtGOX2) and T5V, T5D, T159V, T159D, S213A, S213D, T266A, T266D (ZmGO1) mutated proteins. All constructions were subsequently verified by DNA sequencing using T7 and T7-term primers .Enzyme activities were measured using 5 µg of purified recombinant GOX in 50 mM Tris-HCl, 0.1 mM FMN, pH 8.0 and different glycolate (0.05 to 10 mM), L-lactate (0.3 to 10 mM) and 2-hydroxy-octanoate (0.15 to 5 mM) concentrations by an enzyme-coupled reaction at 30 °C. HAtGOX1, AtGOX2 and ZmGO1 3D-structures were designed based on the spinach GOX (UniProtKB: P05414) 3D-structure  using the SWISS-MODEL server (https://swissmodel.expasy.org/) modelling service. Manipulation of 3D-structures was realized using Deepview (Swiss Pdb-viewer)  and included the compute H-bond formation and mutation tools.


C. elegans that GABAergic motor neurons (D-MNs) bias toward the reward behavior in threat-reward decision-making by retrogradely inhibiting a pair of premotor command interneurons, AVA, that control cholinergic motor neurons in the avoidance neural circuit. This function of D-MNs is mediated by a specific ionotropic GABA receptor (UNC-49) in AVA, and depends on electrical coupling between the two AVA interneurons. Our results suggest that AVA are hub neurons where sensory inputs from threat and reward sensory modalities and motor information from D-MNs are integrated. This study demonstrates at single-neuron resolution how motor neurons may help shape threat-reward choice behaviors through interacting with other neurons.Proper threat-reward decision-making is critical to animal survival. Emerging evidence indicates that the motor system may participate in decision-making but the neural circuit and molecular bases for these functions are little known. We found in  Motor neurons are generally considered to be passive receivers of commands from other neurons. However, this study shows that motor neurons may shape locomotor behaviour by regulating premotor neurons, and that premotor neurons serve to integrate information from sensory neurons and motor neurons. In mice, locomotion modulates neuronal activities in the visual and auditory cortices8. In flies, activities of visual neurons are regulated by walking and flying behaviors10. In worms, head undulation regulates steering movements toward an olfactory attractant through sensorimotor integration12. Although a role of the motor system in decision-making is increasingly appreciated, little is known about the neural circuit and synaptic bases of this function.Perceptual decision-making is a cognitive process of choosing one action from various alternatives based on sensory information. The ability to produce appropriate choice behaviors in face of conflicting environmental factors, such as rewards and threats, is crucial to animal survival. The neural substrates for perceptual decisions may be broadly divided into three components: sensory system, decision-making system, and motor control system. Increasing evidence suggests that the motor system is not a mere passive recipient of commands from the decision-making system, but can interact with the sensory and decision-making systems to shape the final decision. For example, in humans and monkeys, the motor system is an integral component in oculomotor decision-makingC. elegans is a powerful model for investigating the circuit and gene bases of behavior because its complex behaviors are produced by a nervous system of only 302 neurons, for which a complete map of neural connections has been produced. In their natural environments and under laboratory conditions, worms navigate toward food and attractive chemical odors but avoid toxic chemicals and hyperosmotic conditions. The neural circuits mediating many sensory responses have been described. For example, the attractive odor diacetyl is detected by a pair of sensory neurons known as AWA14 whereas a noxious hyperosmotic condition caused by either glycerol or fructose is detected by a pair of sensory neurons known as ASH16. The AWA and ASH sensory neurons then activate downstream neural circuits mediating forward and backward movements, respectively19. Major components of the forward neural circuit include the bilateral pair of AVB premotor interneurons and B-type cholinergic motor neurons (B-MNs) whereas those of the backward circuit include the bilateral pair of AVA premotor interneurons and A-type cholinergic motor neurons (A-MNs)  to AVA27, physiological significance of this synaptic connection is unknown.The nematode Ns) Fig. 20–24. AVIn this study, we investigated synaptic interactions of D-MNs with threat and reward neural circuits, and with premotor interneurons and upstream cholinergic motor neurons using a multifaceted approach. We demonstrate a physiological role of D-MNs in threat-reward decision-making with evidence at the circuit, behavioral, cellular, and molecular levels. At the circuit and behavioral levels, we found that D-MNs tilt the balance of threat-reward decision-making toward reward behavior by inhibiting AVA interneurons, and that electrical synapses between the left and right AVA interneurons help reach this biased decision by balancing inhibitory synaptic signals from D-MNs. At the cellular level, we found that D-MNs are activated by both excitatory inputs from cholinergic MNs and current flow through a stretch receptor in their cell membrane, and that the left and right AVA interneurons differ significantly in electrical properties. At the molecular level, we identified key postsynaptic receptors mediating synaptic transmission from cholinergic MNs to D-MNs and from D-MNs to AVA interneurons, a stretch-activated mechanoreceptor in D-MNs, and an innexin responsible for the electrical coupling between the two AVA interneurons. Collectively, our results depict a circuit and molecular picture showing how a biased threat-reward decision may be reached through sensory integration in AVA interneurons and retrograde regulation of AVA by D-MNs.28. To confirm the role of ACR-12, we determined whether spontaneous postsynaptic currents (PSCs) in D-MNs depend on cholinergic MNs and ACR-12. In VD5, a representative of D-MNs innervating ventral body-wall muscles, spontaneous PSCs occurred at a rate of 5.9 ± 1.3/s and had a mean amplitude of 6.0 ± 0.6 pA in wild type, and knockdown of unc-17 (vesicular acetylcholine (ACh) transporter) specifically in cholinergic neurons caused 65% decrease in spontaneous PSC frequency with the remaining events having a smaller mean amplitude  between wild type and acr-12(ok367), a putative null resulting from a deletion29. We obtained this mutant from the Caenorhabditis Genetics Center, confirmed its molecular lesion by PCR, and outcrossed it with wild type three times before the electrophysiological experiments. Unexpectedly, in both VD5 and DD3, the frequency and mean amplitude of spontaneous PSCs were similar between wild type and the mutant  and lgc-46(ok2949), which are deletion mutants involving one or two exons (www.wormbase.org), ACh-induced whole-cell current was decreased by >70% compared with wild type  promoter, which has activities in 26 neurons, including RME (4), AVL (1), RIS (1), DVB (1), and all the D-MNs (13 VDs and 6 DDs)32. In addition, we examined the effect of optogenetic activation of cholinergic neurons on evoked PSCs in VD5 with worms expressing channelrhodopsin-2 (ChR2) under the control of unc-17 (vesicular ACh transporter). Blue light stimulus evoked a large initial transient current followed by a smaller sustained current in wild type but had a much smaller (~65% less) effect in the lgc-46 RNAi strain  caused by ACh release from AVA, and fast and small events (fPSCs) caused by the activation of an ACh autoreceptor34. Under our experimental conditions, optogenetic activation of an inotropic GABA receptor in VA5 would manifest as an evoked inward current because the Cl− equilibrium potential (−6 mV) was more depolarized than the holding potential (−60 mV), whereas optogenetic inhibition of AVA would cause a reduced frequency of the sPSCs in VA5. We found that optogenetic stimulation of D-MNs did not cause any appreciable inward current but inhibited sPSCs in VA5 profoundly (>90%) and reversibly  mutant, optogenetic activation of D-MNs did not produce the inhibitory effects on either AVA or VA5  in a strain with AVA labeled by mStrawberry34. In transgenic worms, the two AVA interneurons were co-labeled by mStrawberry and GFP  of the bath solution with the ejection pipette tip aimed at VD5 dendrite caused an inward current of 43.5 ± 8.1 pA in wild type but only 5.0 ± 1.9 pA in unc-8(tm2071), a putative null29. The mutant phenotype could be rescued by expressing wild-type UNC-8 specifically in GABAergic neurons (58.9 ± 8.4 pA), and recapitulated in wild-type worms by GABAergic neuron-targeted unc-8 RNAi (6.5 ± 1.7 pA)  and mec-6(u450) double mutant because these two genes genetically interact with unc-8 in producing a defective locomotion phenotype29. VD5 of the double mutant was found to have a normal stretch response . Thus, our results suggest that UNC-8 is a key stretch receptor in D-MNs.We noticed in preliminary experiments that puffing any solution, including the bath solution, could cause inward current in D-MNs if the ejection pressure was sufficiently high , suggesting the presence of a stretch receptor. Stretch responses in worms and flies often result from the activation of degenerin/epithelial sodium channels (DEG/ENaC)pA) Fig. , suggestnse Fig. , which iunc-49 RNAi strain upon puffing (10 psi) the bath solution aimed at VD5  to the vicinity of the nose through a glass pipette Fig. . In wildain Fig. . These runc-49 RNAi strain compared with wild type  AVAR was more hyperpolarized than AVAL in the resting membrane potential  are innervated by D-MNs through one synapse (from VD6) and five synapses , respectivelyial Fig. , suggestial Fig. , suggestial Fig. , which i19 (https://wormwiring.org), we determined whether they are functional by performing dual-neuron voltage-clamp recordings. In this experiment, we applied a series of membrane voltage (Vm) steps  to one AVA interneuron (Neuron 1) from a holding potential of −30 mV while the other AVA interneuron (Neuron 2) was held constant at −30 mV to record junctional current (Ij). Transjunctional voltage (Vj) is defined as “Vm of Neuron 2 − Vm of Neuron 1.” In response to symmetric positive and negative Vj steps, bidirectionally symmetric Ij was observed in both AVAL and AVAR, which gave similar Ij − Vj relationships and identical gap junctional conductance (Gj)  Fig. , indicatant Fig. . The defrms Fig. . These runc-9 mutant than wild type, and this phenotype could be rescued by AVA-targeted expression of wild-type UNC-9 and recapitulated by AVA-targeted unc-9 RNAi . Optogenetic activation of ASH caused a similar inward current in AVAL and AVAR, and disrupting the electrical coupling between AVAL and AVAR had no effect on the evoked current .We also tested whether excitatory synaptic inputs from ASH sensory neurons to AVA differ in functional strength and whether such difference, if any, is equilibrated by the electrical coupling between AVAL and AVAR by analyzing the effect of optogenetic activation of ASH on evoked PSCs in AVA. AVAR is postsynaptic to ASH in 10 chemical synapses , whereas AVAL is postsynaptic to ASH in 12 chemical synapses ent Fig. . These runc-9 RNAi reduced the percentage of escaped worms in the diacetyl and glycerol assay. However, a significant difference occurred at only 3-M glycerol concentration, which is in contrast to the effect of AVA-targeted unc-49 RNAi over a broad glycerol concentration range 26. Our results suggest that AVA are hub neurons where sensory information from both attractant and repellant sensory modalities and motor information from D-MNs are integrated, and this property of AVA allows the motor system to participate in decision-making. This D-AVA circuit might also interact with other neurons in the worm’s locomotion circuit to modulate locomotion behaviors. For example, AVA and AVB may mutually inhibit their activities through reciprocal chemical synapses between them51. A bilateral pair of tyraminergic RIM interneurons, which have synaptic connections with AVA and AVB, and also interact (either directly or indirectly) with primary sensory neurons27, may modulate sensory-motor coupling and multisensory decision-making24. AVA and RIM may interact with other neurons to generate variable sensory responses26. Further studies are needed to determine whether and how the D-AVA circuit interacts with other neurons to control locomotion behavior and threat-reward decision-making.Traditionally, AVA were considered as command interneurons for driving backward locomotionC. elegans body-wall muscle cells are innervated by both excitatory cholinergic MNs and inhibitory D-MNs. D-MNs innervating ventral or dorsal muscles are controlled by cholinergic MNs innervating the contralateral side. This wiring relationship has led to the suggestion that synaptic transmission from cholinergic MNs to D-MNs allows the latter to relax antagonistic muscles on the contralateral side to help produce the sinusoidal body bends required for locomotion20. Worms that are either deficient in GABA release or have their D-MNs killed display a shortening of body length in response to a mechanical touch on the head52. Selective ablation of VDs and DDs causes ventral and dorsal navigational biases, respectively, due to somewhat deeper ventral or dorsal flexures53. The behavioral phenotypes observed in these studies reflect a summed effect of D-MN deficiency. It would be interesting to determine in future studies how disruptions of specific synapses with D-MNs, such as those from cholinergic MNs to D-MNs, from D-MNs to body-wall muscle cells, and from D-MNs to AVA, may affect body bending and navigational behavior.22. This preference for forward movement is beneficial to worm survival because feeding occurs mainly during forward movement24. Given that AVA neurons are required for long reversals50, the tonic activity of the D-AVA circuit might help suppress unnecessary long reversals to favor efficient feeding.Our results showed that the D-AVA circuit was tonically active, and modulated worm locomotion in either the absence or presence of an attractant and/or a repellant. Conceivably, the D-AVA circuit may also have tonic activities under natural conditions and play a generally important role in modulating forward vs. backward locomotion. What might be the physiological significance of a tonically active D-AVA circuit? Wild-type worms spend much more time on forward than backward movement54. Bilateral asymmetry is also a remarkable feature of the worm brain, with evidence mainly from two pairs of sensory neurons, AWC and ASE, which differ between the left and right in gene expression and function61 (reviews63). Similarly, although the two AVA interneurons are bilaterally symmetric in gross morphology18, this study reveals that they differ in functional properties including the resting membrane potential, membrane resistance, and strength of inhibitory synaptic inputs from D-MNs. The higher membrane resistance in AVAL than AVAR is expected to partially compensate for the weaker synaptic inputs from D-MNs to AVAL than AVAR. In addition, our results reveal that gap junctions between the two AVA neurons serve to balance the inhibitory synaptic inputs from D-MNs. This balancing act between the left and right sides of the “brain” contributes to the biased threat-reward decision-making. In mammals, a massive fiber bundle known as corpus callosum runs between the left and right hemispheres to allow both sides of the brain work together. Because there is no obvious corpus callosum-like structure in worms, gap junctions between bilateral pairs of neurons may fulfill at least some of the functions of coordinating the left and right “brains.”Left-right nervous system asymmetry is observed across animal species. For example, in humans, cortical structures for language functions are usually located in the left hemisphere whereas those for spatial recognition mainly in the right hemisphere64. In C. elegans, 14 of 25 innexins are expressed in neurons, and most neurons express multiple innexins and form gap junctions with several other neurons65. It is a standing puzzle why each neuron needs to express multiple innexins66. The results of this study suggest that UNC-7 and UNC-9 in AVA do not associate with innexins in other neurons indiscriminately but rather in a cell-specific manner to form molecularly and functionally different gap junctions.The two AVA interneurons also form gap junctions with some other neurons, including A-MNs. While gap junctions between AVAL and AVAR are non-rectifying UNC-9 homotypic gap junctions, those between AVA and A-MNs are strongly rectifying heterotypic gap junctions consisting of UNC-7 in AVA and UNC-9 in A-MNsC. elegans body-wall muscles do not have structures like the mammalian proprioceptors. It was speculated many years ago that MNs may have proprioceptive properties18. This possibility was confirmed by a recent study showing that C. elegans B-MNs are proprioceptive, and that this property depends on a stretch receptor67 although molecular identity of the putative stretch receptor remains mysterious. Our results indicate that stretch-dependent modulation of locomotion behavior is not a property unique to B-MNs, and that the D-AVA circuit may be activated by both chemical synaptic transmission from cholinergic MNs and activation of the UNC-8 stretch receptor in D-MNs. Under physiological conditions, the UNC-8 stretch receptor in D-MNs is likely activated by worm body bending, like the putative stretch receptor in B-type cholinergic MNs67. Thus, D-MNs might integrate excitatory synaptic signals from cholinergic MNs and UNC-8 stretch receptor-sensed body bending information to modulate locomotion behavior and threat-reward decision-making through the D-AVA circuit.In mammals, proprioceptors in muscles (muscle spindles and Golgi tendon organs) play important roles in muscle function and locomotion. Sensory signals from these proprioceptors are conducted by specific neural fiber tracks to the cerebellum to coordinate movement. However, 28. This function of ACR-12 was concluded based on the observations that acr-12 mutants showed an increased sensitivity to an immobilizing effect of aldicarb (a cholinesterase inhibitor), a reduced frequency of spontaneous PSCs recorded from body-wall muscle cells, an abnormal locomotion waveform, and partial co-localization between a synaptic vesicle marker expressed in cholinergic MNs and GFP-tagged ACR-12 expressed in D-MNs28. Consistently, subsequent studies showed that ACR-12 is localized to spine-like structures in D-MNs70. Unexpectedly, our direct recoding of spontaneous PSCs from D-MNs showed that ACR-12 does not seem to play a role in mediating synaptic transmission from cholinergic MNs. Instead, we found that LGC-46 performs this function in D-MNs, based on the analysis of spontaneous and optogenetically evoked PSCs in D-MNs. Nevertheless, we cannot definitely exclude the reported role of ACR-12. In our dissected worm preparations, some of the laterally projecting commissures that allow synaptic interactions between cholinergic MNs and D-MNs were disrupted, which could have restricted our detection of spontaneous PSCs to a subset of the synaptic events. Further studies are needed to resolve the reported function of ACR-12 in the synaptic transmission from cholinergic MNs to D-MNs.An earlier study identified ACR-12 as a postsynaptic receptor in D-MNs that mediates synaptic transmission from cholinergic MNs34. This function and its newly identified function in D-MNs are presumably due to conduction of inward current. However, it has also been reported that LGC-46 may function as an anion channel at presynaptic sites of cholinergic MNs to inhibit neurotransmitter release71. The different functions of LGC-46 receptors likely result from different molecular compositions of heteromeric receptors. There is evidence suggesting that the LGC-46 receptor at presynaptic sites in cholinergic MNs also contains ACC-4 as a component71. Although LGC-46 alone may form a homomeric ACh receptor in the Xenopus oocyte heterologous expression system34, the 50-fold difference in the decay time constants between LGC-46-mediated spontaneous PSCs in VA5 and VD5  agar plates seeded with OP50 unc-49 is expressed in AVA interneurons, we coinjected four plasmids, wp1427 (Punc-49::GFP), wp1339 (Pgpa-14::Cre), wp1392 (Pflp-18::loxP::LacZ::STOP::loxP::mStrawberry), and lin-15(+) into the lin-15(n765) strain. The expression of wp1339 and wp1392 results in mStrawberry labeling of the two AVA interneurons81. The expression patterns of GFP and mStrawberry were imaged with an inverted microscope  enhanced GFP/fluorescein isothiocyanate and mCherry/Texas Red filter sets  and a Hamamatsu ORCA-Flash4.0 digital camera (Model C11440-22CU). Pflp-18 and Pgpa-14 were gifts from Dr Alexander Gottschalk81 whereas Punc-49 was cloned from genomic DNA of the Bristol N2 strain by PCR using primers listed in Supplementary Table To test whether Punc-4 and Punc-47 were used for expression in A-MNs and D-MNs, respectively, whereas Pgpa-14 and Pflp-18 were used for AVA-specific expression. The primers for amplifying the target mRNA fragments in the sense direction were listed in Supplementary Table Cell-specific RNAi was achieved by expressing plasmids encoding complementary sense and antisense mRNA fragments under specific promoters. 2 5, MgCl2 5, dextrose 11, and HEPES 5 (pH 7.2). The pipette solution contained (in mM) 120 KCl, 20 KOH, 5 Tris, 0.25 CaCl2, 4 MgCl2, 36 sucrose, 5 EGTA, and 4 Na2ATP (pH 7.2) except for experiments of recording outward whole-cell current, in which the pipette solution contained (in mM) 6.8 KCl, 113.2 Kgluconate, 20 KOH, 5 Tris, 0.25 CaCl2, 4 MgCl2, 36 sucrose, 5 EGTA, and 4 Na2ATP (pH 7.2). All current-clamp recordings were performed without current injection.All electrophysiological experiments were performed with young adult hermaphrodites. Briefly, a worm was immobilized on a glass coverslip by applying Vetbond Tissue Adhesive  in a drop of the bath solution. Application of the glue was restricted to the dorsal side of either the head region (for recording from head neurons) or the mid-anterior portion (for recording from motor neurons) of the worm, which allowed either the tail or both the head and tail to sway freely during the experiment. A short (200–300 µm) longitudinal incision was made by cutting through the glued portion using a diamond dissecting tool  to expose neurons of interest. The cuticle flap was folded back and glued to the coverslip. The dissected worm preparation was treated with collagenase A  for 10–15 s before being flushed away by five- to tenfold of the bath solution. Borosilicate glass pipettes (tip resistance ~20 MΩ) were used as electrodes. Motor neurons were identified based on their anatomical locations whereas interneurons based on labeling by a fluorescent protein. Whole-cell current- and voltage-clamp recordings were performed on a Nikon FN1 microscope with 4× and 40× objectives with a Multiclamp 700B amplifier  and Clampex software  on pressure ejections of ACh, glycerol, diacetyl, and the control solution were done with a Picospritzer III microinjector  connected to a glass pipette with a tip diameter of ~2 µm at 2–4 psi. Mechanical activation of stretch receptors was achieved by puffing the bath solution using the Picospritzer microinjection at 10 psi. The bath solution contained (in mM) NaCl 140, KCl 5, CaCl® (Sutter Instrument). Light intensity was adjusted by applying the three standard neural density filters  of the Nikon FN1 microscope in various combinations, which resulted in eight different light intensities ranging from 0.01 to 4.38 mW/mm2. The maximal light intensity was used in all experiments except those assessing the effect of light intensity-dependent activation of ASH neurons on AVA membrane potential. Light intensities were measured with an optical power meter  equipped with a photodiode power sensor . The on-and-off of light stimulation was controlled by NIS-Elements imaging software (version 4.51) through the SmartShutter in the Lambda XL light source.Worms expressing ChR2 first grew to L1-L2 stage on standard NGM plates. They were then transferred to new NGM plates either with or without (for negative control) all-trans retinal 2 days before experiments. The retinal plates were prepared by spotting each plate (60-mm diameter with 10-ml agar) with 200-µl OP50 containing 2-mM retinal . In most experiments, blue light pulses  were generated by a Lambda XL light source  with a 470 ± 20-nm excitation filter  and SmartShutterTrack-A-Worm35.All behavioral assays were performed with young adult hermaphrodites on 6-cm diameter NGM plates without food. To quantify locomotion behavior, a single worm was transferred to the center of a NGM plate. After a 30-s recovery period from the transfer procedure, the worm was imaged for 1 min at 15 frames/s. Both imaging acquisition and subsequent quantitative analyses were performed using an automated worm-tracking system, 48. Briefly, a glycerol ring was created by applying 10-μl glycerol solution to the surface of a NGM plate by tracing a circle (1 cm in diameter) drawn on its back. In multisensory assays, two drops of 1-μl diluted diacetyl (1:1000 in water) were added to opposite sides (near the edge) of the NGM plate outside of the glycerol ring. After a 5-min equilibration period, worms were transferred to the center of the glycerol ring. In this procedure, 50–100 young adult worms were first transferred from a standard NGM plate with food to a NGM plate without food. After adding M9 buffer (100 μl) onto the plate, ~20 μl of the added M9 solution  was pipetted into the glycerol ring. Worms outside and inside the glycerol ring were counted 15 min later.Osmotic avoidance and multisensory assays were performed based on established procedures17. Two parallel lines were drawn on the back of a NGM plate to divide the plate into four regions  of an equal height. One microliter diluted diacetyl (1:1000) and 1-μl water were applied to regions A and D, respectively, followed by adding 1-μl NaN3 (0.1 M) to both regions. About ten worms were transferred to the center of the plate using the procedures described above. Worms in the various regions were counted 15 min later to calculate the CI: Glycerol chemotaxis assay was performed following similar procedures of previous studiesDiacetyl (Sigma-Aldrich) was diluted in water to reach the 1:1000 ratio. Glycerol  was diluted to final concentrations in a buffer containing 100-mM NaCl, 10-mM KCl, and 30-mM Tris [PH 7.5]. ACh  and gabazine  were first dissolved in water to make aliquots of 10-mM frozen stocks, which were diluted to final concentrations using the bath solution before use.Frequencies and amplitudes of spontaneous PSCs were quantified using MiniAnalysis . For spontaneous PSCs in VA5, only the slow and large events (sPSCs), which were identified based on thresholds of amplitude (>5 pA) and decay time (>5 ms), were used for statistical analyses. The resting membrane potential and PSCs caused by glycerol, diacetyl, optogenetic and mechanical stimuli, and exogenous ACh were quantified with Clampfit . The duration and charge transfer of PSC bursts were quantified with Clampfit, while the frequency of PSC bursts was manually counted. Amplitudes of whole-cell current in response to voltage steps were determined from the mean current during the last 100 ms of the 1.2-s voltage steps using the Clampfit. Junctional current and membrane voltage were quantified by measuring the mean amplitude during the last 100 ms of transjunctional voltage steps and current injection steps, respectively, using Clampfit.t-test (paired or unpaired) was used for statistical comparisons as specified in figure legends.Data graphing were performed with Origin 2019 . Statistical analyses with performed with either Origin or SPSS . All data are shown as mean values ± SEM. Either ANOVA (one-way or two-way) or Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The mycotoxin zearalenone (ZEN), which frequently contaminates cereal-based human food and animal feed, is known to have an estrogenic effect. The biological response associated with exposure to ZEN has rarely been reported in organs other than the reproductive system. In the intestine, several studies suggested that ZEN might stimulate molecular changes related to the activation of early carcinogenesis, but the molecular mechanisms behind these events are not yet known. In this study, we investigated gene expression and changes in protein abundance induced by acute exposure to ZEN in the jejunum of castrated male pigs using an explant model. Our results indicate that ZEN induces the accumulation of ERα but not ERβ, modulates Wnt/β-catenin and TGF-β signaling pathways, and induces molecular changes linked with energy sensing and the antimicrobial activity without inducing inflammation. Our results confirm that the intestine is a target for ZEN, inducing changes that promote cellular proliferation and could contribute to the onset of intestinal pathologies. Fusarium genus that frequently contaminate crops such as corn, barley, wheat, rice, oats, and sorghum, particularly in temperate regions [Zearalenone (ZEN) is a mycotoxin synthesized by some fungi of the  regions . ZEN is  regions . Like ot regions . At the  regions . Indeed, regions ,5,6,7. T regions ,9, resul regions ,11. At l regions ,11,12, a regions ,13,14. E regions ,16,17. T regions . ZEN can regions . Consequ regions . In the  regions ,22. Estr regions ,24,25. AThe toxic effect of ZEN in the intestine has been investigated in the past, mostly using pig as model for humans due to their similar sensitivity and toxicity . The maiIn this study, we investigated the activation/repression of Wnt/β-catenin and TGF-β signaling pathways, the immune status and metabolism by ZEN to understand the effect of this toxin on the intestine of castrated male pigs. Our results confirmed that the small intestine is a target for ZEN, and that its toxic effect could contribute to the development or aggravation of severe intestinal pathologies such as cancer.p < 0.05; The abundance of 68 gene transcripts and 14 proteins were evaluated using RT-qPCR and western blot, respectively, in pig jejunal explants from castrated male pigs exposed for 1 h or 4 h to 100 µM ZEN. Differences in gene expression were found for six transcripts after 1-h treatment and 22 transcripts after 4 h of exposure , cofilin-1 (CFL1), β-catenin, vimentin (VIM), and chemerin receptor (CMKLR1) at 4 h (p < 0.01), while ERβ abundance did not change at any time-point. Relative protein quantification by western blot showed a significant up-accumulation of ERα after 4 h of exposure of ZEN . This leads to a cascade of events that allows the stabilization of cytoplasmic β-catenin and its translocation of the nucleus through a process that depends on the activity of glycogen synthase kinase 3 beta (GSK3β) and is inhibited by the proteins adenomatous polyposis coli (APC) and axins. In the nucleus, β-catenin associates with T-cell factor/lymphoid enhancer-binding transcription factors (TCF/LEF) to induce the expression of target genes, such as Cyclin 1, Zinc Finger E-Box Binding Homeobox 1 and 2 (Zeb1/2), Snail1, Snail2, and c-Myc among others. The whole process is inhibited by DKK-1 .p < 0.05; showing a similar trend at the protein level, although the up-accumulation was only almost significant; p = 0.06), Cyclin D1 (p < 0.05), c-Myc (p < 0.05), Zeb1 (p < 0.05) and Snail2 (p < 0.01), whereas TCF1 was up-regulated at 1 h of exposure (p < 0.05). A significant down-regulation was observed at 1 h for TCF4 (p < 0.01), and at 4 h (p < 0.05) for Wnt11 (p < 0.05), DKK1 (p < 0.05), APC (p < 0.01). Nearly significant down-regulation of axin2 (p = 0.06) and APC (p = 0.06) at 1 h was also observed. The gene expression of ATPase H+ transporting accessory protein 2 (ATP6ap2), a facilitation of the transmission of β-catenin signaling [p < 0.05). Several genes of this signaling cascade were up-regulated by 4 h of exposure to ZEN, namely β-catenin  and Vimentin , whose expression was significantly up-regulated at 4 h of exposure. VIM was also significantly up-accumulated at 4 h of exposure at the protein level (p < 0.001).To link the activation of Wnt/β-catenin with cell proliferation, we investigated two markers of cell proliferation, minichromosome maintenance complex component 3 , ZO-1 (p < 0.05) and α-catenin (p < 0.05) gene expression was observed at 4 h of exposure. No differences in occludin and ZO-1 were observed at the protein level, whereas significant up-accumulation of the actin cytoskeleton protein CFL1 was found at 4 h of exposure. (p < 0.05).Among the expected consequences of β-catenin transcriptional activation is the repression of the expression of the tight junctional proteins Zonula occludens-1 (ZO-1), claudins, occludins as well as the adherens junctional proteins cadherins and α-catenin . No chanpBD1; p = 0.05), down-regulation of the expression of β-defensin 3  together with a nearly significant over-expression of lysozyme at the transcript level (p = 0.06). The abundance of regenerating islet-derived protein 3 gamma (RegIIIγ) was investigated by western blot, and an accumulation was observed, although it was only nearly significant (p = 0.07) at 4 h.Paneth cells are small-intestine epithelial cells located at the bottom of the crypts. They contain secretory granules containing antimicrobial proteins and peptides, but also play a role in regulating proliferation in the crypt. As their quantity and maturation depends on Wnt/β-catenin signaling ,38, we iTaken together, our results provide evidence that ZEN can activate the Wnt/β-catenin signaling pathway without affecting the intestinal barrier, and induce the expression of proliferation biomarkers as well as antimicrobial proteins.In the intestine, the Wnt signaling pathway interplays with other pathways, including the TGF-β signaling pathway. TGF-β signaling is mediated through transmembrane receptors that use Smad2 and Smad3 proteins to transduce their signals from the cell surface to the nucleus in a process that requires the presence of Smad4 for DNA binding and transcriptional activation of target genes, and is inhibited by Smad 7 ,39. p < 0.05), but no difference was found at 4 h. The expression of all the Smads studied  remained unchanged at all time points, with the exception of Smad2, whose transcript levels were significantly down-regulated at 1 h (p < 0.05) and 4 h of exposure (p < 0.001). We investigated several target genes in this pathway, and found significant up-regulation of Integrin-alpha 10 at 1 h (p < 0.05), no changes in the expression of the CD44 molecule  nor MMP7, and down-regulation of the expression of fibronectin , as well as MMP2 and MMP9 (p < 0.05) at 4 h of exposure. Gelatinase activity of MMPs leaked to the explant medium was analyzed and whereas no differences were found at 1 h of exposure nor pro-MMP9 at 4 h, the intensity of bands decreased significantly at 4 h of exposure to ZEN for active MMP9 (p < 0.05), pro-MMP2 (p < 0.01) and active MMP2 , and almost significant at 1 h (p < 0.06), of protein S100A10.To investigate if ZEN can induce intestinal inflammation under our conditions, several cytokines and acute phase proteins were analyzed. No differences were found in the expression or protein abundance of all the inflammatory markers investigated, namely interleukins 8, 17, 18, 22, tumor necrosis factor α (TNF-α) and interferon gamma (IFNγ), protein S100A8, protein S100A12, serum amyloid A3 (SAA3), alpha-acid glycoprotein alpha 1 (α-1 AGP) and lipocalin 2 (LCN2). Regarding the complex S100A10/AnnexinA2 (ANXA2), no changes were observed in the gene expression of ANXA2, whereas a marked down-regulation of was significant at 4 h of exposure (p < 0.05).We investigated the expression of peroxisome proliferator activated receptors (PPARs), since ZEN exposure can increase energy production through an increase in the uptake and β-oxidation of fatty acids, in a processes that has been suggested to be mediated by activation of PPARs in Leydig cells . HoweverThese results show that no inflammation was associated with the treatment with ZEN, whereas significant changes were associated with adipokine receptors.Estrogen signaling is important for intestinal homeostasis, but disturbance of the mycoestrogen ZEN in the intestinal molecular pathways that interact with that of estrogen, such as Wnt/β-catenin and TGF-β, is not completely understood. Unfortunately, most of the molecular pathways that regulate intestinal physiology cannot be studied in vitro, since they are compartmentalized or present as a gradient that can increase or decrease along the crypt–villus axis. Here, an ex-vivo model of jejunal explants was used to reflect the complexity of the interaction between the different cell types present in the intestine. The pig is a ZEN-sensitive species , and resThe small intestinal mucosa is a barrier against food contaminants. It is formed by an epithelial cell lining that is continuously renewed in a process that depends on the division of intestinal stem cells (ISCs), located near the base of the crypt ,44. ProlUnder our conditions, significant accumulation of ERα was observed, whereas the levels of ERβ remained unchanged. The accumulation of ERα can explain other molecular changes observed in the present study such as the activation of the Wnt/β-catenin signaling pathway ,16,17. SThere might be a link between the ZEN-induced activation of Wnt/β-catenin signaling and the detected changes in antimicrobial peptides. Although our results were not significant, up-regulation/up-accumulation of the antimicrobial proteins lysozyme and RegIIIγ produced by the Paneth cells was observed. Previous reports report a ZEN-induced increase in Paneth cells in pig . The homIn contrast to the pro-proliferating Wnt/β-catenin signaling, TGF-β maintains a state of growth equilibrium and promotes epithelial differentiation towards the tips of the villus . TGF-β iThe ZEN-mediated induction of inflammation has already been studied in different tissues in immune cells ,11, the We investigated the expression of PPARα, PPARγ as well as that of other genes linked with adipokines, a family of metabolic sensing proteins, of which some members have been found to be regulated by ZEN in the intestine ,40. No dOur results provide new knowledge of the toxicity of ZEN and confirm that exposure to ZEN can promote the development of severe intestinal pathologies. ZEN increase ER-alpha, activate Wnt/β-catenin signaling and repress TGF-β in the small intestine of castrated male pigs, inducing a pro-proliferative/pre-cancerous phenotype, characterized by over-expression of MCM3 and VIM. This suggests that ZEN disturbs the ISC microenvironment, promoting a pro-cancerous, proliferative state in the intestine that has been already described in the literature ,29,30,59Purified zearalenone (ZEN) was purchased from Sigma , and dissolved in dimethylsulfoxide  to obtain a concentration of 20 mM, aliquoted and stored at −20 °C. Six castrated male, 5-week old piglets, were provided by a local farm . The experiment was conducted under the guidelines of the French Ministry of Agriculture for animal research. The Ethics Committee of Pharmacology-Toxicology of Toulouse-Midi-Pyrénées approved all animal experimentation procedures . The collection of jejunum tissue and the preparation of explant cultures was as described previously ,68,69, tThe pig jejunal explants were homogenized using 2 mL plastic bead tubes  in 1 mL of Extract-All reagent  in a Precellys Evolution tissue homogenizer . ARN isolation and RT-qPCR were performed as described elsewhere ,71. DataProteins from jejunal explants were extracted using 2 mL plastic bead tubes  in 0.5 mL of radioimmunoprecipitation assay buffer (RIPA buffer) containing protease- and phosphatase inhibitors  in a Precellys Evolution tissue homogenizer . Total protein content was quantified (Bicinchoninic acid (BCA) protein assay, Thermo Fisher Scientific, Waltham, MA, USA). Total protein extracts (20 µg) were separated in 12.5% or 15% acrylamide-bisacrylamide gels, and transferred onto nitrocellulose membranes. Blots were blocked in RotiBlock  and incubated with the corresponding primary antibody at 4 °C overnight under agitation . DetectiThe explant culture medium was concentrated (approximately 10-fold) using centrifugal filter units . Total protein content was quantified  according to the manufacturer’s instructions. Protein aliquots (10 µg) were separated in 7.5% acrylamide-bisacrylamide gels containing 0.3 mg/mL gelatin from porcine skin  under non-reducing, non-denaturing conditions. Gels were incubated (2 × 30 min) in 2.5% Triton X-100 (Sigma) for protein renaturation and then proteolytic reaction was allowed to take place in 50 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, pH 7.5, for 20 h at 37 °C, followed by staining with colloidal Coomassie blue stain . Gelatinolytic activity was visible as clear zones on a blue background. Images were obtained using a Li-Cor Odyssey Infrared Imager  and analyzed with Image Studio Lite Software v5.2 .p < 0.05.Statistical differences in gene expression and protein abundance were determined using paired t-tests using GraphPad Prism statistical software version 6 . The significance level was set at