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Objective: To investigate the effects and potential mechanism of tumor suppressor dishevelled-binding antagonist of beta-catenin 2 (DACT2) on epithelial-mesenchymal transition of glioma cells. Methods: The expressions of DACT2 in glioma cells U87, U251, A172 and SHG44 were detected by RT-PCR after 5-Aza treatment. The methylation status of DACT2 promoter was detected by methylation specific PCR (MSP). Western blot was used to detect the expression of DACT2 protein. U87 cells overexpressing DACT2 were constructed and verified by Western blot. Transwell assay was used to detect cell migratory and invasive ability. The protein levels of E-cadherin, Vimentin, MMP2 and Wnt/β-cadherin pathway proteins, i.e., active-β-cadherin, p-β-cadherin and total β-cadherin, in cells were detected by Western blot. Results: DACT2 expression was observed in all these cells after 5-Aza treatment; untreated U87 and U251 cells did not express DACT2 while A172 and SHG44 cells showed weak expression. The DACT2 promoter was completely methylated in U87 and U251 cells, and partially methylated in A172 and SHG44 cells. The level of DACT2 in U87 and U251 cells was lower than that in A172 and SHG44 cells (P<0.05). After transfection of pcDNA3.1-DACT2, the expression of DACT2 in U87 cells increased significantly (P<0.05), U87 cells overexpressing DACT2 were successfully constructed. Overexpression of DACT2 could significantly inhibit epithelial-mesenchymal transition, invasion and migration of U87 cells and block Wnt/β-catenin pathway activation (P<0.05). Conclusion: The DACT2 promoter in glioma cells is highly methylated, and the exogenous overexpression of DACT2 can promote the epithelial mesenchymal transition, invasion and migration of U87 cells. The underling mechanism may be related to the regulation of Wnt/β-catenin pathway by DACT2.
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Objective@#To study the different expression of 4 microRNA (miRNA, miR) during the osteogenesis differentiation of bone marrow mesenchymal stem cell (BMSC) cultured in high-fat or normal environment and to explore the relationship of these miRNAs with disheveled 2 during osteogenesis differentiation.@*Methods@#BMSC were cultured with 2 ml normal osteogenic induction (control group) and high-fat osteogenic induction (high-fat group) respectively. On the 3rd, 5th, 7th,14th, 21st day, quantitative real-time PCR (qPCR) was used to analyze expression levels of four miRNAs (miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p), mRNA of disheveled 2, osteogenic related factors such as alkaline phosphatase (ALP), Runt-related transcription gene 2 (Runx2). And the protein was detected by Western blotting. After BMSC were transfected by 50 μl 50 nmol/L miRNA mimics/inhibitors/negative controls respectively, BMSC were put on osteogenic induction, on the 1st, 3rd, 5th, 7th day, ALP activity was detected. On the 7th day, ALP staining was to observe the degree of osteogenesis differentiation, and Western blotting was adopted to analyze the expression of dishevelled 2 and other osteogenic related factors, while qPCR was used to analyze the expression of disheveled 2 mRNA. After 293T cells were co-transfected with disheveled 2 wild-type/mutant firefly luciferase reporter plasmid with either negative control (NC) or a mimic of these four miRNAs respectively for 48 h, luciferase activities were measured.@*Results@#On the 21th day, the expressions of miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p in high-fat groups were higher by 20%, 60%, 340% and 4 420% respectively than those in control groups (P<0.05). The expression of ALP and Runx2 in BMSC decreased after BMSC transfected miR-21-5p and miR-29c-3p mimics, while increased after transfected miR-21-5p and miR-29c-3p inhibitors. The expression of disheveled 2 decreased by 35% after transfected by miR-29c-3p mimic, while it increased by 269% after transfected by miR-29c-3p inhibitor (P<0.05). Transfection of the miR-29c-3p mimics significantly decreased the luciferase activity of wild-type 3'-UTR compared with NC control (P<0.05). There were no statistical significances among other groups.@*Conclusions@#miRNAs had better expression during osteogenesis differentiation of BMSC in high-fat environment; miR-29c-3p could negatively regulate the osteogenesis differentiation of BMSC by targets on dishevelled 2.
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Objective To study the expression of dishevelled-2 (DVL2) in rheumatoid arthritis cartilage and its effect on cartilage destruction.Methods Cartilage DVL2 expression in rat models of rheumatoid arthritis (RA),osteoarthritis(OA) and collagen-induced arthritis(CIA) were tested by Western blotting.DVL2 overexpressed lentivirus was transfected into the knee of CIA rats.Primary chondrocytes were extracted from RA patients by knee arthroplasty and transfected with DVL2 overexpressed lentivirus.Gene expression of related inflammation related cytokines was detected by real-time polymerase chain reaction (PCR).Results Compared with knee articular cartilage in OA patients and normal rats,DVL2 protein was highly expressed in knee cartilage of RA patients and CIA rats (P values 0.041 and 0.032,respectively).DVL2 did not significantly affect the destruction of knee cartilage in CIA rats (P=0.885).DVL2 overexpression in chondrocytes enhanced gene expression of cyclo-oxygenase-2 (COX-2),inducible nitric oxide synthase (NOS),matrix metalloproteinase (MMP) 2,MMP-3,and MMP-9,which could be more pronounced when tumor necrosis factor alpha was added.Conclusions DVL2 is highly expressed in RA articular cartilage and promotes the expression of inflammatory cytokines and MMP gene in chondrocytes by activating Wnt/β-catenin pathway,which involves in the destruction of articular cartilage in RA.
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Dishevelled (Dvl) is a kind of protein widely existing in human tissue, containing three structural domains:Dishevelled and Axin (DIX);postsynaptic density protein-95, disc large tumour suppressor, zonula occludens-1 (PDZ);and Dishevelled, Egl-10 and pleck-strin (DEP). Dvl participates in the Wnt signaling through different structure domains to play a role in nervous system development and neu-rogenesis after nerve injury.
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Objective To optimize the culture method for rheumatoid arthritis fibroblast-like synoviocytes in vitro,and observe the effect of Dishevelled (Dvl) 2 on vascular endothelial growth factor (VEGF) in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS).Methods Synovium from RA patients who underwent knee arthroplasties were cut into small piece,and RA-FLS were isolated and cultured in vitro using tissue block method.Dvl 2 lentivirus overexpressing plasmid was constructed and transfected into RAFLS.Q-polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect the mRNA and protein expression levels of VEGF.Then we used 10 ng/ml tumor necrosis factor (TNF)-α recombinant protein to stimulate the transfected RA-FLS.24 h after stimulation,mRNA and protein expression of VEGF were detected again.Student's t test was used for two group analyses.Results RA-FLS was successfully isolated and cultured in vitro.The multiplicity of infection was 30 and was in conjunction with appropriate concentration of polybrene to promote transfection.Transfection efficiency could meet the test requirements.The mRNA of Dvl 2 increased for 79-fold than the control group.Compared with the control group,Dvl 2 could mildly inhibit RA-FLS secretion of VEGF.After TNF-α stimulation,Dvl 2 could significantly inhibit the VEGF's mRNA (2.15±0.10,2.92±0.47 fold,t=-3.924,P=0.003) and protein [(285±100) pg/ml,(155±61) pg/ml,t=-2.714,P=0.022] expression compared with the control group.Conclusion Dvl 2 can inhibit the effect of TNF-α induced secretion of VEGF in RA-FLS.The specific mechanism needs further study.
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Objective To investigate the effects of Dishevelled (DVL) on apoptosis of diffuse large B-cell lymphoma (DLBCL) cell line OCI-Ly10, and to explore its possible mechanism. Methods Lentivirus plasmid overexpressing DVL2 was constructed, and after virus was packaged, it was transfected into OCI-Ly10 cells. Flow cytometry was used to detect the apoptosis rate of OCI-Ly10 cells with or without the stimulation by TNF-α recombinant protein. Then the gene expression of anti-apoptotic genes, GADD45β and A20, in NF-κB pathway was detected by RT-PCR. Results The virus was sucessfully transfected into OCL-Ly10 cells which overexpressed DVL2. The apoptosis rate of OCL-Ly10 cells overexpressing DVL2 without the stimulation by TNF-α was increased compared with that of the negative control group [(15.46 ±2.37) % vs. (11.72±3.53)%, P=0.03], the A20 mRNA expression level was decreased compared with that of the negative control group [(0.66 ±0.01) vs. 1, P=0.04], and the relative expression level of GADD45β mRNA was not significantly decreased compared with that of the negative control group [(0.79 ±0.15) vs. 1, P=0.642]. The apoptosis rate of DVL2 overexpression OCI-Ly10 cells stimulated by TNF-α was significantly higher than that of the negative control group treated by TNF-α [(22.78±4.56)%vs. (12.79±2.89)%, P=0.007]. The gene expression of A20 and GADD45β in DVL2 overexpression cells stimulated by TNF-α was significantly increased, however, the magnitude of increase in DVL2 overexpression cells was less than that in the negative control group treated by TNF-α [A20: (3.75 ±0.14) times vs. (6.89 ±0.10) times, P=0.008; GADD45β:(4.750±0.21) times vs. (6.14±0.08) times, P=0.03]. Conclusion DVL can promote the apoptosis of OCI-Ly10 cells, and its mechanism may be related with anti-apoptotic genes that inhibits its downstream via NF-κB pathway.
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Objective To investigate the correlation between Dishevelled protein expression and the proliferation and invasion of glioma cells.Methods 67 cases of brain glioma specimens were collected.The expression of Dishevelled protein was detected with immunohistochemical method.The immunoreactivity score (IRS) of Dishevelled protein,and proliferation index (PⅠ) and invasion index (Ⅱ) were measured and their correlations were analyzed.Results The positive rate of Dishevelled protein in glioma was 65.7 ％ (44/67).IRS,PⅠ and Ⅱ were 4.15±3.13,(30.93±17.92) ％,(20.38±13.36) ％,respectively.Both PⅠ and Ⅱ significantly increased with an increase in the pathological grade of brain glioma (P ＜ 0.001).Furthermore,PⅠ and Ⅱ were significantly higher in the Dishevelled protein-positive group than those in the Dishevelled protein-negative group [(38.27±17.60) ％ vs (16.02±8.92) ％ of PⅠ and (30.03±13.81) ％ vs (10.63±4.41) ％ of Ⅱ,respectively,P ＜ 0.001].PⅠ and Ⅱ of glioma cells were positively correlated with IRS of Dishevelled protein (r =0.940 between PⅠ and IRS,and r =0.953 between Ⅱ and IRS,respectively).Conclusion Dishevelled protein plays an important role in the proliferation and invasion of brain malignant glioma.
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Objective To explore the relationship of DVL2 expression and the development of (CCRCC) by comparing the changes of DVL2 mRNA and protein expression in CCRCC specimens and matched normal renal specimens and its clinical significance. Methods DVL2 mRNA expressions in 22 CCRCC tissues, the matched adjacent normal tissues, and 10 CCRCC tissues alone were examined by semiquantitative RT-PCR and fluorescence quantitative PCR (real-time RT-PCR). Meanwhile, the different expression of the CCRCC between TNM Stage Ⅲ + Ⅳ and Stage Ⅰ +Ⅱ was also examined. Furthermore,immunohistochemistry was employed to examine DVL2 protein expression in 22 CCRCC and the matched adjacent normal tissues, and the other 10 CCRCC tissuses without the matched tissues. Results The DVL2 mRNA expression levels in 17 CCRCC tissues were increased by semi-quantitative RT-PCR and by real time RT-PCR compared with that in corresponding adjacent normal tissues, with the difference being significantly different (t = 2.535, P =0.0197). The DVL2 expression of 8 in 13 Ⅲ + ⅣCCRCC was higher than Ⅰ +ⅡCCRCC. Immunohistochemical examination showed that the DVL2 protein was located in cytomembrane and cytoplasm. Moreover, the positive level of DVL2 protein in CCRCC tissues[81.8 % (18/22)]was significantly higher than those in the adjacent tissues. However the expression was not associated with patients' age, gender, TNM stages (Fisher exact frenquently, P >0.05). Conclusion The DVL2 expression in CCRCC is obviously higher than the corresponding normal tissues in the level of mRNA and protein. And the higher DVL2 expression might be closely associated with the development and progression of CCRCC in the level of mRNA, which may be a potential molecular marker of CCRCC development and metastasis mechanism.
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ObjectiveTo explore the relationship between the expression of Dishevelled2 and Vangl2 and the embryonic neural tube defects (NTDs) induced by all-trans retinoic acid (RA)in Kunming mouse. Methods Fifty pregnant mice were randomly divided into control and RA-treated groups.RA-treated mice were fed with 30mg/kg RA dissolved with peanut oil on embryo 7.75 days, while the mice of control group were administrated with an equal volume of peanut oil on the same time. Then all the embryos were sampled from pregnant mice at the 4th, 18th, 42nd, 66th and 90th hour after treatment. In situ hybridization and immunohistochemical staining technique were used to detect the expression of Dishevelled2 and Vangl2 in embryonic neural tube. Results The two proteins both existed in the epithelial tissue of the mouse embryonic neural tube and displayed different expression modes at various developmental stages.Compared with the control group, the RA treated group showed a significant decrease (P≤0.05) at the 18th and 42nd hour and a significant increase (P≤0.05) at the 66th hour in Dishevelled2 protein after maternal treatment, and no significant difference was found at the 90th hour. Compared with the control group, the Vangl2 mRNA expression in the RA treated group displayed a significant decrease (P≤0.05) at the 4th and 18th hour and a significant increase (P≤0.05) at the 66th hour after RA treatment, and no difference was found at the 42nd hour. Compared with the control group, the expression of Vangl2 protein in the RA treated group decreased (P≤0.05) at the 18th and 42nd hour, and increased (P≤0.05) at the 90th hour after RA treatment, no difference was found at the 66th hour. Conclusion Excessive RA may interfere with the normal embryonic neural tube closure by regulating the expression of Dishevelled2 and Vangl2.
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Objective To explore the relationship between the expression of Dishevelled2 and Vangl2 proteins and the development of mouse palate. Methods Twenty-four pregnant mice were randomly divided into eight groups, and the mouse embryos were obtained at eight clock of the pregnant day of thirteen(p13d8h), p13d14h,p13d22h,p14d8h,p14d14h,p14d22h,p15d8h and p15d22h respectively, then paraffin sections were made conventionally.The distrubution and dynamic changes of Dishevelled2 and Vangl2 proteins in the embryonic palatal shelves were detected by immunohistochemistry and image analysis. Results It was found that the two kinds of proteins expressed in the epithelium and mesenchyma of the mouse palatal shelves at different development stages. The expression levels of the Dishevelled2,in both of the epithelium and mesenchyme of the palatal shelves, increased first (p13d8h-p13d22h),then decreased rapidly(p13d22h-p14d14h), and then increased again(p14d14h-p15d22h). The expression of Vangl2 protein in the mesenchyma showed a similar trend to that of the Dishevelled2, but there was no obvious regularity in the epithelium. In addition, the expressive levels of both kinds of proteins in the epithelium were significantly higher than those in mesenchyma of the palatal shelves. Conclusion Dishevelled2 and Vangl2 proteins might directly or indirectly take part in the regulation process of mouse palate morphogenesis.
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Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.
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Animals , Humans , Adaptor Proteins, Signal Transducing/genetics , Blastomeres/cytology , Cell Line , Embryonic Development/genetics , Feedback, Physiological , Frizzled Receptors/genetics , GTP-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Mutation , Phosphoproteins/genetics , Protein Binding , RNA, Small Interfering/genetics , Repressor Proteins/genetics , Transfection , Wnt Proteins/genetics , Xenopus , Xenopus Proteins/geneticsABSTRACT
Glycogen synthase kinase 3 (GSK3) was recently suggested to be a potential target of psychotropics used in psychiatric illnesses such as schizophrenia and bipolar disorder. Relevant studies have found that antipsychotic drugs regulate GSK3 activity via an increase in either inhibitory serine phosphorylation or amount of GSK3 after acute or subchronic treatment. Recent evidence shows that GSK3 is regulated by dopaminergic or serotonergic systems implicated in the pathophysiology and treatment mechanisms of schizophrenia and bipolar disorder. Therefore, antipsychotics may regulate GSK3 via antagonizing dopaminergic or serotonergic activity. However, the signaling pathway that is involved in GSK3 regulation by dopaminergic or serotonergic systems has not been well established. Haloperidol is a typical antipsychotic with potent dopamine D(2) receptor antagonism. Clozapine is an atypical antipsychotic with potent serotonin 5HT(2) receptor antagonism. We injected rats with haloperidol or clozapine and examined the phosphorylation and amount of GSK3alpha/beta and its well-known upstream regulators Akt and Dvl in the rat frontal cortex by Western blotting. Both haloperidol and clozapine induced Ser21/9 phosphorylation of GSK3GSK3alpha/beta. Haloperidol increased the Ser473 phosphorylation of Akt transiently, whereas clozapine maintained the increase for 1 h. Haloperidol did not affect the phosphorylation and amount of Dvl, whereas clozapine increased both phosphorylation and the amount of Dvl. Our results suggest that GSK3 activity may be regulated by both typical and atypical antipsychotics and that Akt or Dvl, depending on the D(2)- or 5HT(2)- receptor antagonism properties of typical and atypical antipsychotics, mediate the regulation differently.