Wnt4 Levels Research Articles

Wnt is coming: A role for serum Wnt2 and 4 in fibrotic remodeling following acute myocardial infarction.

Wnt is coming: A role for serum Wnt2 and 4 in fibrotic remodeling following acute myocardial infarction.

Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 following myocardial infarction.

BackgroundAcute myocardial infarction (AMI)-induced excessive myocardial fibrosis exaggerates cardiac dysfunction. However, serum Wnt2 or Wnt4 level in AMI patients, and the roles in cardiac fibrosis are largely unkown.MethodsAMI and non-AMI patients were enrolled to examine serum Wnt2 and Wnt4 levels by ELISA analysis. The AMI patients were followed-up for one year. MI mouse model was built by ligation of left anterior descending branch (LAD).FindingsSerum Wnt2 or Wnt4 level was increased in patients with AMI, and the elevated Wnt2 and Wnt4 were correlated to adverse outcome of these patients. Knockdown of Wnt2 and Wnt4 significantly attenuated myocardial remodeling and cardiac dysfunction following experimental MI. In vitro, hypoxia enhanced the secretion and expression of Wnt2 and Wnt4 in neonatal rat cardiac myocytes (NRCMs) or fibroblasts (NRCFs). Mechanistically, the elevated Wnt2 or Wnt4 activated β-catenin /NF-κB signaling to promote pro-fibrotic effects in cultured NRCFs. In addition, Wnt2 or Wnt4 upregulated the expression of these Wnt co-receptors, frizzled (Fzd) 2, Fzd4 and (ow-density lipoprotein receptor-related protein 6 (LRP6). Further analysis revealed that Wnt2 or Wnt4 activated β-catenin /NF-κB by the co-operation of Fzd4 or Fzd2 and LRP6 signaling, respectively.InterpretationElevated Wnt2 and Wnt4 activate β-catenin/NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 in fibroblasts, which contributes to adverse outcome of patients with AMI, suggesting that systemic inhibition of Wnt2 and Wnt4 may improve cardiac dysfunction after MI.

Elevated Wnt2 and Wnt4 Activate NF-κB Signaling to Promote Cardiac Fibrosis by Cooperation of Fzd4/2 and LRP6 Following Myocardial Infarction

Background: Acute myocardial infarction (AMI)-induced excessive cardiac fibrosis exaggerates infarcted area and cardiac dysfunction, which is a significant clinical problem. Here, we investigated Wnt2 and Wnt4 levels in AMI patients or MI mouse model, and delineated the potential mechanism in cardiac fibrosis following MI. Methods: We examined serum Wnt2 and Wnt4 levels in AMI and non-AMI patients by ELISA analysis. MI mouse model was built by ligation of left anterior descending branch (LAD). Findings: Serum Wnt2 or Wnt4 level was increased in patients with AMI, and the high serum Wnt2 and Wnt4 levels correlated to adverse outcome of these patients. Elevated serum and cardiac Wnt2 or Wnt4 levels were observed at early-phase after experimental MI. Knockdown of Wnt2 and Wnt4 significantly attenuated myocardial fibrosis and cardiac dysfunction following MI. In vitro, hypoxia enhanced the secretion and expression of Wnt2 and Wnt4 in cardiomyocytes (CMs) or cardiac fibroblasts (CFs). Si-Wnt2 or si-Wnt4 partly abolished the pro-fibrotic effect in cardiac fibroblasts in response to hypoxia. Mechanistically, exogenous Wnt2 or Wnt4 activated β-catenin /NF-κB signaling to promote pro-fibrotic effects in cultured CFs. In cultured CFs, Wnt2 or Wnt4 upregulated Frizzled (Fzd) 2 or Fzd4 and LRP6 expression. Si-Fzd4 or si-Fzd2 significantly attenuated NF-κB activation and the pro-fibrotic effects in CFs treated with Wnt2 or Wnt4, respectively. Knockdown of LRP6 showed the similar results. However, Wnt2 or Wnt4 induced the activation of β-catenin dependently on LRP6 not Fzd4 or Fzd2. Interpretation: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 in fibroblasts, which may contribute to adverse outcome of patients with AMI, suggesting that systemic inhibition of Wnt2 and Wnt4 may improve cardiac dysfunction after MI. Clinical Trial Registration Details: ClinicalTrials.gov Identifier: NCT03342131. Funding Information: This study was supported by National Natural Science Foundation of China (No. 81770239, 81730009, 82070232). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethics Committee of Dalian Medical University/First Affiliated Hospital of Dalian Medical University (PJ-KS-KY-2017-104(x)). Before inclusion in the study, written informed consent was obtained from the patient's family/patient. The investigation conformed to the principles outlined in the Declaration of Helsinki. All experiment protocols about animal in this study were approved by the Animal Care and Use Committee of Zhongshan Hospital, Fudan University, and conformed to the ‘Guidelines for the Care and Use of Laboratory Animals’ published by the National Institutes of Health (No. 85-23, revised 1996).

Evaluating the Expression of Wnt Pathway Related Genes in Mouse Vitrified Preantral Follicles: An Experimental Study

Background:Wnt signaling pathway plays critical role in ovarian follicle development. Therefore, the aim of this study was to evaluate the effects of vitrification on the expression of Wnt pathway related genes in preantral follicles (PFs).Methods:Isolated PFs (n=982) of 14–16 day old female mice (n=45: 15 for each group) were divided into fresh (n=265), toxicity (n=272), and vitrified (n=265). The mRNA levels of Wnt2, Wnt4, Lrp5 and Fzd3 were evaluated by real-time PCR on the 2nd and 6th days of culture period. One-way ANOVA was conducted to analyze the data. Post hoc Tukey’s HSD was used for multiple comparisons and p-value less than 0.05 was considered statistically significant.Results:The developmental parameters of fresh PFs were significantly higher than those of vitrified (p<0.001). There were no differences between fresh and vitrified PFs on the 2nd day of culture (p<0.001). Wnt4 expression levels decreased significantly in vitrified groups compared with fresh ones (p<0.001). Fzd3 and Lrp expression levels increased significantly in vitrified groups compared with those in the fresh group on the 2nd day (p<0.001). On the 6th day of culture period, the expression levels of Wnt2 and Fzd3 increased significantly in vitrified group compared to those of fresh group (p<0.001). Moreover, the expression levels of Wnt4 and Lrp increased significantly in toxicity groups compared to those of the control group (p<0.001).Conclusion:Vitrification increase the expression levels of Wnt2, Lrp and Fzd3 genes of PFs during in vitro culture.

E2F1 regulates testicular descent and controls spermatogenesis by influencing WNT4 signaling.

Cryptorchidism is the most common urologic birth defect in men and is a predisposing factor of male infertility and testicular cancer, yet the etiology remains largely unknown. E2F1 microdeletions and microduplications contribute to cryptorchidism, infertility and testicular tumors. Although E2f1 deletion or overexpression in mice causes spermatogenic failure, the mechanism by which E2f1 influences testicular function is unknown. This investigation revealed that E2f1-null mice develop cryptorchidism with severe gubernacular defects and progressive loss of germ cells resulting in infertility and, in rare cases, testicular tumors. It was hypothesized that germ cell depletion resulted from an increase in WNT4 levels. To test this hypothesis, the phenotype of a double-null mouse model lacking both Wnt4 and E2f1 in germ cells was analyzed. Double-null mice are fertile. This finding indicates that germ cell maintenance is dependent on E2f1 repression of Wnt4, supporting a role for Wnt4 in germ cell survival. In the future, modulation of WNT4 expression in men with cryptorchidism and spermatogenic failure due to E2F1 copy number variations may provide a novel approach to improve their spermatogenesis and perhaps their fertility potential after orchidopexy.

Yiguanjian decoction inhibits macrophage M1 polarization and attenuates hepatic fibrosis induced by CCl4/2-AAF

Context Our previous studies indicated that Yiguanjian decoction (YGJ) has an anti-hepatic-fibrosis effect and could regulate macrophage status. Objective To elucidate the mechanism of YGJ in regulating macrophages. Materials and methods Liver cirrhosis was induced by CCl4 for 12 weeks combined with 2-acetylaminofluorene (2-AAF) for the last 4 weeks in male Wistar rats. YGJ (3.56 mg/kg) orally administered in the last 4 weeks, and SORA (1 mg/kg) as control. In vitro, RAW264.7 cells were treated with lipopolysaccharides (LPSs) to induce macrophage polarization to the M1 phenotype, and they were co-cultured with WB-F344 cells and allocated to M group, YGJ group (2 μg/mL) and WIF-1 group (1 μg/mL) with untreated cells as control. The differentiation direction of WB-F344 cell line was observed in the presence or absence of YGJ. Pathology, fibrosis-related cytokines, macrophage polarization-related components, and Wnt signalling pathway components were detected. Results In vivo, the expression levels of α-SMA, Col (1), OV6, SOX9, EpCAM and M1 macrophage-related components (STAT1, IRF3, IRF5, IRF8, SOCS3) significantly decreased in the YGJ group compared with those in the 2-AAF/CCl4 group (p < 0.01 or 0.05). In vitro, the expression levels of M1 macrophage-related components, including STAT1, NF-κB, IRF3, IRF5, and SOCS3, in RAW264.7 cells decreased significantly in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). The expression levels of Wnt3A, FZD5, LRP-5/-6, and β-catenin significantly increased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). In addition, the expression levels of Wnt-4/-5A/-5B, and FZD2 significantly decreased in the YGJ group compared with those in the M group (p < 0.05 or p < 0.01). Conclusion This study suggests that the anti-cirrhosis effect of YGJ is associated with its ability to inhibit macrophage M1-polarization, which provides a scientific basis for the clinical application of YGJ.

WNT4 secreted by tumor tissues promotes tumor progression in colorectal cancer by activation of the Wnt/\u03b2-catenin signalling pathway

BackgroundWingless and Int-related protein (Wnt) ligands are aberrantly expressed in patients with colorectal cancer (CRC). However, the aberrant level of Wnt ligands in serum have not been explored. Here, we aimed to identify the levels of WNT4 in serum and explored its oncogenic role in CRC.MethodsThe Oncomine database was used to analyze the relationship between WNT4 and the prognosis of CRC. ELISA was performed to measure WNT4 levels in serum and conditioned medium from fresh CRC tissues and adjacent normal tissues. Western blot and immunohistochemistry were carried out to measure the expression of WNT4 in human CRC tissues and adjacent normal tissues. The migration and invasion of CRC cells were determined by trans-well assay, and the effects of WNT4 on CRC invasion and metastasis in vivo were verified by tumor xenograft in nude mice. Cancer-associated fibroblasts (CAFs) and angiogenesis in subcutaneous nodules were detected by immunofluorescence (IF). In addition, the suspended spheres formation and tube formation assay were performed to explore the effects of WNT4 on CAFs and angiogenesis respectively.ResultsWNT4 was significantly upregulated in serum of CRC patients, and CRC tissues were identified as an important source of elevated WNT4 levels in CRC patients. Interestingly, elevated levels of WNT4 in serum were downregulated after tumor resection. Furthermore, we found that WNT4 contributed to epithelial-to-mesenchymal transition (EMT) and activated fibroblasts by activating the WNT4/β-catenin pathway in vitro and in vivo. Moreover, angiogenesis was induced via the WNT4/β-catenin/Ang2 pathway. Those effects could be reversed by ICG-001, a β-catenin/TCF inhibitor.ConclusionOur findings indicated that serum levels of WNT4 may be a potential biomarker for CRC. WNT4 secreted by colorectal cancer tissues promote the progression of CRC by inducing EMT, activate fibroblasts and promote angiogenesis through the canonical Wnt/β-catenin signalling pathway.

Identification of Foxl2 in freshwater mussel Hyriopsis cumingii and its involvement in sex differentiation.

The freshwater mussel Hyriopsis cumingii, is the most important species for pearl culture in China. At present, the mechanisms underlying sex differentiation and determination remain unclear in this species. Herein the open reading frame (ORF) of Foxl2 from H. cumingii (Hc-Foxl2) was cloned, and Hc-Foxl2 expression levels in six tissues (the gonad, gill, adductor muscle, foot, mantle, and kidney) were determined. Further, we performed quantitative real-time PCR to compare expressions levels between 1 and 8months of age and 1-, 2-, and 3-year-old H. cumingii. The localization of Hc-Foxl2 expression in the ovary was analyzed by in situ hybridization, and its function was explored using RNA interference. We found that the ORF region of Hc-Foxl2 was 1215bp in length, encoded 404 amino acids, and contained conserved FH domains. Hc-Foxl2 was expressed in the male and female tissues, with the expression levels being significantly higher in the ovary than in the testis. In 1-8-month-old H. cumingii, Hc-Foxl2 was expressed at the highest level at 5months of age, and the gonads began to differentiate at the same time. Moreover, in 1-, 2-, and 3-year-old individuals, Hc-Foxl2 expression levels in the ovaries gradually decreased, but they were higher than those in the testis. Strong hybridization signals for Hc-Foxl2 were detected on the oocyte membrane in 3-year-old female mussels. We also performed double-stranded RNA (dsRNA) interference experiments using three dsRNA strands, which were injected into 5-month-old H. cumingii; the interference effects were the best at 12h and 48h post-injection. After interference with Hc-Foxl2, the expression levels of Wnt4, which has an antagonistic relationship with Foxl2 during ovarian development, were slightly increased. Thus, we speculate that Hc-Foxl2 is a female-related gene in H. cumingii and that it is involved in sex differentiation and ovarian development.

Effect of sitagliptin on tubulointerstitial Wnt/β-catenin signalling in diabetic nephropathy.

To investigate the effect of sitagliptin on Wnt/β-catenin signalling in the tubulointerstitium of diabetic nephropathy. Forty male Wistar rats were divided into normal control (NC), diabetic model (DM), low and high-dose sitagliptin intervention groups (ST1 and ST2, respectively). Changes in the biochemical parameters and tubulointerstitial fibrosis index were observed. The levels of protein and gene expression of different indicators were detected via immunohistochemistry and real-time polymerase chain reaction. NRK-52E cells were divided into the normal control group, mannitol control group, high glucose group (HG), high glucose plus sitagliptin intervention group (HG + ST) and high glucose plus Wnt/β-catenin inhibitor group (HG + XAV939). The relevant indicators were examined by Western blot or enzyme-linked immunosorbent assay. Compared with the NC group, the blood glucose, glycosylated haemoglobin, 24 h urinary albumin, creatinine clearance and tubulointerstitial fibrosis index were significantly increased in the DM group. These parameters were decreased in the ST1 and ST2 groups compared to the DM group. Compared with the NC group, the levels of Wnt4, β-catenin, dipeptidyl peptidase-4 and α-smooth muscle actin were higher and E-cadherin was lower in the DM group. Sitagliptin treatment reversed these changes. In the high glucose-stimulated NRK-52E cells, sitagliptin and XAV939 inhibited the elevated expression of Wnt4, β-catenin, dipeptidyl peptidase-4, α-smooth muscle actin, transforming growth factor-β and fibronectin and restored E-cadherin activity. Sitagliptin may inhibit the tubulointerstitial Wnt/β-catenin signalling pathway in diabetic nephropathy and provide renal protection by alleviatinge renal tubulointerstitial transdifferentiation and fibrosis.

Salvia miltiorrhiza protects against diabetic nephropathy through metabolome regulation and wnt/β-catenin and TGF-β signaling inhibition.

Diabetic nephropathy (DN) is a complication of diabetes that is caused by uncontrolled high blood sugar. It has been reported that Salvia miltiorrhiza (SM) possesses the ability to prevent kidney damage, although the mechanisms remain unclear. The study was to investigate whether and how SM improved DN injury via regulation of metabolome and the molecular mechanisms. In this study, SD rats were fed a high glucose / high fat diet accompanied by 0.5% glucose water. Three weeks later, the rats were given one intraperitoneal injection of 30 mg/kg STZ each day for three days for DN model. The biochemical indicators and metabolomics of plasma, urine and renal tissue were analyzed. Then the western blotting analysis of renal tissue and glomerular mesangial cells were investigated. The results showed that Salvia miltiorrhiza extracts improved the renal injury and regulation of abnormal glycolipid metabolism. The metabolites in serum, urine and renal tissues have been changed significantly. The involved metabolic pathways mainly include phospholipid, arachidonic acid, and pyrimidine metabolisms. Meanwhile, SM inhibited the relative expression levels of wnt4, β-catenin and TGF-β in renal tissue and high-glucose induced glomerular mesangial cells.

The osteoarthritis-associated gene PAPSS2 promotes differentiation and matrix formation in ATDC5 chondrogenic cells.

3′-Phosphoadenosine 5′-phosphosulfate synthetase 2 (PAPSS2) has been shown to be important in the development of normal skeletal structure. The aim of the present study was to evaluate the role of PAPSS2 in the differentiation of chondrocytes as well as their mechanisms. Using RNA interference-mediated via a lentivirus and a retrovirus, PAPSS2 gene silence and overexpression in ATDC5 chondrogenic cells were performed. Chondrocyte differentiation and chondrogenic-related gene markers associated with extracellular matrix formation were noted. The mRNA and protein expression for Wnt4, β-catenin and SOX9 genes were observed. The PAPSS2 transcript expression levels progressively decline in ATDC5-induced chondrocyte-like cells during differentiation. Silencing of PAPSS2 expression had a significantly attenuating effect on cell differentiation and decreased expression of collagen II and X. In contrast, over-expression of PAPSS2 promoted the differentiation of ATDC5 chondrogenic cells. The mRNA expression levels of Wnt4 and SOX9 decreased significantly in PAPSS2 knock down cells vs. control cells. However, this expression was increased in the cells over-expressing PAPSS2. These data indicate that PAPSS2 regulates aggrecan activity as well as cell differentiation. The findings favor a mechanism by which PAPSS2 induces differentiation in ATDC5 cells via direct regulation of early signaling events that promote formation of collagenous matrix components. This control is probably mediated via extracellular matrix formation Wnt/β-catenin signaling pathways.

Suppression of Wnt/β-catenin signaling by EGF receptor is required for hair follicle development.

Mice that lack the epidermal growth factor receptor (EGFR) fail to develop a hair coat, but the mechanism responsible for this deficit is not completely understood. Here, we show that EGFR plays a critical role to attenuate wingless-type MMTV integration site family member (Wnt)/β-catenin signaling during postnatal hair follicle development. Genetic ablation of EGFR in mice resulted in increased mitotic activity in matrix cells, apoptosis in hair follicles, and impaired differentiation of epithelial lineages that form hair. EGFR is activated in wild-type hair follicle stem cells marked with SOX9 or NFATc1 and is essential to restrain proliferation and support stem cell numbers and their quiescence. We observed elevated levels of Wnt4, 6, 7b, 10a, 10b, and 16 transcripts and hyperactivation of the β-catenin pathway in EGFR knockout follicles. Using primary keratinocytes, we linked ligand-induced EGFR activation to suppression of nascent mRNA synthesis of Wnt genes. Overexpression of the Wnt antagonist sFRP1 in mice lacking EGFR demonstrated that elevated Wnts are a major cause for the hair follicle defects. Colocalization of transforming growth factor α and Wnts regulated by EGFR in stem cells and progeny indicates that EGFR autocrine loops control Wnts. Our findings define a novel mechanism that integrates EGFR and Wnt/β-catenin pathways to coordinate the delicate balance between proliferation and differentiation during development.

Effect of human umbilical cord blood derived CD34+ hematopoietic stem cell on the expression of Wnt4 and P53 genes in a rat model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary liver malignancy. Chronic liver injuries as chronic hepatitis C and hepatitis B viruses, aflatoxins consumption and nonalcoholic fatty liver disease are well-established causes of HCC. HCC is associated with a series of molecular changes, as alternation in glypican-3, P53 expression and Wnt/β-catenin pathway. Hepatic cancer progenitor cells could contribute to HCC development. This research aimed to study the effectiveness of human CD34+ hematopoietic stem cell on Wnt4 and P53 genes expression, histopathological grading and hepatic progenitor cells percentage in HCC rat model. HCC was induced in the experimental group of outbred Sprague Dawley rats by administration of 50 mg/L N-nitroso-Di-Ethylamine (DEN) in drinking water for 15 weeks. Forty-six animals were used in total, they were initially subdivided into two groups; control (n = 6) and experimental (n = 40), the latter consisting of 4 DEN-unaffected, 6 DEN-lethalities and 30 surviving DEN-animals with elevated AFP. These 30 animals with elevated AFP were then subdivided into a new HCC control group (n = 15) and the stem cell treated group (n = 15). The latter group was injected with CD34+ human hematopoietic stem cell (1 × 106 cells/rat) in the rat's tail vein. Cyclosporine A (10 mg/kg) was injected intraperitoneal, starting 24 h before human stem cell transplantation. After 20 weeks passing since the beginning of the experiment, all rats were sacrificed and liver specimens were subjected to histopathological examination, RT-PCR in order to examine Wnt4 and P53 gene expression and flow cytometry to measure hepatic progenitor OV6 positive cells percentage. The saline-treated HCC group (with prior 15 week DEN exposure) showed higher levels of wnt4 and p53 gene expression (1.59 and 1.36 fold, respectively) and increased percentage in OV6+ progenitor cells (+4.9% in absolute terms) compared to saline-treated controls (p < 0.01, ANOVA). Compared with the saline HCC-group, transplantation with CD34+ human hematopoietic stem cells produced a further increase in the levels of wnt4 (+19.4%) and p53 gene expression (+53%), a 2-fold increase in the percentage of cancer progenitor cells and increased HCC pathology grading (all p < 0.01). The positive correlation between p53 and HCC grade (Spearman rho +0.73, p < 0.05) and negative correlation between wnt and OV6+% levels (rho -0.65, p < 0.05) in the saline-HCC group were not observed in the CD34+ HCC group. Human CD34+ cells transplantation has a deteriorating effect on HCC.

Psoralen promotes the expression of cyclin D1 in chondrocytes via the Wnt/β-catenin signaling pathway.

Psoralen (PSO), the active ingredient of Fructus Psoraleae (FP) the dried ripe fruit of Psoralea corylifolia L., has been commonly used in traditional Chinese medicine (TCM) for the treatment of osteoarthritis (OA). We found that PSO activates cartilaginous cellular functions of rat chondrocytes in vitro. However, the effect of PSO on chondrocyte proliferation and the precise mechanisms involved remain to be elucidated. We investigated the effects of PSO on chondrocytes isolated from Sprague-Dawley (SD) rats and evaluated involvement of the Wnt/β-catenin signaling pathway. The viability of chondrocytes treated with PSO was increased in a dose- and time-dependent manner, as assessed by MTT assay. We found that the gene expression and protein levels of Wnt-4, Frizzled-2, β-catenin and cyclin D1 in the PSO-treated chondrocytes were significantly upregulated, while the gene expression and protein level of glycogen synthase kinase-3β (GSK-3β) were downregulated, compared with the untreated chondrocytes. By immunofluorescence, we also found that PSO induced β-catenin nuclear translocation. Importantly, the expression of β-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/β-catenin signaling pathway. Additionally, Col-II expression in chondrocytes was increased after treatment with PSO. Taken together, these results indicate that PSO promotes chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway, and it may play an important role in the treatment of OA.

Decreased Wnt4 expression inhibits thymoma development through downregulation of FoxN1.

The Wnt signaling pathway controls the development of thymic epithelial cells by regulating the expression of FoxN1. Thymoma is a type of malignant tumor arising from the thymic epithelial cells. To determine whether Wnt4 and FoxN1 are involved in the pathogenesis of thymoma, this study determined the mRNA and protein levels of Wnt4 and Foxn1 in thymoma, and analyzed the effect of thymoma cell apoptosis and tumor growth in nude mice after Wnt4 and FoxN1 downregulation. Wnt4 and FoxN1 mRNA and protein levels in thymoma tissues were analyzed by RT-qPCR and immunohistochemistry, respectively. Thymoma cells were cultured and transfected with siRNA targeting the Wnt4, JNK, and FoxN1 genes. Apoptosis of thymoma cells were analyzed after Wnt4 and FoxN1 downregulation. In addition, thymoma cells were inoculated into nude mice and tumor growth was analyzed. The rates of expression of Wnt4 and FoxN1 protein were 64.3% and 58.9%, while the levels of mRNA expression were 2.56±0.04 and 1.83±0.11, respectively. With increasing malignancy of thymoma, the rates of positivity for Wnt4 and FoxN1 mRNA and protein expression gradually increased. Upon interfering with Wnt4, JNK, and FoxN1 gene expression by using siRNA technology, the inhibition rates were 56.7%, 72.6%, and 63.2%, respectively. The expression of FoxN1 mRNA and protein was decreased after Wnt4 and JNK downregulation. After downregulation of Wnt4 and FoxN1 gene expression, the apoptosis rate of thymoma cells increased and the tumor volume decreased in nude mice. High expression of Wnt4 and FoxN1 may play an important role in the generation and development of thymoma. The FoxN1 gene produced a marked downstream effect through the regulation of Wnt4. Determining the positivity for both Wnt4 and FoxN1 can help us to evaluate the level of malignancy of thymoma.

Silencing Med12 Gene Reduces Proliferation of Human Leiomyoma Cells Mediated via Wnt/β-Catenin Signaling Pathway.

Uterine fibroids, or leiomyoma, are the most common benign tumors in women of reproductive age. In this work, the effect of silencing the mediator complex subunit 12 (Med12) gene in human uterine fibroid cells was evaluated. The role of Med12 in the modulation of Wnt/β-catenin and cell proliferation-associated signaling was evaluated in human uterine fibroid cells. Med12 was silenced in the immortalized human uterine fibroid cell line (HuLM) using a lentivirus-based Med12 gene-specific RNA interference strategy. HuLM cells were infected with lentiviruses carrying Med12-specific short hairpin RNA (shRNA) sequences or a nonfunctional shRNA scrambled control with green fluorescence protein. Stable cells that expressed low levels of Med12 protein were characterized. Wnt/β-catenin signaling, sex steroid receptor signaling, cell cycle-associated, and fibrosis-associated proteins were measured. Med12 knockdown cells showed significantly (P < 0.05) reduced levels of Wnt4 and β-catenin proteins as well as cell proliferation, as compared with scrambled control cells. Med12 knockdown cells also showed reduced levels of cell cycle-associated cyclin D1, Cdk1, and Cdk2 proteins as well as reduced activation of p-extracellular signal-regulated kinase, p-protein kinase B, and transforming growth factor (TGF)-β signaling pathways as compared with scrambled control cells. Moreover, TGF-β-regulated fibrosis-related proteins such as fibronectin, collagen type 1, and plasminogen activator inhibitor-1 were significantly (P < 0.05) reduced in Med12 knockdown cells as compared with scrambled control cells. Together, these results suggest that Med12 plays a key role in the regulation of HuLM cell proliferation through the modulation of Wnt/β-catenin, cell cycle-associated, and fibrosis-associated protein expression.

Dysfunction of WNT4/WNT5A in deciduas: possible relevance to the pathogenesis of preeclampsia.

Preeclampsia affects 5-7% of all human pregnancies worldwide. It is characterized primarily by hypertension and proteinuria. Although the pathogenesis of preeclampsia is still not fully understood, the deficiency in decidualization is considered to have a role in this disease. WNT4 and WNT5A are highly expressed in the deciduas of mice and human beings; their deficiency causes the abnormality of mouse decidualization, and consequently affects the development of the placenta and the fetus. We aimed to define the roles of WNT4 and WNT5A in human decidulization and their relationship with preeclampsia. In this study, we investigated the expression of WNT4 and WNT5A in human deciduas and their functions during decidualization, as well as their relationship with preeclampsia. We obtained deciduas from 20 women whose pregnancies were complicated by severe preeclampsia and from 20 women with uncomplicated pregnancies, as well as human endometrial stromal cells (hESCs). The levels of WNT4 and WNT5A were examined by real-time PCR, immunohistochemistry, and western blotting in deciduas. The expression of WNT4 and WNT5A in cultured hESCs was determined during decidualization. Furthermore, the roles of WNT4, WNT5A, and BMP2 on decidualization as well as their interactions were studied in cultured hESCs by interference of their expression with specific small interfering RNA, respectively. WNT4 and WNT5A were present in the decidual tissues of women with preeclampsia and women with uncomplicated pregnancies. Their expression levels of mRNA and protein in women with severe preeclampsia were significantly lower compared with women with uncomplicated pregnancies (P < 0.05). WNT4 and WNT5A were upregulated in decidualized hESCs. Knocking down of BMP2 or WNT4 in hESCs resulted in a significant increase of WNT5A mRNA and a significant reduction in the transcription of decidualization markers, insulin-like growth factor binding protein1 and prolactin (P < 0.05). In addition, silencing of WNT5A resulted in a reduction of the transcription of insulin-like growth factor binding protein1 and prolactin mRNA (P < 0.05). Our data suggest that WNT4 and WNT5 are relevant to normal decidualization, and thus trophoblast invasion and implantation. Their deficiency is likely involved in the development of preeclampsia.

Yi Qi Qing Re Gao-containing serum inhibits lipopolysaccharide-induced rat mesangial cell proliferation by suppressing the Wnt pathway and TGF-β1 expression.

The aim of the present study was to investigate the effect of Yi Qi Qing Re Gao-containing serum (YQ-S) on rat mesangial cell (MC) proliferation and to investigate the underlying mechanism. MCs were divided into the control, lipopolysaccharide (LPS)-stimulated, YQ-S and fosinopril-containing serum (For-S) groups, and cultured for 48 h. An MTT assay was used to evaluate the proliferation of MCs. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were conducted to detect the expression levels of Wnt4, β-catenin and transforming growth factor (TGF)-β1 in MCs. The results indicated that YQ-S inhibited LPS-induced MC proliferation. The Wnt4 and TGF-β1 mRNA expression levels were reduced in the YQ-S group (P<0.01 or P<0.05). Furthermore, the Wnt4, β-catenin and TGF-β1 protein expression levels were suppressed in the YQ-S group (P<0.01 or P<0.05). Therefore, YQ-S appears to inhibit MC proliferation, and its mechanism may involve the inhibition of the Wnt signaling pathway and downregulation of TGF-β1 expression.

Vitamin D3 Inhibits Wnt/β-Catenin and mTOR Signaling Pathways in Human Uterine Fibroid Cells.

Somatic mutations in the Med12 gene are known to activate Wnt/β-catenin signaling in human uterine fibroids (UFs). The objective of the study was to examine the role of vitamin D3 in the modulation of Wnt/β-catenin and mammalian target of rapamycin (mTOR) signaling in human UF cells. Immortalized human UF cells (HuLM) and human primary UF (PUF) cells were treated with increasing concentrations of vitamin D3 and thereafter analyzed using Western blots and immunocytochemistry. Wnt/β-catenin and mTOR signaling proteins in cultured HuLM and PUF cells were measured. UF tumors with Med12 somatic mutations showed an up-regulation of Wnt4 and β-catenin as compared with adjacent myometrium. Vitamin D3 administration reduced the levels of Wnt4 and β-catenin in both HuLM and PUF cells. Vitamin D3 also reduced the expression/activation of mTOR signaling in both cell types. In contrast, vitamin D3 induced the expression of DNA damaged-induced transcription 4 (an inhibitor of mTOR) and tuberous sclerosis genes (TSC1/2) in a concentration-dependent manner in HuLM cells. Furthermore, we observed a concentration-dependent reduction of Wisp1 (Wnt induced signaling protein 1) and flap endonuclease 1 proteins in HuLM cells. Additionally, abrogation of vitamin D receptor expression (by silencing) in normal myometrial cells induces Wnt4/β-catenin as well as prompts a fibrotic process including an increase in cell proliferation and increased extracellular matrix production. Together these results suggest that vitamin D3 functions as an inhibitor of Wnt4/β-catenin and mTOR signaling pathways, which may play major roles in fibroid pathogenesis. Vitamin D3 may have utility as a novel long-term therapeutic and/or preventive option for uterine fibroids.

Wnt4 signaling is associated with the decrease of proliferation and increase of apoptosis during age-related thymic involution.

The thymus is a central lymphoid organ that is responsible for T-lymphocyte development and maturation. Through negative and positive selection, lymphoid progenitor cells, which initiate from the bone marrow, develop into mature T cells in the thymus, and are subsequently involved in peripheral cell immunity. It has been reported that the Wnt signaling pathway exists widely in thymic epithelial cells and T lymphocytes. Wnt signaling affects the shape and function of thymic epithelial cells and has an important role in maintaining pro‑T‑cells, and in the subsequent T‑cell differentiation. Previous studies have demonstrated that the Wnt signaling pathway participates in age‑associated thymic involution. In the present study alterations in proliferation and apoptosis were investigated in murine thymic cells during aging. The results of the present study demonstrated that the aged thymus was characterized by markedly decreased cell numbers, as well as decreased proliferation and increased apoptosis. Concurrently, age‑associated changes in thymic cell number and function were accompanied by a decrease in the transcription levels of Wnt4, and downregulation of forkhead box N1 and B‑cell lymphoma‑extra large, which are two target genes of the Wnt4 signaling pathway. In vitro studies demonstrated that activation of the Wnt4 signaling pathway promotes mouse thymus epithelial cell 1 (MTEC1) cell proliferation, and that Wnt4 signaling modulation alleviates dexamethasone‑mediated MTEC1 cell apoptosis. These results suggest that normal expression levels of Wnt4 have a critical role in maintaining the balance between cell proliferation and apoptosis. Alterations in the Wnt signaling pathway may disrupt the epithelial network structure of the thymus, eventually leading to microenvironmental damage. Therefore, further studies regarding the effects of the Wnt signaling pathway on thymus development and age-related thymic involution, may be beneficial for improving the health conditions of the elderly.