Inhibition of GSK‐3β enhances neural differentiation in unrestricted somatic stem cells

GSK‐3β is a key molecule in several signalling pathways, including the Wnt/β‐catenin signalling pathway. There is increasing evidence suggesting Wnt/β‐catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/β‐catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/β‐catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6‐bromoindirubin‐3′‐oxime), a specific GSK‐3β inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (β‐tubulin III). Moreover, the expression of pGSK‐3β and stabilized β‐catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK‐3β in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/β‐catenin signalling pathway towards neural fate.     

miR‐193b represses influenza A virus infection by inhibiting Wnt/β‐catenin signalling

Due to an increasing emergence of new and drug‐resistant strains of the influenza A virus (IAV), developing novel measures to combat influenza is necessary. We have previously shown that inhibiting Wnt/β‐catenin pathway reduces IAV infection. In this study, we aimed to identify antiviral human microRNAs (miRNAs) that target the Wnt/β‐catenin signalling pathway. Using a miRNA expression library, we identified 85 miRNAs that up‐regulated and 20 miRNAs that down‐regulated the Wnt/β‐catenin signalling pathway. Fifteen miRNAs were validated to up‐regulate and five miRNAs to down‐regulate the pathway. Overexpression of four selected miRNAs (miR‐193b, miR‐548f‐1, miR‐1‐1, and miR‐509‐1) that down‐regulated the Wnt/β‐catenin signalling pathway reduced viral mRNA, protein levels in A/PR/8/34‐infected HEK293 cells, and progeny virus production. Overexpression of miR‐193b in lung epithelial A549 cells also resulted in decreases of A/PR/8/34 infection. Furthermore, miR‐193b inhibited the replication of various strains, including H1N1 (A/PR/8/34, A/WSN/33, A/Oklahoma/3052/09) and H3N2 (A/Oklahoma/309/2006), as determined by a viral reporter luciferase assay. Further studies revealed that β‐catenin was a target of miR‐193b, and β‐catenin rescued miR‐193b‐mediated suppression of IAV infection. miR‐193b induced G0/G1 cell cycle arrest and delayed vRNP nuclear import. Finally, adenovirus‐mediated gene transfer of miR‐193b to the lung reduced viral load in mice challenged by a sublethal dose of A/PR/8/34. Collectively, our findings suggest that miR‐193b represses IAV infection by inhibiting Wnt/β‐catenin signalling.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.     

Silencing long non‐coding RNA MEG3 accelerates tibia fraction healing by regulating the Wnt/β‐catenin signalling pathway

As fracture healing is related to gene expression, fracture healing is prospected to be implicated in long non‐coding RNAs (lncRNAs). This study focuses on the effects of epigenetic silencing of long non‐coding RNA maternally expressed gene 3 (lncRNA MEG3) on fracture healing by regulating the Wnt/β‐catenin signalling pathway. Genes expressed in fracture were screened using bioinformatics and the subcellular location of MEG3 was determined using FISH. Next, we successfully established tibia fracture (TF) models of C57BL/6J and Col2a1‐ICAT mice and the effect of silencing lncRNA MEG3 on fracture healing was detected after TF mice were treated with phosphate buffer saline (PBS), MEG3 siRNA and scramble siRNA. X‐ray imaging, Safranin‐O/fast green and haematoxylin‐eosin (HE) staining and histomorphometrical and biomechanical analysis were adopted to observe and to detect the fracture healing conditions. Additionally, the positive expression of collagen II and osteocalcin was examined using immunohistochemistry. At last, in the in vitro experiment, the relationship of MEG3 and the Wnt/β‐catenin signalling pathway in fraction healing was investigated. MEG3 was located in the cell nucleus. In addition, it was found that MEG3 and the Wnt/β‐catenin signalling pathway were associated with fraction healing. Moreover, silencing MEG3 was proved to elevate callus area and maximum bending load and to furthermore enhance the recanalization of bone marrow cavity. Finally, MEG3 knockdown elevated levels of Col10a1, Runx2, Osterix, Osteocalcin, Wnt10b and β‐catenin/β‐catenin whereas it reduced p‐GSK‐3β/GSK‐3β levels. Taken together, our data supported that epigenetic silencing of lncRNA MEG3 could promote the tibia fracture healing by activating the Wnt/β‐catenin signalling pathway.     

β‐Catenin and Rho GTPases as downstream targets of TGF‐β1 during pulp repair

TGF‐β1 (transforming growth factor‐β1) plays a central role in regulating proliferation, migration and differentiation of dental pulp cells during the repair process after tooth injury. Our previous study showed that p38 mitogen‐activated protein kinase may act downstream of TGF‐β1 signalling to effect the differentiation of dental pulp cells. However, the molecular mechanisms that trigger and regulate the process remain to be elucidated. TGF‐β1 interacts with signalling pathways such as Wnt/β‐catenin and Rho to induce diverse biological effects. TGF‐β1 activates β‐catenin signalling, increases β‐catenin nuclear translocation and interacts with LEF/TCF to regulate gene expression. Morphologic changes in response to TGF‐β1 are associated with activation of Rho GTPases, but are abrogated by inhibitors of Rho‐associated kinase, a major downstream target of Rho. These results suggest that the Wnt/β‐catenin and Rho pathways may mediate the downstream events of TGF‐β1 signalling.     

MicroRNA‐140‐5p elevates cerebral protection of dexmedetomidine against hypoxic–ischaemic brain damage via the Wnt/β‐catenin signalling pathway

Hypoxia–ischaemia (HI) remains a major cause of foetal brain damage presented a scarcity of effective therapeutic approaches. Dexmedetomidine (DEX) and microRNA‐140‐5p (miR‐140‐5p) have been highlighted due to its potentially significant role in the treatment of cerebral ischaemia. This study was to investigate the role by which miR‐140‐5p provides cerebral protection using DEX to treat hypoxic–ischaemic brain damage (HIBD) in neonatal rats via the Wnt/β‐catenin signalling pathway. The HIBD rat models were established and allocated into various groups with different treatment plans, and eight SD rats into sham group. The learning and memory ability of the rats was assessed. Apoptosis and pathological changes in the hippocampus CA1 region and expressions of the related genes of the Wnt/β‐catenin signalling pathway as well as the genes responsible of apoptosis were detected. Compared with the sham group, the parameters of weight, length growth, weight ratio between hemispheres, the rate of reaching standard, as well as Bcl‐2 expressions, were all increased. Furthermore, observations of increased levels of cerebral infarction volume, total mortality rate, response times, total response duration, expressions of Wnt1, β‐catenin, TCF‐4, E‐cadherin, apoptosis rate of neurons, and Bax expression were elevated. Following DEX treatment, the symptoms exhibited by HIBD rats were ameliorated. miR‐140‐5p and si‐Wnt1 were noted to attenuate the progression of HIBD. Our study demonstrates that miR‐140‐5p promotes the cerebral protective effects of DEX against HIBD in neonatal rats by targeting the Wnt1 gene through via the negative regulation of the Wnt/β‐catenin signalling pathway.     

Ginsenoside Rb1 ameliorates CKD‐associated vascular calcification by inhibiting the Wnt/β‐catenin pathway

Vascular calcification (VC) is a pathological process underpinning major cardiovascular conditions and has attracted public attention due to its high morbidity and mortality. Chronic kidney disease (CKD) is a common disease related to VC. Ginsenoside Rb1 (Rb1) has been reported to protect the cardiovascular system against vascular diseases, yet its role in VC and the underlying mechanisms remain unclear. In this study, we established a CKD‐associated VC rat model and a β‐glycerophosphate (β‐GP)‐induced vascular smooth muscle cell (VSMC) calcification model to investigate the effects of Rb1 on VC. Our results demonstrated that Rb1 ameliorated calcium deposition and VSMC osteogenic transdifferentiation both in vivo and in vitro. Rb1 treatment inhibited the Wnt/β‐catenin pathway by activating peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), and confocal microscopy was used to show that Rb1 inhibited β‐catenin nuclear translocation in VSMCs. Furthermore, SKL2001, an agonist of the Wnt/β‐catenin pathway, compromised the vascular protective effect of Rb1. GW9662, a PPAR‐γ antagonist, reversed Rb1's inhibitory effect on β‐catenin. These results indicate that Rb1 exerted anticalcific properties through PPAR‐γ/Wnt/β‐catenin axis, which provides new insights into the potential theraputics of VC.     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.     

miR‐150‐5p suppresses the stem cell‐like characteristics of glioma cells by targeting the Wnt/β‐catenin signaling pathway

Glioma is the most common brain tumor malignancy with high mortality and poor prognosis. Emerging evidence suggests that cancer stem cells are the key culprit in the development of cancer. MicroRNAs have been reported to be dysregulated in many cancers, while the mechanism underlying miR‐150‐5p in glioma progression and proportion of stem cells is unclear. The expression levels of miR‐150‐5p and catenin beta 1 (CTNNB1, which encodes β‐catenin) were measured by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot. The expression levels of downstream genes of the Wnt/β‐catenin pathway and stem cell markers were detected by qRT‐PCR. Tumorigenesis was investigated by cell viability, colony formation, and tumor growth in vitro and in vivo. The interaction between miR‐150‐5p and β‐catenin was explored via bioinformatics analysis and luciferase activity assay. We found that miR‐150‐5p was downregulated in glioma and its overexpression inhibited cell proliferation, colony formation, and tumor growth. Moreover, miR‐150‐5p directly suppressed CTNNB1 and negatively regulated the abundances of downstream genes of the Wnt/β‐catenin pathway and stem cell markers. Furthermore, miR‐150‐5p expression was decreased and β‐catenin level was enhanced in CD133+ glioma stem cells. Knockdown of miR‐150‐5p contributed to CD133? cells with stem cell‐like phenotype, whereas overexpression of miR‐150‐5p suppressed CD133+ glioma stem cell‐like characteristics. In conclusion, miR‐150‐5p inhibited the progression of glioma by controlling stem cell‐like characteristics via regulating the Wnt/β‐catenin pathway, providing a novel target for glioma treatment.     

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/β‐catenin pathway and attenuate GSK3β activity

The canonical Wnt/β‐catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co‐receptor for Wnt/β‐catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β‐mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane‐anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6‐ICD) can activate the Wnt/β‐catenin pathway in a β‐catenin and TCF/LEF‐1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6‐ICD to attenuate GSK3β activity and modulate activation of the Wnt/β‐catenin pathway requires phosphorylation of the LRP6‐ICD PPP(S/T)P motifs, in a manner similar to the membrane‐anchored LRP6 intracellular domain. Here we provide evidence that the LRP6‐ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β‐catenin resulting in activation of TCF/LEF‐1 and the Wnt/β‐catenin pathway. LRP6‐ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β‐mediated phosphorylation of β‐catenin and tau to the same extent. These data indicate that the LRP6‐ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6‐ICD may be distinct from membrane‐anchored LRP6, and that release of the LRP6‐ICD may provide a complimentary signaling cascade capable of modulating Wnt‐dependent gene expression. J. Cell. Biochem. 108: 886–895, 2009. © 2009 Wiley‐Liss, Inc.     

DKK3 blocked translocation of β‐catenin/EMT induced by hypoxia and improved gemcitabine therapeutic effect in pancreatic cancer Bxpc‐3 cell

The Wnt/β‐catenin signalling pathway is activated in pancreatic cancer initiation and progression. Dickkopf‐related protein 3 (DKK3) is a member of the human Dickkopf family and an antagonist of Wnt ligand activity. However, the function of DKK3 in this pathway in pancreatic cancer is rarely known. We examined the expression of DKK3 in six human pancreatic cancer cell lines, 75 pancreatic cancer and 75 adjacent non‐cancerous tissues. Dickkopf‐related protein 3 was frequently silenced and methylation in pancreatic cancer cell lines (3/6). The expression of DKK3 was significantly lower in pancreatic cancer tissues than in adjacent normal pancreas tissues. Further, ectopic expression of DKK3 inhibits nuclear translocation of β‐catenin induced by hypoxia in pancreatic cancer Bxpc‐3 cell. The forced expression of DKK3 markedly suppressed migration and the stem cell‐like phenotype of pancreatic cancer Bxpc‐3 cell in hypoxic conditions through reversing epithelial–mesenchymal transition (EMT). The stable expression of DKK3 sensitizes pancreatic cancer Bxpc‐3 cell to gemcitabine, delays tumour growth and augments gemcitabine therapeutic effect in pancreatic cancer xenotransplantation model. Thus, we conclude from our finding that DKK3 is a tumour suppressor and improved gemcitabine therapeutic effect through inducing apoptosis and regulating β‐catenin/EMT signalling in pancreatic cancer Bxpc‐3 cell.     

MicroRNA‐708 modulates Hepatic Stellate Cells activation and enhances extracellular matrix accumulation via direct targeting TMEM88

Transmembrane protein 88 (TMEM88) is a potential 2‐transmembrane‐type protein that interacts with the PDZ domain of Dishevelled‐1 (DVL‐1), a crucial component of Wnt signalling pathway through its C‐terminal Val‐Trp‐Val (VWV) motif in Xenopus embryo cells. Since the significant function of β‐catenin in liver fibrosis, it is urgent to study the TMEM88 mechanism in liver fibrosis. The current research was for evaluating the function of TMEM88 in the process of the liver fibrosis and clarifying the inherent mechanism. The study found that TMEM88 is decreased in human fibrotic liver tissues. Functionally, TMEM88 significantly reduced the expression levels of α‐smooth muscle actin (α‐SMA) and collagen type I (Col.I) and repressed extracellular matrix (ECM) accumulation by restoring the balance between matrix metalloproteinases (MMPs) and TIMPs (tissue inhibitor of metalloproteinases). TMEM88 inhibited HSCs proliferation and evaluated the apoptosis of activated LX‐2 cells by regulating Wnt3a, Wnt2b and β‐catenin of Wnt/β‐catenin signalling pathway. Moreover, we demonstrated that miR‐708 particularly targeted TMEM88 3′‐UTR regions and down‐regulated the expression level of TMEM88 in TGF‐β1‐stimulated LX‐2 cells. MiR‐708 promoted the generation of ECM and cell activation in activated LX‐2 cells. These results determined that miR‐708 could promote HSCs activation and enhance ECM accumulation via direct targeting TMEM88 by Wnt/β‐catenin signalling pathway. This will provide a potential target for future research in the process of liver fibrosis.     

Nrf2 attenuates inflammatory response in COPD/emphysema: Crosstalk with Wnt3a/β‐catenin and AMPK pathways

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and abnormal inflammatory response. Wnt/β‐catenin and AMP‐activated protein kinase (AMPK) have been shown to modulate lung inflammatory responses and injury. However, it remains elusive whether Wnt/β‐catenin and AMPK modulate nuclear factor erythroid‐2 related factor‐2 (Nrf2)‐mediated protective responses during the development of emphysema. Here we showed that treatment with a Wnt pathway activator (LiCl) reduced elastase‐induced airspace enlargement and cigarette smoke extract (CSE)‐induced lung inflammatory responses in WT mice, which was associated with increased activation of Nrf2 pathway. Interestingly, these effects of LiCl were not observed in Nrf2^?/? mice exposed to elastase. In normal human bronchial epithelial (NHBE) cells, Wnt3a overexpression up‐regulated, whereas Wnt3a knockdown further down‐regulated the levels of Nrf2 and its target proteins heme oxygenase‐1 (HO‐1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) by CSE treatment. In contrast, Nrf2 deficiency did not have any effects on Wnt/β‐catenin pathway in mouse lungs and NHBE cells. Both elastase and CSE exposures reduced AMPK phosphorylation. A specific AMPK activator metformin increased Wnt3a, β‐catenin, Nrf2 phosphorylation and activation but reduced the levels of IL‐6 and IL‐8 in NHBE cells and mouse lungs exposed to CSE. Furthermore, Nrf2 deficiency abolished the protection of metformin against CSE‐induced increase in IL‐6 and IL‐8 in NHBE cells. In conclusion, Nrf2 mediates the protective effects of both Wnt3a/β‐catenin and AMPK on lung inflammatory responses during the development of COPD/emphysema. These findings provide potential therapeutic targets for the intervention of COPD/emphysema.