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.     

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.     

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.     

WNT unrelated activities in commercially available preparations of recombinant WNT3a

WNT signaling pathways play an important role in both development and disease. By analyzing the signaling capabilities of commercially available WNT3a preparations towards the PI3K/AKT/GSK3 signaling pathway, we discovered unexpected inconsistencies from lot to lot of recombinant WNT3a. We provide evidence that: (1) The ability to trigger AKT/GSK3 signaling varies dramatically between different lots of WNT3a, without any variation in their ability to activate the canonical WNT/β‐catenin signaling. (2) sFRP1, a WNT signaling inhibitor, is unable to interfere with the activation of AKT/GSK3 signaling induced by some of the WNT3a lots. (3) Pharmacological inhibition of AKT/GSK3 phosphorylation by PI3K inhibitors fails to affect the stabilization of β‐catenin, the central effector of the canonical WNT/β‐catenin signaling pathway. In summary, while all tested lots of recombinant WNT3a activated WNT/β‐catenin pathway, our results suggest that individual lots of recombinant WNT3a activate the PI3K/AKT/GSK3 pathway in a WNT‐independent manner, hampering thus the analysis of regulation of PI3K/AKT/GSK3 by WNT ligand. J. Cell. Biochem. 111: 1077–1079, 2010. © 2010 Wiley‐Liss, Inc.     

GSKIP,an inhibitor of GSK3β, mediates the N‐cadherin/β‐catenin pool in the differentiation of SH‐SY5Y cells

Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3β interaction protein (GSKIP) able to negatively regulate GSK3β in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH‐SY5Y cells treated with retinoic acid (RA) to differentiate to neuron‐like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH‐SY5Y cells. GSKIP may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases β‐catenin in the nucleus and raises the level of cyclin D1 to promote cell‐cycle progression in SH‐SY5Y cells. Additionally, overexpression of GSKIP downregulates N‐cadherin expression, resulting in decreased recruitment of β‐catenin. Moreover, depletion of β‐catenin by small interfering RNA, neurite outgrowth is blocked in SH‐SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3β/β‐catenin, β‐catenin/cyclin D1, and β‐catenin/N‐cadherin pool during RA signaling in SH‐SY5Y cells. J. Cell. Biochem. 108: 1325–1336, 2009. © 2009 Wiley‐Liss, Inc.     

Zbed3 promotes proliferation and invasion of lung cancer partly through regulating the function of Axin‐Gsk3β complex

Our previous work showed that Zbed3 is overexpressed in nonsmall cell lung cancer and that down‐regulation of Zbed3 inhibited β‐catenin expression and cancer cell proliferation and invasiveness. Here, we investigated Zbed3's ability to promote lung cancer cell proliferation and invasion and the involvement of the Axin/TPC/glycogen synthase kinase 3β (Gsk‐3β) complex to the response. Coimmunoprecipitation assays showed that wild‐type Zbed3 bound to Axin but a Zbed3 mutant lacking the Axin binding site did not. In A549 and H1299 lung cancer cells, Zbed3 overexpression promoted cancer cell proliferation and invasiveness, as well as Wnt signalling and expression of downstream mediators, including β‐catenin, cyclin D1 and MMP7 (P < 0.05). In contrast, the Zbed3 mutant failed to enhance β‐catenin expression (P > 0.05), and its ability to promote cancer cell proliferation and invasiveness was much less than wild‐type Zbed3 (P < 0.05). The ability of Zbed3 to increase β‐catenin levels was abolished by Axin knockdown in A549 cells (P > 0.05). Similarly, treating the cells with a GSK‐3β inhibitor abolished Zbed3's ability to increase β‐catenin levels and Wnt signalling. These results indicate that Zbed3 enhances lung cancer cell proliferation and invasiveness at least in part by inhibiting Axin/adenomatous polyposis coli/GSK‐3β‐mediated negative regulation of β‐catenin levels.     

miR‐145 is differentially regulated by TGF‐β1 and ischaemia and targets Disabled‐2 expression and wnt/β‐catenin activity

The effect of wnt/β‐catenin signalling in the response to acute myocardial infarction (AMI) remains controversial. The membrane receptor adaptor protein Disabled‐2 (Dab2) is a tumour suppressor protein and has a critical role in stem cell specification. We recently demonstrated that down‐regulation of Dab2 regulates cardiac protein expression and wnt/β‐catenin activity in mesenchymal stem cells (MSC) in response to transforming growth factor‐β₁ (TGF‐β₁). Although Dab2 expression has been shown to have effects in stem cells and tumour suppression, the molecular mechanisms regulating this expression are still undefined. We identified putative binding sites for miR‐145 in the 3′‐UTR of Dab2. In MSC in culture, we observed that TGF‐β₁ treatment led to rapid and sustained up‐regulation of pri–miR‐145. Through gain and loss of function studies we demonstrate that miR‐145 up‐regulation was required for the down‐regulation of Dab2 and increased β‐catenin activity in response to TGF‐β₁. To begin to define how Dab2 might regulate wnt/β‐catenin in the heart following AMI, we quantified myocardial Dab2 as a function of time after left anterior descending ligation. There was no significant Dab2 expression in sham‐operated myocardium. Following AMI, Dab2 levels were rapidly up‐regulated in cardiac myocytes in the infarct border zone. The increase in cardiac myocyte Dab2 expression correlated with the rapid and sustained down‐regulation of myocardial pri–miR‐145 expression following AMI. Our data demonstrate a novel and critical role for miR‐145 expression as a regulator of Dab2 expression and β‐catenin activity in response to TGF‐β₁ and hypoxia.     

Imperatorin promotes osteogenesis and suppresses osteoclast by activating AKT/GSK3 β/β‐catenin pathways

Osteoporosis is caused by disturbance in the dynamic balance of bone remodelling, a physiological process, vital for maintenance of healthy bone tissue in adult humans. In this process, a new bone is formed by osteoblasts and the pre‐existing bone matrix is resorbed by osteoclasts. Imperatorin, a widely available and inexpensive plant extract with antioxidative and apoptotic effects, is reported to treat osteoporosis. However, the underlying mechanism and specific effects on bone metabolism have not been elucidated. In this study, we used rat bone marrow‐derived mesenchymal stem cells and found that imperatorin can activate RUNX2, COL1A1 and osteocalcin by promoting the Ser9 phosphorylation of GSK3β and entry of β‐catenin into the nucleus. Imperatorin also enhanced the production of phospho‐AKT (Ser473), an upstream factor that promotes the Ser9 phosphorylation of GSK3β. We used ipatasertib, a pan‐AKT inhibitor, to inhibit the osteogenic effect of imperatorin, and found that imperatorin promotes osteogenesis via AKT/GSK3β/β‐catenin pathway. Next, we used rat bone marrow‐derived monocytes, to check whether imperatorin inhibits osteoclast differentiation via AKT/GSK3β/β‐catenin pathway. Further, we removed the bilateral ovaries of rats to establish an osteoporotic model. Intragastric administration of imperatorin promoted osteogenesis and inhibited osteoclast in vivo. Our experiments showed that imperatorin is a potential drug for osteoporosis treatment.     

Calcitonin gene‐related peptide (CGRP) inhibits apoptosis in human osteoblasts by β‐catenin stabilization

Transgenic mice over‐expressing calcitonin gene‐related peptide (CGRP) in osteoblasts have increased bone density due to increased bone formation, thus suggesting that CGRP plays a role in bone metabolism. In this study we determined the relationship between CGRP, the canonical Wnt signaling and apoptosis in human osteoblasts (hOBs) in consideration of the well‐documented involvement of this pathway in bone cells. Primary cultures of hOBs were treated with CGRP 10^?8 M. Levels of β‐catenin, which is the cytoplasmic protein mediator of canonical Wnt signaling, and mRNA were determined. CGRP increases both the expression and the levels of cytoplasmic β‐catenin by binding to its receptor, as this effect is blocked by the antagonist CGRP_8–37. This facilitatory action on β‐catenin appears to be mediated by the inhibition of the enzyme GSK‐3β via protein kinase A (PKA) activation. GSK‐3β is a glycogen synthase kinase that, by phosphorylating β‐catenin, promotes its degradation by the proteosomal machinery. Moreover, the peptide is able to inhibit hOBs apoptosis stimulated by dexamethasone or by serum deprivation, possibly through the accumulation of β‐catenin, since the inhibitor of PKA activity H89 partially prevents the antiapoptotic effect of the peptide. In conclusion CGRP, released by nerve fibers, exerts its anabolic action on bone cells by stimulating canonical Wnt signaling and by inhibiting hOBs apoptosis, thus favoring local bone regeneration. J. Cell. Physiol. 225: 701–708, 2010. © 2010 Wiley‐Liss, Inc.     

MicroRNA‐300 promotes apoptosis and inhibits proliferation,migration, invasion and epithelial‐mesenchymal transition via the Wnt/β‐catenin signaling pathway by targeting CUL4B in pancreatic cancer cells

The study aims to verify the hypothesis that up‐regulation of microRNA‐300 (miR‐300) targeting CUL4B promotes apoptosis and suppresses proliferation, migration, invasion, and epithelial‐mesenchymal transition (EMT) of pancreatic cancer cells by regulating the Wnt/β‐catenin signaling pathway. Pancreatic cancer tissues and adjacent tissues were collected from 110 pancreatic cancer patients. Expression of miR‐300, CUL4B, Wnt, β‐catenin, E‐cadherin, N‐cadherin, Snail, GSK‐3β, and CyclinD1 were detected using qRT‐PCR and Western blot. CFPAC‐1, Capan‐1, and PANC‐1 were classified into blank, negative control (NC), miR‐300 mimics, miR‐300 inhibitors, siRNA‐CUL4B, and miR‐300 inhibitors + siRNA‐CUL4B groups. The proliferation, migration, invasion abilities, the cell cycle distribution, and apoptosis rates were measured in CCK‐8 and Transwell assays. Pancreatic cancer tissues showed increased CUL4B expression but decreased miR‐300 expression. When miR‐300 was lowly expressed, CUL4B was upregulated which in‐turn activated the Wnt/β‐catenin pathway to protect the β‐catenin expression and thus induce EMT. When miR‐300 was highly expressed, CUL4B was downregulated which in‐turn inhibited the Wnt/β‐catenin pathway to prevent EMT. Weakened cell migration and invasion abilities and enhanced apoptosis were observed in the CUL4B group. The miR‐300 inhibitors group exhibited an evident increase in growth rate accompanied the largest tumor volume. Smaller tumor volume and slower growth rate were observed in the miR‐300 mimics and siRNA‐CUL4B group. Our study concludes that lowly expressed miR‐300 may contribute to highly expressed CUL4B activating the Wnt/β‐catenin signaling pathway and further stimulating EMT, thus promoting proliferation and migration but suppressing apoptosis of pancreatic cancer cells.     

Wnt/β‐catenin signaling in the mouse embryonic cranial mesenchyme is required to sustain the emerging differentiated meningeal layers

Cranial neural crest cells (CNCCs) give rise to cranial mesenchyme (CM) that differentiates into the forebrain meningeal progenitors in the basolateral and apical regions of the head. This occurs in close proximity to the other CNCC‐CM‐derivatives, such as calvarial bone and dermal progenitors. We found active Wnt signaling transduction in the forebrain meningeal progenitors in basolateral and apical populations and in the non‐meningeal CM preceding meningeal differentiation. Here, we dissect the source of Wnt ligand secretion and requirement of Wnt/β‐catenin signaling for the lineage selection and early differentiation of the forebrain meninges. We find persistent canonical Wnt/β‐catenin signal transduction in the meningeal progenitors in the absence of Wnt ligand secretion in the CM or surface ectoderm, suggesting additional sources of Wnts. Conditional mutants for Wntless and β‐catenin in the CM showed that Wnt ligand secretion and Wnt/β‐catenin signaling were dispensable for specification and proliferation of early meningeal progenitors. In the absence of β‐catenin in the CM, we found diminished laminin matrix and meningeal hypoplasia, indicating a structural and trophic role of mesenchymal β‐catenin signaling. This study shows that β‐catenin signaling is required in the CM for maintenance and organization of the differentiated meningeal layers in the basolateral and apical populations of embryonic meninges.     

Caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis through modulating GSK3β/β-catenin pathway

In this study, we investigate the anti‐cancer activity of caudatin in carcinomic human alveolar basal epithelial cell line A549 and anti‐angiogenic activity in human umbilical vein endothelial cells (HUVECs). We show that caudatin impairs the cell viability and induces G₀/G₁ phase arrest in A549 cells with a dose dependent manner. A549 cells, not HUVECs, dealing with caudatin exhibited typical characteristics of apoptosis, which were accompanied by activation of caspase‐3, caspase‐9 and Poly(ADP–Ribose) Polymerase (PARP). In addition, caudatin treatment resulted in a decrease of β‐catenin and increase of phosphorylation of β‐catenin, and inhibited phosphorylation levels of GSK3β (Ser 9) in A549 cells. Conditional medium of A549 cells‐induced or growth factors‐induced tube formation of HUVECs was markedly inhibited by caudatin treatment, which was associated with the inhibiting VEGF secretion from A549 cells by caudatin. Our findings suggest that caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis by targeting GSK3β/β‐catenin pathway and suppressing VEGF production. J. Cell. Biochem. 113: 3403–3410, 2012. © 2012 Wiley Periodicals, Inc.     

Extracellular vesicle‐mediated crosstalk between NPCE cells and TM cells result in modulation of Wnt signalling pathway and ECM remodelling

Primary open‐angle glaucoma is a leading cause of irreversible blindness, often associated with increased intraocular pressure. Extracellular vesicles (EVs) carry a specific composition of proteins, lipids and nucleotides have been considered as essential mediators of cell‐cell communication. Their potential impact for crosstalk between tissues responsible for aqueous humour production and out‐flow is largely unknown. The study objective was to investigate the effects of EVs derived from non‐pigmented ciliary epithelium (NPCE) primary cells on the expression of Wnt proteins in a human primary trabecular meshwork (TM) cells and define the mechanism underlying exosome‐mediated regulation that signalling pathway. Consistent with the results in TM cell line, EVs released by both primary NPCE cells and NPCE cell line showed diminished pGSK3β phosphorylation and decreased cytosolic levels of β‐catenin in primary TM cells. At the molecular level, we showed that NPCE exosome treatment downregulated the expression of positive GSKβ regulator‐AKT protein but increased the levels of GSKβ negative regulator‐PP2A protein in TM cells. NPCE exosome protein analysis revealed 584 miRNAs and 182 proteins involved in the regulation of TM cellular processes, including WNT/β‐catenin signalling pathway, cell adhesion and extracellular matrix deposition. We found that negative modulator of Wnt signalling miR‐29b was abundant in NPCE exosomal samples and treatment of TM cells with NPCE EVs significantly decreased COL3A1 expression. Suggesting that miR‐29b can be responsible for decreased levels of WNT/β‐catenin pathway. Overall, this study highlights a potential role of EVs derived from NPCE cells in modulating ECM proteins and TM canonical Wnt signalling.     

Porphyromonas gingivalis lipopolysaccharide inhibits the osteoblastic differentiation of preosteoblasts by activating Notch1 signaling

Although Porphyromonas gingivalis lipopolysaccharide (P‐LPS) is known to inhibit osteoblast differentiation, the exact molecular mechanisms underlying this phenomenon remain unclear. Here, we investigated the role of Notch signaling in the osteoblastic differentiation of both MC3T3E‐1 cells and primary mouse bone marrow stromal cells (BMSCs). P‐LPS stimulation activated the Notch1 signaling cascade and increased expression of the Notch target genes HES1 and HEY1. P‐LPS can also act as an inhibitor because it is capable of suppressing Wnt/β‐catenin signaling in preosteoblasts by decreasing both glycogen synthase kinase‐3β (GSK‐3β) phosphorylation and the expression of nuclear β‐catenin. These effects were rescued, however, by inhibiting Notch1 signaling. Furthermore, P‐LPS treatment inhibited osteoblast differentiation in preosteoblasts as demonstrated by reductions in alkaline phosphatase activity, osteoblast gene expression, and mineralization, all of which were rescued by suppression of Notch1 signaling. Moreover, inhibition of GSK‐3β, HES1, or HEY1 partially reversed the P‐LPS‐induced inhibition of osteoblast differentiation. Together, these findings suggest that P‐LPS inhibits osteoblast differentiation by promoting the expression of Notch target genes and suppressing canonical Wnt/β‐catenin signaling. J. Cell. Physiol. 225: 106–114, 2010. © 2010 Wiley‐Liss, Inc.     

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.