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.     

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.     

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.     

Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers

Retinal pigment epithelium (RPE) alterations in age-related macular degeneration occur in patches, potentially involving long-distance communication between damaged and healthy areas. Communication along the epithelium might be mediated by extracellular vesicles (EVs). To test this hypothesis, EVs were collected from supernatants of polarized ARPE-19 and primary porcine RPE monolayers for functional and biochemical assays. EVs from oxidatively stressed donor cells reduced barrier function in recipient RPE monolayers when compared to control EVs. The effect on barrier function was dependent on EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Mass spectrometry-based proteomic analysis of EVs identified HDAC6, which is known to reduce tight junction stability. Activity assays confirmed the presence of HDAC6 in EVs, and EV transfer assays using HDAC6 inhibitors confirmed its effect in monolayers. These findings demonstrate that EVs can communicate stress messages to healthy RPE cells, potentially contributing to RPE dysfunction.     

Tumour necrosis factor‐α‐induced protein 8‐like 2 is a novel regulator of proliferation,migration, and invasion in human rectal adenocarcinoma cells

Tumour necrosis factor‐α‐induced protein 8‐like 2 (TIPE2) is a tumour suppressor in many types of cancer. However, the mechanism of action of TIPE2 on the growth of rectal adenocarcinoma is unknown. Our results showed that the expression levels of TIPE2 in human rectal adenocarcinoma tissues were higher than those in adjacent non‐tumour tissues. Overexpression of TIPE2 reduced the proliferation, migration, and invasion of human rectal adenocarcinoma cells and down‐regulation of TIPE2 showed reverse effects. TIPE2 overexpression increased apoptosis through down‐regulating the expression levels of Wnt3a, phospho (p)‐β‐Catenin, and p‐glycogen synthase kinase‐3β in rectal adenocarcinoma cells, however, TIPE2 knockdown exhibited reverse trends. TIPE2 overexpression decreased autophagy by reducing the expression levels of p‐Smad2, p‐Smad3, and transforming growth factor‐beta (TGF‐β) in rectal adenocarcinoma cells, however, TIPE2 knockdown showed opposite effects. Furthermore, TIPE2 overexpression reduced the growth of xenografted human rectal adenocarcinoma, whereas TIPE2 knockdown promoted the growth of rectal adenocarcinoma tumours by modulating angiogenesis. In conclusion, TIPE2 could regulate the proliferation, migration, and invasion of human rectal adenocarcinoma cells through Wnt/β‐Catenin and TGF‐β/Smad2/3 signalling pathways. TIPE2 is a potential therapeutic target for the treatment of rectal adenocarcinoma.     

Mechanisms of extracellular vesicle uptake in stressed retinal pigment epithelial cell monolayers

PurposeExtracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells.MethodsRPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H₂O₂, others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry.ResultsClathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided.ConclusionsIn summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease.     

Proteomic Signature of Mesenchymal Stromal Cell‐Derived Small Extracellular Vesicles

Small extracellular vesicles (EVs) are 50–200 nm vesicles secreted by most cells. They are considered as mediators of intercellular communication, and EVs from specific cell types, in particular mesenchymal stem/stromal cells (MSCs), offer powerful therapeutic potential, and can provide a novel therapeutic strategy. They appear promising and safe (as EVs are non‐self‐replicating), and eventually MSC‐derived EVs (MSC‐EVs) may be developed to standardized, off‐the‐shelf allogeneic regenerative and immunomodulatory therapeutics. Promising pre‐clinical data have been achieved using MSCs from different sources as EV‐producing cells. Similarly, a variety EV isolation and characterization methods have been applied. Interestingly, MSC‐EVs obtained from different sources and prepared with different methods show in vitro and in vivo therapeutic effects, indicating that isolated EVs share a common potential. Here, well‐characterized and controlled, publicly available proteome profiles of MSC‐EVs are compared to identify a common MSC‐EV protein signature that might be coupled to the MSC‐EVs’ common therapeutic potential. This protein signature may be helpful in developing MSC‐EV quality control platforms required to confirm the identity and test for the purity of potential therapeutic MSC‐EVs.     

Temporal and contextual orchestration of cardiac fate by WNT‐BMP synergy and threshold

Cardiomyogenic development proceeds with a cascade of intricate signalling events that operate in a temporo‐spatial fashion to specify cardiac cell fate during early embryogenesis. In fact, conflicting reports exist regarding the role of Wnt/β‐catenin signalling during cardiomyogenesis. Here, we describe a dose‐dependent and temporal effect of Wnt/β‐catenin signalling on in vitro cardiomyogenesis using embryonic stem cells (ESCs) as a model system. We could demonstrate that canonical Wnt activation during early stage of differentiation either through ligand or by GSK3β inhibition helped in maintaining Oct4 and Nanog expressions, and in parallel, it promoted mesoderm and endoderm inductions. In contrast, it led to attenuation in cardiomyogenesis that was reversed by moderate concentration of DKK1, but not soluble Fz8. However, higher DKK1 could also block cardiomyogenesis, suggesting thereby governance of a particular signalling threshold underlying this developmental event. Interestingly, Wnt signalling activation at early stage modulated BMP4 expression in a stage‐specific manner. Wnt activation, synchronized with BMP4 and brachyury up‐regulation at early stage, correlated well with mesoderm induction. Conversely, Wnt activation led to BMP4 and Wnt5a down‐regulation at late stage culminating in cardiomyogenic attenuation. Our findings suggested the existence of precise regulatory machinery with context‐dependent role of Wnt for fine tuning mesoderm induction and its derivatives, through establishment of Wnt gradient during ESCs’ differentiation. Moreover, contrary to mere activation/inhibition, a specific threshold of Wnt and BMP and their synergy seemed necessary for providing the guiding cues in orchestrating mesoderm induction and subsequent cardiomyogenesis.     

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.     

Mitochondrial DNA in extracellular vesicles declines with age

The mitochondrial free radical theory of aging suggests that accumulating oxidative damage to mitochondria and mitochondrial DNA (mtDNA) plays a central role in aging. Circulating cell‐free mtDNA (ccf‐mtDNA) isolated from blood may be a biomarker of disease. Extracellular vesicles (EVs) are small (30–400 nm), lipid‐bound vesicles capable of shuttling proteins, nucleic acids, and lipids as part of intercellular communication systems. Here, we report that a portion of ccf‐mtDNA in plasma is encapsulated in EVs. To address whether EV mtDNA levels change with human age, we analyzed mtDNA in EVs from individuals aged 30–64 years cross‐sectionally and longitudinally. EV mtDNA levels decreased with age. Furthermore, the maximal mitochondrial respiration of cultured cells was differentially affected by EVs from old and young donors. Our results suggest that plasma mtDNA is present in EVs, that the level of EV‐derived mtDNA is associated with age, and that EVs affect mitochondrial energetics in an EV age‐dependent manner.     

A gene trap mutagenesis screen for genes underlying cellular response to the mood stabilizer lithium

Identifying the biological pathways mediating the action of a therapeutic compound may help the development of more specific treatments while also increasing our understanding of the underlying disease pathology. Salts of the metal lithium are commonly used as a front‐line mood stabilizing treatment for bipolar disorder. Lithium's action has been variously linked to inositol phosphate metabolism and the WNT/Glycogen Synthase Kinase 3β (GSK3β)/β‐Catenin signalling cascade, but, to date, little is known about which of these provides the principal therapeutic benefit for patients and, more specifically, which constituent genes, through presumed sequence variation, determine differences in patient response to treatment. Here, we describe a functional screen in which SH‐SY5Y neuroblastoma cells were randomly mutated through genomic integration of the pMS1 poly A ‘gene trap’ plasmid vector. Lithium normally induces differentiation of neuroblastoma cells, but a small proportion of mutated cells continued to proliferate and formed colonies. Rapid amplification of cDNA ends (RACE)‐PCR was used to identify the ‘trapped’ gene in each of these lithium‐resistant colonies. Heterozygous, gene trap integrations were identified within ten genes, eight of which are likely to produce loss‐of‐function mutations including MED10, MSI2 and three long intergenic non‐coding (LINC) RNAs. Both MED10 and MSI2 have been previously linked with WNT/GSK3β/β‐Catenin pathway function suggesting that this is an important mediator of lithium action in this screen. The methodology applied here provides a rapid, objective and economic approach to define the genetic contribution to drug action, but could also be readily adapted to any desired in vitro functional selection/screening paradigm.     

PGE2 inhibits MMP expression by suppressing MKK4–JNK MAP kinase–c‐JUN pathway via EP4 in human articular chondrocytes

Prostaglandin E2 (PGE2) is one of pro‐inflammatory mediators. PGE2 maintains the homeostasis of many organs including articular cartilage, and a previous report showed that continuous inhibition of PGE2 accelerates the progression of osteoarthritis (OA). While PGE2 inhibits matrix metalloprotease (MMP) expression in several types of cells, little is known on direct effects of PGE2 on MMP expression in articular chondrocytes. The objective of this study was to investigate direct effects of PGE2 on IL‐1β‐induced MMP‐1 and MMP‐13 expression and the intracellular signaling in articular chondrocytes. PGE2 showed inhibitory effects on IL‐1β‐induced MMP‐1 and MMP‐13 expression demonstrated by immunoblotting both in OA and normal chondrocytes, which was further confirmed by enzyme‐linked immunosorbent assay and immunohistochemistry of explant cultures of articular cartilages. An EP4 agonist, ONO‐AE1‐329, mimicked the inhibitory effect of PGE2, while an EP4 antagonist, ONO‐AE3‐208, blocked the effects. PGE2 suppressed the phosphorylation of JNK and ERK MAP kinases, but only knockdown of JNK by specific siRNA mimicked the effect of PGE2. PGE2 further inhibited the phosphorylation of MKK4 without suppression of MKK7 phosphorylation, and of c‐JUN to decrease expression levels of MMP‐1 and MMP‐13. These results demonstrate that PGE2 inhibits IL‐1β‐induced MMP‐1 and MMP‐13 productions via EP4 by suppressing MKK4–JNK MAP kinase–c‐JUN pathway. J. Cell. Biochem. 109: 425–433, 2010. © 2009 Wiley‐Liss, Inc.     

Molecular effects of lithium exposure during mouse and chick gastrulation and subsequent valve dysmorphogenesis

BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK‐3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/β‐catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells‐c1 (NFATc1), both key regulators of valve development. Li‐exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK‐3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/β‐catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up‐regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK‐3 directly to augment Wnt signaling. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Trolox‐induced cardiac differentiation is mediated by the inhibition of Wnt/β‐catenin signaling in human embryonic stem cells

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions, wherein the regulation of Wnt/β‐catenin pathway is often desirable. Here, we examined the effect of trolox, a vitamin E analog, on the cardiac differentiation of human embryonic stem cells (hESCs). 6‐Hydroxy‐2,5,7,8‐tetramethylchromane‐2‐carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time‐ and dose‐dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/β‐catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/β‐catenin regulator.     

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.