The role of adherens junctions in the developing neocortex

The disproportional enlargement of the neocortex through evolution has been instrumental in the success of vertebrates, in particular mammals. The neocortex is a multilayered sheet of neurons generated from a simple proliferative neuroepithelium through a myriad of mechanisms with substantial evolutionary conservation. This developing neuroepithelium is populated by progenitors that can generate additional progenitors as well as post-mitotic neurons. Subtle alterations in the production of progenitors vs. differentiated cells during development can result in dramatic differences in neocortical size. This review article will examine how cadherin adhesion proteins, in particular α-catenin and N-cadherin, function in regulating the neural progenitor microenvironment, cell proliferation, and differentiation in cortical development.     

β-Cells retain a pool of insulin-containing secretory vesicles regulated by adherens junctions and the cadherin-binding protein p120 catenin

The β-cells of the islets of Langerhans are the sole producers of insulin in the human body. In response to rising glucose levels, insulin-containing vesicles inside β-cells fuse with the plasma membrane and release their cargo. However, the mechanisms regulating this process are only partly understood. Previous evidence indicated reductions in α-catenin elevate insulin release, while reductions in β-catenin decrease insulin release. α- and β-catenin contribute to cellular regulation in a range of ways but one is as members of the adherens junction complex. Therefore, we investigated the effects of adherens junctions on insulin release. We show in INS-1E β-cells knockdown of either E- or N-cadherin had only small effects on insulin secretion, but simultaneous knockdown of both cadherins resulted in a significant increase in basal insulin release to the same level as glucose-stimulated release. This double knockdown also significantly attenuated levels of p120 catenin, a cadherin-binding partner involved in regulating cadherin turnover. Conversely, reducing p120 catenin levels with siRNA destabilized both E- and N-cadherin, and this was also associated with an increase in levels of insulin secreted from INS-1E cells. Furthermore, there were also changes in these cells consistent with higher insulin release, namely reductions in levels of F-actin and increased intracellular free Ca²⁺ levels in response to KCl-induced membrane depolarization. Taken together, these data provide evidence that adherens junctions play important roles in retaining a pool of insulin secretory vesicles within the cell and establish a role for p120 catenin in regulating this process.     

Type 3 muscarinic acetylcholine receptor stimulation is a determinant of endothelial barrier function and adherens junctions integrity: role of protein-tyrosine phosphatase 1B

The main purpose of this study was to investigate whether type 3 muscarinic acetylcholine receptor (M3R) dysfunction induced vascular hyperpermeability. Transwell system analysis showed that M3R inhibition by selective antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and small interfering RNA both increased endothelial permeability. Using coimmunoprecipitation and Western blot assay, we found that M3R inhibition increased VE-cadherin and β-catenin tyrosine phosphorylation without affecting their expression. Using PTP1B siRNA, we found that PTP1B was required for maintaining VE-cadherin and β-catenin protein dephosphorylation. In addition, 4-DAMP suppressed PTP1B activity by reducing cyclic adenosine monophosphate (cAMP), but not protein kinase Cα (PKCα). These data indicate that M3R preserves the endothelial barrier function through a mechanism potentially maintaining PTP1B activity, keeping the adherens junction proteins (AJPs) dephosphorylation. [BMB Reports 2014; 47(10): 552-557]     

N-cadherin-based adherens junction regulates the maintenance,proliferation, and differentiation of neural progenitor cells during development

This review addresses our current understanding of the regulatory mechanism by which N-cadherin, a classical cadherin, affects neural progenitor cells (NPCs) during development. N-cadherin is responsible for the integrity of adherens junctions (AJs), which develop in the sub-apical region of NPCs in the neural tube and brain cortex. The apical domain, which contains the sub-apical region, is involved in the switching from symmetric proliferative division to asymmetric neurogenic division of NPCs. In addition, N-cadherin-based AJ is deeply involved in the apico-basal polarity of NPCs and the regulation of Wnt-β-catenin, hedgehog (Hh), and Notch signaling. In this review, we discuss the roles of N-cadherin in the maintenance, proliferation, and differentiation of NPCs through components of AJ, β-catenin and αE-catenin.     

Macrophage Migration Inhibitory Factor Promotes Proliferation and Neuronal Differentiation of Neural Stem/Precursor Cells through Wnt/β-Catenin Signal Pathway

Macrophage migration inhibitory factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects. Recent studies demonstrated that the receptors of MIF, including CD44, CXCR2, CXCR4 and CD74, are expressed in the neural stem/progenitor cells (NSPCs). The potential regulatory effect of MIF on NSPCs proliferation and neuronal differentiation, however, is largely unknown. Here, we investigated the effect of MIF on NSPC proliferation and neuronal differentiation, and further examined the signal pathway by which MIF transduced these signal effects in mouse NSPCs in vitro. The results showed that both Ki67-positive cells and neurosphere volumes were increased in a dose-dependent manner following MIF treatment. Furthermore, the expression of nuclear β-catenin was significantly stronger in MIF-stimulated groups than that in control groups. Conversely, administration of IWR-1, the inhibitor of Wnt/β-catenin pathway, significantly inhibited the proliferative effect of MIF on NSPCs. Immunostaining and Western blot further indicated that doublecortin (DCX) and Tuj 1, two neuronal markers, were evidently increased with MIF stimulation during NSPC differentiation, and there were more Tuj1-positive cells migrated out from neurospheres in MIF-stimulated groups than those in control groups. During NSPC differentiation, MIF increased the activity of β-galactosidase that responds to Wnt/β-catenin signaling. Wnt1 and β-catenin proteins were also up-regulated with MIF stimulation. Moreover, the expression of DCX and Tuj 1 was inhibited significantly by IWR-1. Taken together, the present study indicated that MIF enhances NSPC proliferation and promotes the neuronal differentiation, by activating Wnt/β-catenin signal pathway. The interaction between MIF and Wnt/β-catenin signal pathway may play an important role in modulating NSPC renewal and fate during brain development.     

Enterocytic differentiation is modulated by lipid rafts-dependent assembly of adherens junctions

Integrity of the epithelial barrier is determined by apical junctional complexes which also participate in the signalling pathways inducing intestinal cell differentiation. Lipid rafts (LR) have been proposed to play a role in the organization and the function of these intercellular complexes. This study investigated potential mechanisms by which LR could participate in the establishment of adherens junctions (AJ) and the initiation of enterocytic differentiation.We showed that the differentiation of epithelial cells in rat colons correlates with the emergence of LR. Using HT-29 cells we demonstrated that during the differentiation process, LR are required for the recruitment and the association of p120ctn to E-cadherin. Silencing of flotillin-1, a LR component, alters the recruitment of AJ proteins in LR and delays the expression of differentiation markers. Furthermore, the ability of p120ctn/E-cadherin complexes to support cell differentiation is altered in HT-29 Rac1N17 cells. These results show a contributory role of LR in the enterocytic differentiation process, which serve as signalling platforms for Rac1-mediated organization of AJ. A better understanding of the mechanism involved in the establishment of junctional complex and their role in enterocytic differentiation provides new insights into the regulation of intestinal homeostasis.     

Subcellular distribution of Wnt-1 at adherens junctions and actin-rich densities in endothelial cells

The Wnt family of signaling proteins functions in embryonic development and mammalian oncogenesis. It is unknown whether these molecules have a role in normal, postdevelopmental, homeostatic processes. Possessing a putative signal sequence and potential glycosylation sites, Wnt-1 is believed to be secreted and remain associated with the cell surface and extracellular matrix. While it has been suggested that Wnt proteins may target cytoskeletal structures more directly, no definitive studies have identified an intracellular association and function for these molecules. Here, we report that Western blots of lysates from retinoic-acid-differentiated P19 cells and bovine endothelial cells indicate the presence of a 45-kDa Wnt-1 protein. In endothelium, Wnt-1 was present in both the Triton X soluble and the insoluble cell fractions. Immunocytochemical labeling localized Wnt-1 to adherens junctions, codistributing with beta-catenin. Wnt-1 also was detected at actin-rich densities (ARDs) within basal cell regions. In wounded monolayers, ARDs delineated the distal margins of cells undergoing directed migration. Transfection with antisense oligonucleotides to Wnt-1 resulted in reduced cohesion of wound edge cells, abnormal protrusive activity, and random movement. Our data indicate that Wnt-1 protein is present in postdevelopmental endothelial cells where it associates with cytoskeletal elements and may retain function as a tissue polarity gene.     

Integrin α1β1 Mediates a Unique Collagen-dependent Proliferation Pathway In Vivo

Activation of integrins upon binding to extracellular matrix proteins is believed to be a crucial step for the regulation of cell survival and proliferation. We have used integrin α1-null mice to investigate the role of this collagen receptor in the regulation of cell growth and survival in vivo. α1-deficient animals, which are viable and fertile, have a hypocellular dermis and a deficiency in dermal fibroblast proliferation as embryos. In vitro analysis of α1-null embryonic fibroblasts has revealed that their proliferation rate is markedly reduced when plated on collagenous substrata, despite normal attachment and spreading. Moreover, on the same collagenous matrices, α1-null fibroblasts fail to recruit and activate the adaptor protein Shc. The failure to activate Shc is accompanied by a downstream deficiency in recruitment of Grb2 and subsequent mitogen-activated protein kinase activation. Taken together with the growth deficiency observed on collagens, this finding indicates that the α1β1 is the sole collagen receptor which can activate the Shc mediated growth pathway. Thus, integrin α1 has a unique role among the collagen receptors in regulating both in vivo and in vitro cell proliferation in collagenous matrices.     

NEK2 plays an active role in Tumorigenesis and Tumor Microenvironment in Non-Small Cell Lung Cancer

Abnormal expression and dysfunction of Never-in-mitosis-A-related kinase 2 (NEK2) result in tumorigenesis. High levels of NEK2 are related to malignant progression, drug resistance, and poor prognosis. However, the relationship between NEK2 levels and the occurrence of non-small cell lung cancer (NSCLC) remains unknown. This study aimed to explore the impacts of NEK2 on the oncogenesis of NSCLC and the tumor microenvironment. Downregulation of NEK2 inhibited A549 and H1299 cell proliferation, migration, and invasion, blocking cell cycle at the G0/G1 phase. Loss of NEK2 inhibited the release of IL-10 from tumor cells, M2-like polarization of macrophages, angiogenesis, and vascular endothelial cell migration. Furthermore, NEK2 deficiency inhibited tumor growth in vivo. Taken together, NEK2 knockdown inhibited the occurrence and development of NSCLC, M2 polarization of macrophages, and angiogenesis. The abnormal expression of NEK2 might not only indicate tumor progression and patient prognosis but also serve as a potential molecular therapeutic target with great development prospects.     

p120 catenin associates with kinesin and facilitates the transport of cadherin-catenin complexes to intercellular junctions

p120 catenin (p120) is a component of adherens junctions and has been implicated in regulating cadherin-based cell adhesion as well as the activity of Rho small GTPases, but its exact roles in cell-cell adhesion are unclear. Using time-lapse imaging, we show that p120-GFP associates with vesicles and exhibits unidirectional movements along microtubules. Furthermore, p120 forms a complex with kinesin heavy chain through the p120 NH2-terminal head domain. Overexpression of p120, but not an NH2-terminal deletion mutant deficient in kinesin binding, recruits endogenous kinesin to N-cadherin. Disruption of the interaction between N-cadherin and p120, or the interaction between p120 and kinesin, leads to a delayed accumulation of N-cadherin at cell-cell contacts during calcium-initiated junction reassembly. Our analyses identify a novel role of p120 in promoting cell surface trafficking of cadherins via association and recruitment of kinesin.     

Pantothenate Kinase 1 Inhibits the Progression of Hepatocellular Carcinoma by Negatively Regulating Wnt/β-catenin Signaling

Hyperactivation of Wnt/β-catenin signaling has been reported in hepatocellular carcinoma (HCC). However, the mechanisms underlying the hyperactivation of Wnt/β-catenin signaling are incompletely understood. In this study, Pantothenate kinase 1 (PANK1) is shown to be a negative regulator of Wnt/β-catenin signaling. Downregulation of PANK1 in HCC correlates with clinical features. Knockdown of PANK1 promotes the proliferation, growth and invasion of HCC cells, while overexpression of PANK1 inhibits the proliferation, growth, invasion and tumorigenicity of HCC cells. Mechanistically, PANK1 binds to CK1α, exerts protein kinase activity and cooperates with CK1α to phosphorylate N-terminal serine and threonine residues in β-catenin both in vitro and in vivo. Additionally, the expression levels of PANK1 and β-catenin can be used to predict the prognosis of HCC. Collectively, the results of this study highlight the crucial roles of PANK1 protein kinase activity in inhibiting Wnt/β-catenin signaling, suggesting that PANK1 is a potential therapeutic target for HCC.     

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the ¹⁵N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.     

Leucine Zipper Tumor Suppressor 2 Inhibits Cell Proliferation and Regulates Lef/Tcf-dependent Transcription through Akt/GSK3β Signaling Pathway in Lung Cancer

Leucine zipper tumor suppressor 2 (LZTS2) is implicated in several cancers; however, its biological mechanisms in non-small cell lung cancer (NSCLC) are not yet understood. We found that low levels of LZTS2 in NSCLC were correlated with tumor and nodal status. LZTS2 could inhibit cell proliferation and cell cycle transition at the G1/S phase and was implicated in the regulation of proteins associated with the canonical Wnt pathway, including GSK3β and β-catenin through inactivating the Akt pathway. These results provide novel mechanistic insight into the biological roles of LZTS2 in lung cancer cells.     

Phosphorylation state regulates the localization of Scribble at adherens junctions and its association with E-cadherin-catenin complexes

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin–catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin–catenin system and may control cadherin-mediated cell–cell adhesion.     

CTHRC1 promotes growth,migration and invasion of trophoblasts via reciprocal Wnt/β-catenin regulation

Preeclampsia (PE) is a pregnancy complication that is characterized by high blood pressure and is associated with high maternal and fetal morbidities. At a mechanistic level, PE is characterized by reduced invasion ability of trophoblasts. Collagen triple helix repeat containing-1 (CTHRC1) is a well-known tumor-promoting factor in several malignant tumors, but its role in trophoblasts remains unknown. In this study, we characterized the expression of CTHRC1 in placenta tissue samples from PE pregnancies and from normal pregnancies. We used the trophoblasts cell lines HTR-8/SVneo and JEG-3 to investigate the role of CTHRC1 in cell migration, invasion and proliferation. Western blot, PCR and TOP/FOP luciferase activity assays were used to investigate the molecular mechanisms underlying these cell behaviors. Placenta tissue samples obtained from pregnant women with PE expressed lower levels of CTHRC1 than those of placenta tissues from women with normal pregnancies. Down-regulation of CTHRC1 impaired cell proliferation, migration and invasion of trophoblasts, while CTHRC1 overexpression promoted nuclear translocation of β-catenin, a result that was further confirmed by TOP/FOP luciferase activity assay. Our findings suggest that CTHRC1 promotes migration and invasion of trophoblasts via reciprocal Wnt/β-catenin signaling pathway. Down-regulation of CTHRC1 may be a potential mechanism underpinning the development of preeclampsia.     

Role of Wnt signaling in fracture healing

The Wnt signaling pathway is well known to play major roles in skeletal development and homeostasis. In certain aspects, fracture repair mimics the process of bone embryonic development. Thus, the importance of Wnt signaling in fracture healing has become more apparent in recent years. Here, we summarize recent research progress in the area, which may be conducive to the development of Wnt-based therapeutic strategies for bone repair. [BMB Reports 2014; 47(12): 666-672]