CCL2 disrupts the adherens junction: implications for neuroinflammation

Alterations to blood-brain barrier (BBB) adhesion molecules and junctional integrity during neuroinflammation can promote central nervous system (CNS) pathology. The chemokine CCL2 is elevated during CNS inflammation and is associated with endothelial dysfunction. The effects of CCL2 on endothelial adherens junctions (AJs) have not been defined. We demonstrate that CCL2 transiently induces Src-dependent disruption of human brain microvascular endothelial AJ. β-Catenin is phosphorylated and traffics from the AJ to PECAM-1 (platelet endothelial cell adhesion molecule-1), where it is sequestered at the membrane. PECAM-1 is also tyrosine-phosphorylated, an event associated with recruitment of the phosphatase SHP-2 (Src homology 2 domain-containing protein phosphatase) to PECAM-1, β-catenin release from PECAM-1, and reassociation of β-catenin with the AJ. Surface localization of PECAM-1 is increased in response to CCL2. This may enable the endothelium to sustain CCL2-induced alterations in AJ and facilitate recruitment of leukocytes into the CNS. Our novel findings provide a mechanism for CCL2-mediated disruption of endothelial junctions that may contribute to BBB dysfunction and increased leukocyte recruitment in neuroinflammatory diseases.     

Platelet-activating factor regulates cadherin-catenin adhesion system expression and beta-catenin phosphorylation during Kaposi's sarcoma cell motility

In the present study, we evaluated whether motility of Kaposi's sarcoma (KS) cells induced by platelet-activating factor (PAF) is dependent on the regulation of adherens junctions components. The results obtained indicate that PAF dose and time dependently reduced the endogenous expression of the main components of the adherens junctions: VE-cadherin, alpha-catenin, and beta-catenin. In addition, PAF initiated events that directly or indirectly up-regulated both the tyrosine and serine/threonine phosphorylation pathways, and both types of phosphorylation of beta-catenin were involved in the motility of KS cells. This motility was abrogated by addition of the tyrosine kinase inhibitor genistein, suggesting that this phosphorylation is an important signal responsible for breaking down the adherens junctions and diminishing the ability of neighboring cells to interact. Furthermore, immunofluorescence analysis showed that beta-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of controls to a diffuse pattern with gap formation in PAF-treated KS cells. In conclusion, the data presented here indicate that PAF induces tumor cell motility by altering cell-cell adhesion through beta-catenin phosphorylation.     

Expression of endometrial glycogen synthase kinase-3beta protein throughout the menstrual cycle and its regulation by progesterone

Glycogen synthase kinase-3beta (GSK-3beta) is a serine/threonine kinase that plays a role in glycogen synthesis by inhibiting glycogen synthase (GS) through phosphorylation. We hypothesized that GSK-3beta by virtue of its role in glycogen synthesis through the inhibition of GS will play a role in the preparation of the endometrium for blastocyst implantation. Immunohistochemical (IHC) analysis and Western blot analysis (WBA) detected GSK-3beta in the endometrium, myometrium, Fallopian tube and ovary. WBA showed more than 5-fold higher endometrial expression of the phosphorylated GSK-3beta (pGSK-3beta) isoform (inactive) in the secretory phase as compared with the proliferative phase (P < 0.001), whereas no differences in total GSK-3beta expression were detected. IHC analysis confirmed the WBA and showed marked expression of pGSK-3beta predominantly in glandular epithelial cells in early and mid secretory endometrium with scant expression during the proliferative phase. In in vitro experiments using human endometrial-derived epithelial cell line (HES), progesterone did not alter total GSK mRNA or protein expression. However, progesterone induced a dose-dependent increase in the expression of pGSK-3beta, which could be blocked by RU486. Cyclic expression of GSK-3beta's active and inactive forms in the endometrium suggests that sex hormones regulate the expression of this enzyme. In vitro experiments demonstrate that progesterone through receptor-mediated mechanisms induces phosphorylation of endometrial GSK-3beta.     

糖原合酶激酶-3β信号分子在心肌肥厚中的作用

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine protein kinase, which takes part not only in glycogen metabolism, but also in cell proliferation, differentiation and apoptosis. GSK-3β is inhibited by growth factors and hypertrophic stimuli through phosphorylation of its N-terminal end serine (Ser9) residue. It is also activated by phosphorylation of its tyrosine (Tyr216) residue. GSK-3β is profoundly inactivated in mice with hypertrophic cardiomyopathy (HCM) and plays a secondary role in myosin heavy chain mutation. However, the role of GSK-3β in HCM was controversial. Recent studies have demonstrated that the activation of GSK-3β inhibits the myocardial hypertrophy, and is regulated by Wnt/Frizzld and PI3-K/Akt signaling pathways. This article introduces the molecular role of glycogen synthase kinase-3β signaling in myocardial hypertrophy and the different pathways on the activity of glycogen synthase kinase-3β (GSK-3β)     

Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localisation of the kinase.

Glycogen synthase kinase-3 (GSK-3) reduced the mobility of human tau on SDS-PAGE, prevented binding of the monoclonal antibody (mAb), Tau.1, and induced binding of the mAb 8D8. Recombinant tau phosphorylated by GSK-3 aligned on SDS-PAGE with the abnormally phosphorylated tau (PHF-tau) associated with the paired helical filaments in Alzheimer's disease brain. Phosphorylated serine396 (numbering of the largest human brain tau isoform) was identified as a binding site on tau for mAb 8D8. The localisation of GSK-3 within granular structures in pyramidal cells indicates that GSK-3 alpha and GSK-3 beta may have a role in the production of PHF-tau in Alzheimer's disease.     

Wnt5a modulates glycogen synthase kinase 3 to induce phosphorylation of receptor tyrosine kinase Ror2

The receptor tyrosine kinase Ror2 plays important roles in mediating non-canonical Wnt5a signaling by activating the Wnt-JNK pathway and inhibiting the beta-catenin-TCF pathway. It has been shown that Ror2 is phosphorylated and activated by casein kinase Iepsilon when both molecules are over-expressed in cultured cells. However, it remains unknown whether or not Ror2 is phosphorylated upon Wnt5a stimulation. Here we show that Ror2 is phosphorylated on serine/threonine residues upon stimulation of cultured cells, expressing Ror2 endogenously, with Wnt5a, but not Wnt3a. It was found that treatment of cells with glycogen synthase kinase-3 (GSK-3) inhibitors (LiCl and SB216763) or small interfering RNAs (siRNAs) for GSK-3 (mainly GSK-3alpha) can inhibit Wnt5a-induced phosphorylation of Ror2. Immunoprecipitated Ror2 can also be phosphorylated by purified GSK-3alpha or GSK-3betain vitro, and ectopic co-expression of Ror2 and GSK-3 (mainly GSK-3alpha) in cultured cells results in Ror2 phosphorylation, irrespective of Wnt5a, that is sensitive to SB216763. These results indicate that GSK-3 is involved in Wnt5a-induced phosphorylation of Ror2. Moreover, it was found that Wnt5a-induced cell migration can be inhibited by SB216763 or by siRNA-mediated suppression of GSK-3alpha (and GSK-3beta) expression, further emphasizing the role(s) of GSK-3 in Wnt5a-induced signaling.     

Interaction between the transforming growth factor-beta type II receptor/Smad pathway and beta-catenin during transforming growth factor-beta1-mediated adherens junction disassembly

The aim of the current study was to examine the influence of transforming growth factor (TGF)-beta 1 on proximal tubular epithelial cell-cell interaction, with particular emphasis on the regulation of adherens junction complex formation. Stimulation of the proximal tubular cell line HK-2 cells by TGF-beta 1 led to loss of cell-cell contact and disassembly of both adherens and tight junctional complexes. Adherens junction disassembly was associated with reduction of both Triton-soluble and Triton-insoluble E-cadherin, and an increase in detergent-soluble beta-catenin. Under these conditions, immunoprecipitation and Western analysis demonstrated decreased association of beta-catenin, both with E-cadherin, alpha-catenin, and the cell cytoskeleton. Confocal microscopy after immunostaining, showed decreased intensity of peripheral E-cadherin staining, and redistribution of beta-catenin expression to a perinuclear location. Tight junction disassembly was manifest by a reduction in the expression of Triton-soluble occludin and ZO-1 by Western analysis and their disassociation manifested by immunostaining and confocal microscopy. Loss of cell-cell contact and disassembly of adherens junctions were seen after addition of TGF-beta 1 to the basolateral aspect of the cells. Immunoprecipitation experiments demonstrated co-localization of E-cadherin, beta-catenin, and TGF-beta 1 RII in unstimulated cells. After TGF-beta 1 stimulation, the TGF-beta 1 RII no longer associated with either E-cadherin or beta-catenin. Dissociation of the adherens junction protein from the TGF-beta 1 receptor was associated with increased beta-catenin tyrosine phosphorylation and decreased threonine phosphorylation. Furthermore after receptor ligand binding, beta-catenin became associated with the TGF-beta 1-signaling molecules Smad3 and Smad4.     

Cdc42 is crucial for the establishment of epithelial polarity during early mammalian development.

To study the role of Cdc42 in the establishment of epithelial polarity during mammalian development, we generated murine Cdc42-null embryonic stem cells and analyzed peri-implantation development using embryoid bodies (EBs). Mutant EBs developed endoderm and underlying basement membrane, but exhibited defects of cell polarity, cell-cell junctions, survival, and cavitation. These defects corresponded to a decreased phosphorylation and membrane localization of aPKC, a reduced phosphorylation of GSK3beta, and a diminished activity of Rac1. However, neither Rac1 nor the kinase function of GSK3beta seem to contribute to cell polarization and cell-cell contacts. In contrast, EBs expressing dominant-negative (dn) PKCzeta mimicked well the phenotype of Cdc42-null EBs, suggesting a major role of aPKC in mediating cell polarization downstream of Cdc42. Finally, aggregation experiments with endodermal cell lines suggested that Cdc42 might affect formation of adherens and tight junctions by PKCzeta-dependent regulation of the protein levels of p120 catenin and E-cadherin.     

Extracellular matrix,junctional integrity and matrix metalloproteinase interactions in endothelial permeability regulation

Vascular endothelial permeability is maintained by the regulated apposition of adherens and tight junctional proteins whose organization is controlled by several pharmacological and physiological mediators. Endothelial permeability changes are associated with: (1) the spatial redistribution of surface cadherins and occludin, (2) stabilization of focal adhesive bonds and (3) the progressive activation of matrix metalloproteinases (MMPs). In response to peroxide, histamine and EDTA, endothelial cells sequester VE-cadherin and alter its cytoskeletal binding. Simultaneously, these mediators enhance focal adhesion to the substratum. Oxidants, cytokines and pharmacological mediators also trigger the activation of matrix metalloproteinases (MMPs) in a cytoskeleton and tyrosine phosphorylation dependent manner to degrade occludin, a well-characterized tight junction element. These related in vitro phenomena appear to co-operate during inflammation, to increase endothelial permeability, structurally stabilize cells while also remodelling cell junctions and substratum.     

beta-Catenin activity negatively regulates bacteria-induced inflammation

Wild-type (WT) Salmonella typhimurium causes acute intestinal inflammation by activating the nuclear factor kappa B (NF-kappaB) pathway. Interestingly, WT Salmonella infection also causes degradation of beta-catenin, a regulator of cellular proliferation. Regulation of beta-catenin and the inhibitor of NF-kappaB, IkappaBalpha, is strikingly similar, involving phosphorylation at identical sites, ubiquitination by the same E3 ligase, and subsequent proteasomal degradation. However, how beta-catenin directly regulates the NF-kappaB pathway during bacteria-induced inflammation in vivo is unknown. Using streptomycin-pretreated mice challenged with Salmonella, we demonstrated that WT Salmonella stimulated beta-catenin degradation and decreased the physical association between NF-kappaB and beta-catenin. Accordingly, WT Salmonella infection decreased the expression of c-myc, a beta-catenin-regulated target gene, and increased the levels of IL-6 and TNF-alpha, the NF-kappaB-regulated target genes. Bacterial infection directly stimulated phosphorylation of beta-catenin, both in vivo and in vitro. Closer examination revealed that glycogen synthase kinase 3beta (GSK-3beta) kinase activity was increased in response to WT Salmonella, whereas non-virulent Salmonella had no effect. siRNA of GSK-3beta was able to stabilize IkappaBalpha in response to WT Salmonella. Pretreatment for 24 h with LiCl, an inhibitor of GSK-3beta, reduced WT Salmonella induced IL-8 secretion. Additionally, cells expressing constitutively active beta-catenin showed IkappaBalpha stabilization and inhibition of NF-kappaB activity not only after WT Salmonella infection but also after commensal bacteria (Escherichia coli F18) and TNF-alpha treatment. This study suggests a new role for beta-catenin as a negative regulator of inflammation.     

Protection against LPS-induced pulmonary edema through the attenuation of protein tyrosine phosphatase-1B oxidation

One hallmark of acute lung injury is the disruption of the pulmonary endothelial barrier. Such disruption correlates with increased endothelial permeability, partly through the disruption of cell-cell contacts. Protein tyrosine phosphatases (PTPs) are known to affect the stability of both cell-extracellular matrix adhesions and intercellular adherens junctions (AJs). However, evidence for the role of select PTPs in regulating endothelial permeability is limited. Our investigations noted that the inhibition of PTP1B in cultured pulmonary endothelial cells (ECs), as well as in the vasculature of intact murine lungs via the transient overexpression of a catalytically inactive PTP1B, decreased the baseline resistance of cultured EC monolayers and increased the formation of edema in murine lungs, respectively. In addition, we observed that the overexpression of wild-type PTP1B enhanced basal barrier function in vitro. Immunohistochemical analyses of pulmonary ECs and the coimmunoprecipitation of murine lung homogenates demonstrated the association of PTP1B with the AJ proteins β-catenin, p120-catenin, and VE-cadherin both in vitro and ex vivo. Using LPS in a model of sepsis-induced acute lung injury, we showed that reactive oxygen species were generated in response to LPS, which correlated with enhanced PTP1B oxidation, inhibited phosphatase activity, and attenuation of the interactions between PTP1B and β-catenin, as well as enhanced β-catenin tyrosine phosphorylation. Finally, the overexpression of a cytosolic PTP1B fragment, shown to be resistant to nicotinamide adenine dinucleotide phosphate-reduced oxidase-4 (Nox4)-mediated oxidation, significantly attenuated LPS-induced endothelial barrier dysfunction and the formation of lung edema, and preserved the associations of PTP1B with AJ protein components, independent of PTP1B phosphatase activity. We conclude that PTP1B plays an important role in maintaining the pulmonary endothelial barrier, and PTP1B oxidation appears to contribute to sepsis-induced pulmonary vascular dysfunction, possibly through the disruption of AJs.     

Insights into monocyte-driven osteoclastogenesis and its link with hematopoiesis: regulatory roles of PECAM-1 (CD31) and SHP-1

Osteoclasts are derived from hematopoietic cells of monocyte-macrophage lineage. Osteoclastogenesis is orchestrated by the migration of monocytic osteoclast progenitor cells in close proximity to bone surfaces destined for resorption. Although the overall roles of monocyte migratory behavior in osteoclastogenesis remain enigmatic, impaired monocyte migration can lead to either decreased or increased osteoclastogenesis, which appears contingent upon the roles of migration in either fusion events required for osteoclast formation or terminal differentiation of osteoclasts. The cell adhesion molecule PECAM-1 (platelet endothelial cell adhesion molecule 1), in concert with the tyrosine phosphatase SHP-1 (Src homology 2-containing protein tyrosine phosphatase 1) and tyrosine kinase Syk-1 (spleen tyrosine kinase 1), functions as a negative regulator of osteoclastogenesis. Both PECAM-1 (CD31) and SHP-1 knockout mice exhibit not only increased osteoclastogenesis but also abnormal hematopoiesis, which is suggestive of the intricate interplay between hematopoiesis and osteoclastogenesis. Interestingly, the most pronounced effect of PECAM-1 deficiency on hematopoiesis is reflected by excessive megakaryocytopoiesis. Emerging data have suggested the role of megakaryocytes in bone remodeling. Megakaryocytopoiesis-osteoclastogenesis interactions are discussed herein, reconciling the discrepancies shown by different studies in this area. PECAM-1 and non-receptor tyrosine phosphatase polymorphisms have been revealed in a spectrum of diseases. The complex regulatory roles of PECAM-1 and SHP-1 in vivo suggest the potential utilization of polymorphisms of these genes for diagnostic purposes.     

Wnt-induced dephosphorylation of Axin releases β-catenin from the Axin complex

The stabilization of beta-catenin is a key regulatory step during cell fate changes and transformations to tumor cells. Several interacting proteins, including Axin, APC, and the protein kinase GSK-3beta are implicated in regulating beta-catenin phosphorylation and its subsequent degradation. Wnt signaling stabilizes beta-catenin, but it was not clear whether and how Wnt signaling regulates the beta-catenin complex. Here we show that Axin is dephosphorylated in response to Wnt signaling. The dephosphorylated Axin binds beta-catenin less efficiently than the phosphorylated form. Thus, Wnt signaling lowers Axin's affinity for beta-catenin, thereby disengaging beta-catenin from the degradation machinery.     

Phosphorylation and disorganization of vascular-endothelial cadherin in interaction between breast cancer and vascular endothelial cells.

Adherens junctions of the endothelium play a key role in the maintenance of endothelial permeability and are composed of the vascular endothelial (VE)-cadherin/catenin adhesion complex. We report that following tumour cell (MDA MB231 cells) adherence to the HUVECs, there was a rapid (within 5 min) redistribution of VE-cadherin, resulting in its transient loss from regions of endothelial cell-cell contact. The molecule gradually reorganised within the endothelial cell contacts after this time. It was further shown that the overall expression of VE-cadherin did not change, however, the amount of alpha- and beta-catenins coprecipitated with VE-cadherin markedly decreased after 5 min of tumour cell adhesion to the HUVECs. Immunoprobing of these samples with anti-phosphotyrosine antibodies demonstrated that the tyrosine phosphorylation of VE-cadherin was significantly increased following 5 min of tumour cell adhesion. Together, these results suggest that the adhesion of tumour cells to HUVEC promotes the redistribution of VE-cadherin from interendothelial adherens junctions, an effect that may be attributed to the increase in tyrosine phosphorylation of members of the VE-cadherin/catenin adhesion complex. This, in turn, may increase vascular endothelial permeability and facilitate the transendothelial migration of tumour cells during extravasation.     

Aging alters vascular mechanotransduction: pressure-induced regulation of p70S6k in the rat aorta

Physical forces are important regulators of vascular structure and function though it is unknown how aging may affect the ability of the vasculature to respond to mechanical stimuli. We investigated the pressure-induced activation of ribosomal S6-kinase (p70S6k) and its pathway-related proteins (Akt, GSK-3beta, SHP-2, PTEN) in aortae from young adult (6 month), aged (30 month), and very aged (36 month) Fischer 344 x Brown Norway F1 hybrid rats. With aging, the aortic tissue content of Akt. SHP-2, and PTEN was significantly increased while total p70S6k and GSK-3beta were unchanged. By comparison, the basal phosphorylation of p70S6k at Thr 389 and Thr 421/Ser 424 was increased ( approximately 40%) and unchanged, respectively, while Akt decreased (approximately 37%), GSK-3beta was unchanged, SHP-2 increased (approximately 73.5%), and PTEN increased (approximately 120%) in the aortae of very aged rats. Acute pressurization of aortae resulted in similar increases in phosphorylation of Akt among the different age groups. By comparison, pressure-induced phosphorylation of p70S6k at Thr 389, GSK-3beta and SHP-2 decreased; whereas, PTEN dephosphorylation was increased in 36-month versus 6-month aortae. The results indicate marked alterations in the p70S6k signaling pathway with aging. The implications of these findings on age-associated vessel remodeling are discussed.