PECAM-1 (CD31) functions as a reservoir for and a modulator of tyrosine-phosphorylated beta-catenin

1999 ◽  
Vol 112 (18) ◽  
pp. 3005-3014 ◽  
Author(s):  
N. Ilan ◽  
S. Mahooti ◽  
D.L. Rimm ◽  
J.A. Madri
Keyword(s):  
Endothelial Cell ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Nuclear Translocation ◽  
Adherens Junction ◽  
Cell Contact ◽  
Beta Catenin ◽  
Dependent Manner

Catenins function as regulators of cellular signaling events in addition to their previously documented roles in adherens junction formation and function. Evidence to date suggests that beta and gamma catenins can act as signaling molecules, bind transcriptional factors and translocate to the nucleus. Beta- and gamma-catenin are also major substrates for protein tyrosine kinases, and tyrosine phosphorylation of junctional proteins is correlated with decreased adhesiveness. One way in which catenin functions are modulated is by dynamic incorporation into junctional complexes which controls, in part, the cytoplasmic levels of catenins. Here we show that: (1) vascular endothelial growth factor (VEGF) induces beta-catenin tyrosine phosphorylation in a time-, and dose-dependent manner and that VEGF receptors co-localize to areas of endothelial cell-cell contact in vitro and in vivo. (2) Platelet-endothelial cell adhesion molecule (PECAM)-1 can function as a reservoir for, and modulator of, tyrosine phosphorylated beta-catenin. (3) PECAM-1 can prevent beta-catenin nuclear translocation in transfected SW480 colon carcinoma cells. We suggest that PECAM-1 may play a role in modulating beta-catenin tyrosine phosphorylation levels, localization and signaling and by doing so, functions as an important modulator of the endothelium.

scholarly journals Polymorphonuclear leukocyte adhesion triggers the disorganization of endothelial cell-to-cell adherens junctions.

1996 ◽  
Vol 135 (2) ◽  
pp. 497-510 ◽  
Author(s):  
A Del Maschio ◽  
A Zanetti ◽  
M Corada ◽  
Y Rival ◽  
L Ruco ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Leukocyte Adhesion ◽  
Adherens Junction ◽  
Immunohistochemical Analysis ◽  
Cell Junctions ◽  
Beta Catenin ◽  
Cell Extracts ◽  
Beta 2

Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE-cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE-cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen-reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.

scholarly journals FER-mediated tyrosine phosphorylation and PIK3R2/p85β recruitment on IRS4 promotes the PI3K-AKT signaling pathway and tumorigenesis in ovarian cancer

2020 ◽  
Author(s):  
Yanchun Zhang ◽  
Xuexue Xiong ◽  
Qi Zhu ◽  
Jiali Zhang ◽  
Yuetong Wang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Cells ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Regulatory Subunit ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Kinase Substrate

AbstractTyrosine phosphorylation, orchestrated by tyrosine kinases and phosphatases, modulates a multi-layered signaling network in a time and space dependent manner. Dysregulation of this post-translational modification is inevitably associated with pathological diseases. Our previous work has demonstrated that non-receptor tyrosine kinase FER is upregulated in ovarian cancer. Knockdown of the kinase attenuates metastatic phenotypes in tumor cells. Here we employed mass spectrometry and biochemical approaches to identify IRS4 as a novel substrate of FER. Using a proximity-based tagging system, we determined that FER-mediated phosphorylation of Tyr779 enables IRS4 to recruit PIK3R2/p85β, the regulatory subunit of PI-3K, and activate the PI3K-AKT pathway. Rescuing IRS4-null ovarian tumor cells with phosphorylation-defective mutant, but not WT IRS4, delayed tumor cell proliferation both in vitro and in vivo. Overall, we revealed a kinase-substrate regulatory mode between FER and IRS4, and the pharmacological inhibition of FER kinase may be beneficial for ovarian cancer patients with PI3K-AKT hyperactivation.

scholarly journals Phaseolin Attenuates Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells and Zebrafish

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 420
Author(s):  
Su-Jung Hwang ◽  
Ye-Seul Song ◽  
Hyo-Jong Lee
Keyword(s):  
Nitric Oxide ◽  
Nuclear Translocation ◽  
Raw 264.7 Cells ◽  
Network Pharmacology ◽  
Raw 264.7 ◽  
Dependent Manner ◽  
Bioactive Constituents

Kushen (Radix Sophorae flavescentis) is used to treat ulcerative colitis, tumors, and pruritus. Recently, phaseolin, formononetin, matrine, luteolin, and quercetin, through a network pharmacology approach, were tentatively identified as five bioactive constituents responsible for the anti-inflammatory effects of S. flavescentis. However, the role of phaseolin (one of the primary components of S. flavescentis) in the direct regulation of inflammation and inflammatory processes is not well known. In this study, the beneficial role of phaseolin against inflammation was explored in lipopolysaccharide (LPS)-induced inflammation models of RAW 264.7 macrophages and zebrafish larvae. Phaseolin inhibited LPS-mediated production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), without affecting cell viability. In addition, phaseolin suppressed pro-inflammatory mediators such as cyclooxygenase 2 (COX-2), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) in a dose-dependent manner. Furthermore, phaseolin reduced matrix metalloproteinase (MMP) activity as well as macrophage adhesion in vitro and the recruitment of leukocytes in vivo by downregulating Ninjurin 1 (Ninj1), an adhesion molecule. Finally, phaseolin inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB). In view of the above, our results suggest that phaseolin could be a potential therapeutic candidate for the management of inflammation.

scholarly journals A mechanism of Rap1-induced stabilization of endothelial cell–cell junctions

2011 ◽  
Vol 22 (14) ◽  
pp. 2509-2519 ◽  
Author(s):  
Jian J. Liu ◽  
Rebecca A. Stockton ◽  
Alexandre R. Gingras ◽  
Ararat J. Ablooglu ◽  
Jaewon Han ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Small Gtpases ◽  
Cell Junctions ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Cardiovascular Development ◽  
Cavernous Malformations ◽  
Cell Cell

Activation of Rap1 small GTPases stabilizes cell–cell junctions, and this activity requires Krev Interaction Trapped gene 1 (KRIT1). Loss of KRIT1 disrupts cardiovascular development and causes autosomal dominant familial cerebral cavernous malformations. Here we report that native KRIT1 protein binds the effector loop of Rap1A but not H-Ras in a GTP-dependent manner, establishing that it is an authentic Rap1-specific effector. By modeling the KRIT1–Rap1 interface we designed a well-folded KRIT1 mutant that exhibited a ∼40-fold-reduced affinity for Rap1A and maintained other KRIT1-binding functions. Direct binding of KRIT1 to Rap1 stabilized endothelial cell–cell junctions in vitro and was required for cardiovascular development in vivo. Mechanistically, Rap1 binding released KRIT1 from microtubules, enabling it to locate to cell–cell junctions, where it suppressed Rho kinase signaling and stabilized the junctions. These studies establish that the direct physical interaction of Rap1 with KRIT1 enables the translocation of microtubule-sequestered KRIT1 to junctions, thereby supporting junctional integrity and cardiovascular development.

scholarly journals Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4495-4501 ◽  
Author(s):  
T Tauchi ◽  
JE Damen ◽  
K Toyama ◽  
GS Feng ◽  
HE Broxmeyer ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Tyrosine Phosphatase ◽  
Erythropoietin Receptor ◽  
Sh2 Domains ◽  
Proliferation And Differentiation ◽  
Stimulation Of

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.

The JAK Inhibitor INCB20 Induces Antiproliferative and Apoptotic Effects in Human Myeloma Cells In Vitro and In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3357-3357
Author(s):  
Renate Burger ◽  
Steven Legouill ◽  
Yu-Tzu Tai ◽  
Reshma Shringarpure ◽  
Klaus Podar ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Jak Inhibitor ◽  
Synthetic Compound ◽  
Akt Phosphorylation ◽  
Ic50 Values ◽  
Dose Dependent

Abstract In multiple myeloma (MM), IL-6 plays an important role for tumor cell growth, survival, and drug resistance. Janus kinases (JAKs) are protein tyrosine kinases and constitutively associated with the gp130 chain of the IL-6 receptor complex. Their activation is one of the first steps in cytokine receptor-mediated signaling and critical for virtually all subsequent downstream signaling cascades. INCB20 is a small-molecule synthetic compound which, in biochemical assays, potently inhibited all four JAKs with IC50 values between 0.3 nM and 1.2 nM (for comparison, IC50 of AG490, another JAK inhibitor, was >50 μM). Consistent with the central role of JAKs in gp130-mediated signaling, INCB20 inhibited IL-6 induced phosphorylation of SHP-2, STAT1, STAT3, ERK1/2, and AKT in MM1.S cells. In contrast, AKT phosphorylation induced by IGF-1 remained unchanged. Evaluation of the cellular efficacy of INCB20 was performed using the IL-6 dependent INA -6 cell line. Growth of INA-6 cells was inhibited in a dose-dependent manner with an IC50 of approx. 0.5 μM, as measured by [3H]-thymidine uptake and an MTS-based assay (for comparison, the cellular IC50 of AG490 was 15–20 μM). This correlated with an increase in the percentage of apoptotic cells, as evaluated by Apo2.7 staining after 48 hours. Importantly, INA-6 growth was inhibited in the presence of bone marrow stromal cells accompanied by a decrease in phospho-STAT3 levels. Furthermore, in a subcutaneous INA-6-SCID model, INCB20 inhibited tumor growth (and phosphorylated STAT3) in a dose-dependent manner. Our studies provide the conceptual basis for the use of JAK inhibitors as a therapeutic approach in MM.

scholarly journals Vialinin A, an Edible Mushroom-Derived p-Terphenyl Antioxidant, Prevents VEGF-Induced Neovascularization In Vitro and In Vivo

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Himangshu Sonowal ◽  
Kirtikar Shukla ◽  
Sumedha Kota ◽  
Ashish Saxena ◽  
Kota V. Ramana
Keyword(s):  
Nuclear Translocation ◽  
Vascular Endothelial Cell ◽  
Natural Antioxidants ◽  
Edible Mushroom ◽  
Tube Formation ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
In Vivo Models

Increased side toxicities and development of drug resistance are the major concern for the cancer chemotherapy using synthetic drugs. Therefore, identification of novel natural antioxidants with potential therapeutic efficacies is important. In the present study, we have examined how the antioxidant and anti-inflammatory activities of vialinin A, a p-terphenyl compound derived from Chinese edible mushroomT. terrestrisandT. vialis, prevents human umbilical vascular endothelial cell (HUVEC) neovascularization in vitro and in vivo models. Pretreatment of HUVECs with vialinin A prevents vascular endothelial growth factor- (VEGF) induced HUVEC cell growth in a dose-dependent manner. Further, vialinin A also inhibits VEGF-induced migration as well as tube formation of HUVECs. Treatment of HUVECs prevents VEGF-induced generation of reactive oxygen species (ROS) and malondialdehyde (MDA) and also inhibits VEGF-induced NF-κB nuclear translocation as well as DNA-binding activity. The VEGF-induced release of various angiogenic cytokines and chemokines in HUVECs was also significantly blunted by vialinin A. Most importantly, in a mouse model of Matrigel plug assay, vialinin A prevents the formation of new blood vessels and the expression of CD31 and vWF. Thus, our results indicate a novel role of vialinin A in the prevention of neovascularization and suggest that anticancer effects of vialinin A could be mediated through its potent antioxidant and antiangiogenic properties.

scholarly journals Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

Development ◽  
2021 ◽  
Author(s):  
Sophie H. L. Austin ◽  
Rut Gabarró-Solanas ◽  
Piero Rigo ◽  
Oana Paun ◽  
Lachlan Harris ◽  
...  
Keyword(s):  
Stem Cell ◽  
In Vitro Models ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Direct Role ◽  
Adult Nscs ◽  
Hippocampal Networks ◽  
Dose Dependent

Adult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage, but whether it has a direct role in the regulation of NSCs remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of NSCs in an active state and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that deletion of β-catenin in NSCs did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which reconciles some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

scholarly journals Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

2020 ◽  
Author(s):  
Sophie H. L. Austin ◽  
Lachlan Harris ◽  
Oana Paun ◽  
Piero Rigo ◽  
François Guillemot ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Direct Role ◽  
Adult Nscs ◽  
Hippocampal Networks ◽  
Dose Dependent

AbstractAdult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage and has been shown to promote the proliferation of intermediate progenitor cells. However, whether it has a direct role in the regulation of NSCs still remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that both active and quiescent adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of active NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that inhibiting NSCs response to Wnt, by conditionally deleting β-catenin, did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which could reconcile some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

The Tyrophostin Adaphostin (NSC680410) Inhibits Multiple Myeloma Bone Marrow Angiogenesis In Vitro and In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2507-2507 ◽  
Author(s):  
Klaus Podar ◽  
Jing Zhang ◽  
Marc S. Raab ◽  
Sonia Vallet ◽  
Mariateresa Fulciniti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Dependent Manner ◽  
Antiangiogenic Effect ◽  
Myelocytic Leukemia ◽  
Endothelial Cell Growth

Abstract Our own and other previous studies demonstrate marked anti-proliferative activity of the tyrophostin adaphostin (NSC680410) in a variety of hematologic malignancies including chronic myelocytic leukemia (CML), chronic lymphcytic leukemia (CLL), acute myelocytic leukemia (AML), and Multiple Myeloma. Here we show that adaphostin (NSC680410), similar to bortezomib, additionally inhibits tumor angiogenesis within the MM bone marrow (BM) microenvironment. This effect is elicited both indirectly by inhibition of VEGF production and secretion in MM cells, as well as directly by abrogation of endothelial cell growth. Specifically, adaphostin triggers marked downregulation of nuclear c-Myc expression in MM cells. Both adaphostin, as well as specific downregulation of c-Myc using siRNA, lead to a decrease in cobalt chloride- induced Hif-1alpha- expression and Hif-1alpha activity, as evidenced by western blot analysis and expression of Hif-1alpha- driven luciferase, respectively. Indeed secretion of the Hif-1alpha target gene VEGF is markedly inhibited in a dose- and time- dependent manner. Importantly, neither knockdown of c-Abl expression nor exogenous overexpression of caspase- cleavage- induced c-Abl fragment abrogates drug- induced Hif-1alpha downregulation or inhibition of its activity. Taken together, these results indicate the existence of a c-Myc/ Hif-1alpha- dependent, but c-Abl- independent, pathway modulating MM cell production and secretion of VEGF. In contrast, we demonstrate a direct antiangiogenic effect of adaphostin on endothelial cells, similar to H2O2, is mediated via c-Jun upregulation, inhibition of cell proliferation, and the induction of cell apoptosis. Moreover, our data further demonstrate activity of adaphostin within the BM microenvironment. Adaphostin, similar to bortezomib, significantly inhibits VEGF secretion triggered by adhesion of MM cells to BMSCs and endothelial cells. Consequently, conditioned medium derived from adaphostin- treated co-cultures markedly inhibits endothelial cell growth and tubule formation in a dose- dependent manner. Finally, we confirmed these in vitro results using an in vivo xenograft mouse model of human MM. Specifically, western blot analysis, as well as immunohistochemistry, demonstrate marked downregulation of both Hif-1alpha and CD31 in tumors isolated from adaphostin- treated animals versus control animals, confirming the in vivo antiangiogenic effect of adaphostin. Similar effects were obtained using a SCIDhu mouse model as well as a significant decrease of MM- related bone disease, due to anti- VEGF activity of adaphostin. Taken together, these data provide the rationale for the clinical evaluation of adaphostin to target both MM cells and the BM milieu to improve patient outcome in Multiple Myeloma.

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