scholarly journals Involvement of Src Family Kinases in N-Cadherin Phosphorylation and β-Catenin Dissociation during Transendothelial Migration of Melanoma Cells

2006 ◽  
Vol 17 (3) ◽  
pp. 1261-1272 ◽  
Author(s):  
Jianfei Qi ◽  
Junfu Wang ◽  
Olena Romanyuk ◽  
Chi-Hung Siu
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Nuclear Translocation ◽  
Temporal Correlation ◽  
Transendothelial Migration ◽  
Cytoplasmic Domain ◽  
Melanoma Cells ◽  
Src Family Kinases ◽  
Dominant Negative

N-cadherin is recruited to the heterotypic contact during transendothelial migration of melanoma cells in a coculture system with tumor cells seeded on top of a monolayer of endothelial cells. However, β-catenin dissociates from N-cadherin and redistributes to the nucleus of transmigrating melanoma cells to activate gene transcription. In this report, we demonstrate that Src becomes activated at the heterotypic contact between the transmigrating melanoma cell and neighboring endothelial cells. Src activation shows close temporal correlation with tyrosine phosphorylation of N-cadherin. Expression of a dominant-negative Src in melanoma cells blocks N-cadherin phosphorylation, β-catenin dissociation, and nuclear translocation in transmigrating cells, consistent with the involvement of Src family kinases. In in vitro binding assays, Src-mediated phosphorylation of the N-cadherin cytoplasmic domain results in a significant reduction in β-catenin binding. Although five phospho-tyrosine residues can be identified on the N-cadherin cytoplasmic domain by mass spectrometry, site-specific mutagenesis indicates that Tyr-860 is the critical amino acid involved in β-catenin binding. Overexpression of N-cadherin carrying the Y860F mutation inhibits the transmigration of transfected cells across the endothelium. Together, the data suggest a novel role for tyrosine phosphorylation of N-cadherin by Src family kinases in the regulation of β-catenin association during transendothelial migration of melanoma cells.

scholarly journals Transendothelial Migration of Melanoma Cells Involves N-Cadherin-mediated Adhesion and Activation of the β-Catenin Signaling Pathway

2005 ◽  
Vol 16 (9) ◽  
pp. 4386-4397 ◽  
Author(s):  
Jianfei Qi ◽  
Ning Chen ◽  
Junfu Wang ◽  
Chi-Hung Siu
Keyword(s):  
Endothelial Cells ◽  
Cancer Cells ◽  
Cancer Metastasis ◽  
Transendothelial Migration ◽  
Melanoma Cells ◽  
Dominant Negative ◽  
Vitro Assay ◽  
Multistep Process ◽  
Adhesive Interactions

Cancer metastasis is a multistep process involving many types of cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of cancer cells. Transendothelial migration of cancer cells was investigated using an in vitro assay, in which melanoma cells were seeded on top of a monolayer of endothelial cells. Attachment of melanoma cells on the endothelium induced a twofold increase in N-cadherin expression in melanoma cells and the redistribution of N-cadherin to the heterotypic contacts. Transendothelial migration was inhibited when N-cadherin expression was repressed by antisense RNA, indicating a key role played by N-cadherin. Whereas N-cadherin and β-catenin colocalized in the contact regions between melanoma cells and endothelial cells during the initial stages of attachment, β-catenin disappeared from the heterotypic contacts during transmigration of melanoma cells. Immunolocalization and immunoprecipitation studies indicate that N-cadherin became tyrosine-phosphorylated, resulting in the dissociation of β-catenin from these contact regions. Concomitantly, an increase in the nuclear level of β-catenin occurred in melanoma cells, together with a sixfold increase in β-catenin-dependent transcription. Transendothelial migration was compromised in cells expressing a dominant-negative form of β-catenin, thus supporting a regulatory role of β-catenin signaling in this process.

scholarly journals Involvement of Integrin αvβ3and Cell Adhesion Molecule L1 in Transendothelial Migration of Melanoma Cells

2001 ◽  
Vol 12 (9) ◽  
pp. 2699-2710 ◽  
Author(s):  
Evelyn B. Voura ◽  
Ravi A. Ramjeesingh ◽  
Anthony M.P. Montgomery ◽  
Chi-Hung Siu
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Polyclonal Antibodies ◽  
Transendothelial Migration ◽  
Melanoma Cells ◽  
Vitro Assay ◽  
Adhesive Interactions

Tumor metastasis involves many stage-specific adhesive interactions. The expression of several cell adhesion molecules, notably the integrin αvβ3, has been associated with the metastatic potential of tumor cells. In this study, we used a novel in vitro assay to examine the role of αvβ3 in the transmigration of melanoma cells through a monolayer of human lung microvascular endothelial cells. Confocal microscopy revealed the presence of the integrin αvβ3 on melanoma membrane protrusions and pseudopods penetrating the endothelial junction. αvβ3 was also enriched in heterotypic contacts between endothelial cells and melanoma cells. Transendothelial migration of melanoma cells was inhibited by either a cyclic Arg-Gly-Asp peptide or the anti-αvβ3monoclonal antibody LM609. Although both platelet endothelial cell adhesion molecule-1 and L1 are known to bind integrin αvβ3, only L1 serves as a potential ligand for αvβ3 during melanoma transendothelial migration. Also, polyclonal antibodies against L1 partially inhibited the transendothelial migration of melanoma cells. However, addition of both L1 and αvβ3 antibodies did not show additive effects, suggesting that they are components of the same adhesion system. Together, the data suggest that interactions between the integrin αvβ3 on melanoma cells and L1 on endothelial cells play an important role in the transendothelial migration of melanoma cells.

scholarly journals EGF-R signaling through Fyn kinase disrupts the function of integrin α6β4 at hemidesmosomes

2001 ◽  
Vol 155 (3) ◽  
pp. 447-458 ◽  
Author(s):  
Agnese Mariotti ◽  
Paul A. Kedeshian ◽  
Michael Dans ◽  
Anna Maria Curatola ◽  
Laurent Gagnoux-Palacios ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitors ◽  
Egf Receptor ◽  
Lung Metastases ◽  
Squamous Carcinoma ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Squamous Carcinoma Cells ◽  
Integrin Α6β4

We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin α6β4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the β4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of β4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with α6β4. By contrast, Src and Lck do not associate with α6β4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of β4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the β4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.

scholarly journals LPS induces pp60c-src-mediated tyrosine phosphorylation of Hsp90 in lung vascular endothelial cells and mouse lung

2013 ◽  
Vol 304 (12) ◽  
pp. L883-L893 ◽  
Author(s):  
Nektarios Barabutis ◽  
Vaishali Handa ◽  
Christiana Dimitropoulou ◽  
Ruslan Rafikov ◽  
Connie Snead ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Phosphorylation ◽  
Anticancer Agents ◽  
Posttranslational Modifications ◽  
Hsp90 Inhibitor ◽  
Dominant Negative ◽  
Mouse Lung ◽  
Barrier Dysfunction

Heat shock protein 90 (Hsp90) inhibitors were initially developed as anticancer agents; however, it is becoming increasing clear that they also possess potent anti-inflammatory properties. Posttranslational modifications of Hsp90 have been reported in tumors and have been hypothesized to affect client protein- and inhibitor-binding activities. In the present study we investigated the posttranslational modification of Hsp90 in inflammation. LPS, a prototypical inflammatory agent, induced concentration- and time-dependent tyrosine (Y) phosphorylation of Hsp90α and Hsp90β in bovine pulmonary arterial and human lung microvascular endothelial cells (HLMVEC). Mass spectrometry identified Y309 as a major site of Y phosphorylation on Hsp90α (Y300 of Hsp90β). LPS-induced Hsp90 phosphorylation was prevented by the Hsp90 inhibitor 17-allyl-amino-demethoxy-geldanamycin (17-AAG) in vitro as well as in lungs from LPS-treated mice, in vivo. Furthermore, 17-AAG prevented LPS-induced pp60src activation. LPS-induced Hsp90 phosphorylation was also prevented by the pp60src inhibitor PP2. Additionally, Hsp90 phosphorylation was induced by infecting cells with a constitutively active pp60src adenovirus, whereas either a dominant-negative pp60src adenovirus or reduced expression of pp60src by a specific siRNA prevented the LPS-induced Y phosphorylation of Hsp90. Transfection of HLMVEC with the nonphosphorylatable Hsp90β Y300F mutant prevented LPS-induced Hsp90β tyrosine phosphorylation but not pp60src activation. Furthermore, the Hsp90β Y300F mutant showed a reduced ability to bind the Hsp90 client proteins eNOS and pp60src and HLMVEC transfected with the mutant exhibited reduced LPS-induced barrier dysfunction. We conclude that inflammatory stimuli cause posttranslational modifications of Hsp90 that are Hsp90-inhibitor sensitive and may be important to the proinflammatory actions of Hsp90.

scholarly journals Activation of Nuclear Factor κb and bcl-x Survival Gene Expression by Nerve Growth Factor Requires Tyrosine Phosphorylation of IκBα

2001 ◽  
Vol 152 (4) ◽  
pp. 753-764 ◽  
Author(s):  
Nguyen Truc Bui ◽  
Antonia Livolsi ◽  
Jean-Francois Peyron ◽  
Jochen H.M. Prehn
Keyword(s):  
Gene Expression ◽  
Tyrosine Phosphorylation ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Factor ◽  
Human Neuroblastoma ◽  
Tnf Α

NGF has been shown to support neuron survival by activating the transcription factor nuclear factor-κB (NFκB). We investigated the effect of NGF on the expression of Bcl-xL, an anti–apoptotic Bcl-2 family protein. Treatment of rat pheochromocytoma PC12 cells, human neuroblastoma SH-SY5Y cells, or primary rat hippocampal neurons with NGF (0.1–10 ng/ml) increased the expression of bcl-xL mRNA and protein. Reporter gene analysis revealed a significant increase in NFκB activity after treatment with NGF that was associated with increased nuclear translocation of the active NFκB p65 subunit. NGF-induced NFκB activity and Bcl-xL expression were inhibited in cells overexpressing the NFκB inhibitor, IκBα. Unlike tumor necrosis factor-α (TNF-α), however, NGF-induced NFκB activation occurred without significant degradation of IκBs determined by Western blot analysis and time-lapse imaging of neurons expressing green fluorescent protein–tagged IκBα. Moreover, in contrast to TNF-α, NGF failed to phosphorylate IκBα at serine residue 32, but instead caused significant tyrosine phosphorylation. Overexpression of a Y42F mutant of IκBα potently suppressed NFG-, but not TNF-α–induced NFκB activation. Conversely, overexpression of a dominant negative mutant of TNF receptor-associated factor-6 blocked TNF-α–, but not NGF-induced NFκB activation. We conclude that NGF and TNF-α induce different signaling pathways in neurons to activate NFκB and bcl-x gene expression.

The Rac activator Tiam1 controls efficient T-cell trafficking and route of transendothelial migration

Blood ◽  
2009 ◽  
Vol 113 (24) ◽  
pp. 6138-6147 ◽  
Author(s):  
Audrey Gérard ◽  
Rob A. van der Kammen ◽  
Hans Janssen ◽  
Saskia I. Ellenbroek ◽  
John G. Collard
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
Transendothelial Migration ◽  
Contact Hypersensitivity ◽  
Cell Trafficking ◽  
T Cell Trafficking ◽  
Cell Arrest

Abstract Migration toward chemoattractants is a hallmark of T-cell trafficking and is essential to produce an efficient immune response. Here, we have analyzed the function of the Rac activator Tiam1 in the control of T-cell trafficking and transendothelial migration. We found that Tiam1 is required for chemokine- and S1P-induced Rac activation and subsequent cell migration. As a result, Tiam1-deficient T cells show reduced chemotaxis in vitro, and impaired homing, egress, and contact hypersensitivity in vivo. Analysis of the T-cell transendothelial migration cascade revealed that PKCζ/Tiam1/Rac signaling is dispensable for T-cell arrest but is essential for the stabilization of polarization and efficient crawling of T cells on endothelial cells. T cells that lack Tiam1 predominantly transmigrate through individual endothelial cells (transcellular migration) rather than at endothelial junctions (paracellular migration), suggesting that T cells are able to change their route of transendothelial migration according to their polarization status and crawling capacity.

scholarly journals TRPC6 is the endothelial calcium channel that regulates leukocyte transendothelial migration during the inflammatory response

2015 ◽  
Vol 212 (11) ◽  
pp. 1883-1899 ◽  
Author(s):  
Evan W. Weber ◽  
Fei Han ◽  
Mohammad Tauseef ◽  
Lutz Birnbaumer ◽  
Dolly Mehta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Response ◽  
Calcium Channel ◽  
Transient Receptor Potential ◽  
Leukocyte Adhesion ◽  
Transendothelial Migration ◽  
Dominant Negative ◽  
Ion Concentration ◽  
Free Calcium ◽  
Leukocyte Transendothelial Migration

Leukocyte transendothelial migration (TEM) is a tightly regulated, multistep process that is critical to the inflammatory response. A transient increase in endothelial cytosolic free calcium ion concentration (↑[Ca2+]i) is required for TEM. However, the mechanism by which endothelial ↑[Ca2+]i regulates TEM and the channels mediating this ↑[Ca2+]i are unknown. Buffering ↑[Ca2+]i in endothelial cells does not affect leukocyte adhesion or locomotion but selectively blocks TEM, suggesting a role for ↑[Ca2+]i specifically for this step. Transient receptor potential canonical 6 (TRPC6), a Ca2+ channel expressed in endothelial cells, colocalizes with platelet/endothelial cell adhesion molecule-1 (PECAM) to surround leukocytes during TEM and clusters when endothelial PECAM is engaged. Expression of dominant-negative TRPC6 or shRNA knockdown in endothelial cells arrests neutrophils apically over the junction, similar to when PECAM is blocked. Selectively activating endothelial TRPC6 rescues TEM during an ongoing PECAM blockade, indicating that TRPC6 functions downstream of PECAM. Furthermore, endothelial TRPC6 is required for trafficking of lateral border recycling compartment membrane, which facilitates TEM. Finally, mice lacking TRPC6 in the nonmyeloid compartment (i.e., endothelium) exhibit a profound defect in neutrophil TEM with no effect on leukocyte trafficking. Our findings identify endothelial TRPC6 as the calcium channel mediating the ↑[Ca2+]i required for TEM at a step downstream of PECAM homophilic interactions.

scholarly journals An Autocrine Wnt5a Loop Promotes NF-κB Pathway Activation and Cytokine/Chemokine Secretion in Melanoma

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1060 ◽  
Author(s):  
Gastón Barbero ◽  
María Victoria Castro ◽  
María Belén Villanueva ◽  
María Josefina Quezada ◽  
Natalia Brenda Fernández ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Epithelial Mesenchymal Transition ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Melanoma Cells ◽  
Dominant Negative ◽  
Migration And Invasion ◽  
Soluble Receptor ◽  
Mesenchymal Transition ◽  
Cytokines And Chemokines

Wnt5a signaling has been implicated in the progression of cancer by regulating multiple cellular processes, largely migration and invasion, epithelial-mesenchymal transition (EMT), and metastasis. Since Wnt5a signaling has also been involved in inflammatory processes in infectious and inflammatory diseases, we addressed the role of Wnt5a in regulating NF-κB, a pivotal mediator of inflammatory responses, in the context of cancer. The treatment of melanoma cells with Wnt5a induced phosphorylation of the NF-κB subunit p65 as well as IKK phosphorylation and IκB degradation. By using cDNA overexpression, RNA interference, and dominant negative mutants we determined that ROR1, Dvl2, and Akt (from the Wnt5a pathway) and TRAF2 and RIP (from the NF-κB pathway) are required for the Wnt5a/NF-κB crosstalk. Wnt5a also induced p65 nuclear translocation and increased NF-κB activity as evidenced by reporter assays and a NF-κB-specific upregulation of RelB, Bcl-2, and Cyclin D1. Further, stimulation of melanoma cells with Wnt5a increased the secretion of cytokines and chemokines, including IL-6, IL-8, IL-11, and IL-6 soluble receptor, MCP-1, and TNF soluble receptor I. The inhibition of endogenous Wnt5a demonstrated that an autocrine Wnt5a loop is a major regulator of the NF-κB pathway in melanoma. Taken together, these results indicate that Wnt5a activates the NF-κB pathway and has an immunomodulatory effect on melanoma through the secretion of cytokines and chemokines.

LRP8 activates STAT3 to induce PD-L1 expression in osteosarcoma

2020 ◽  
pp. 030089162095287
Author(s):  
Shiqin Zheng ◽  
Yuxi Wei ◽  
Yu Jiang ◽  
Yi Hao
Keyword(s):  
Cell Line ◽  
Culture System ◽  
Nuclear Translocation ◽  
Interleukin 2 ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
Osteosarcoma Cell Line ◽  
Osteosarcoma Cell ◽  
Normal Tissues

Purpose: Targeting programmed death-ligand 1 (PD-L1) may be an effective intervention for osteosarcoma and PD-L1 expression is controlled by diverse regulatory factors. Low-density lipoprotein receptor-related protein 8 (LRP8) regulates osteoblast differentiation and it is unclear whether and how LRP8 could contribute to osteosarcoma pathogenesis. In this study, we investigated the LRP8/signal transducer and activator of transcription 3 (STAT3)/PD-L1 network in osteosarcoma. Methods: The expression of LRP8, STAT3, and PD-L1 was measured in osteosarcoma tissues and paired normal tissues. The effects of LRP8 on STAT3 and PD-L1 expression were investigated in an osteosarcoma cell line. The effects on immunosuppression were investigated in an in vitro co-culture system with Jurkat cell line and osteosarcoma cell line. The effects of LRP8 were blocked by a LRP8 neutralizing antibody, dominant-negative STAT3, or STAT3 inhibitor. Results: LRP8 was overexpressed in osteosarcoma compared to normal tissues and its level was correlated with phospho-STAT3 (p-STAT3) level in osteosarcoma tissues. In osteosarcoma cell lines, LRP8 increased p-STAT3 level and promoted nuclear translocation of STAT3. STAT3 activation also increased PD-L1 mRNA, protein, and promoter activity. In addition, LRP8 enhanced PD-L1 expression via STAT3. In a co-culture system, LRP8 overexpression in an osteosarcoma cell line impaired viability and interleukin-2 secretion of Jurkat cells and induced apoptosis of Jurkat cells. The effects of LRP8 could be blocked by neutralizing LRP8 antibody or STAT3 inhibitor. Blocking LRP8 inhibits proliferation and induces apoptosis of osteosarcoma cells. Conclusions: Our results provide evidence for a novel regulation network of LRP8/STAT3/PD-L1 in osteosarcoma and LRP8 may be a potential therapeutic target in osteosarcoma.

scholarly journals Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin

1997 ◽  
Vol 137 (3) ◽  
pp. 703-714 ◽  
Author(s):  
Timothy D. Garver ◽  
Qun Ren ◽  
Shmuel Tuvia ◽  
Vann Bennett
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Phosphorylation ◽  
Adhesion Molecules ◽  
Tyrosine Kinases ◽  
Cell Adhesion Molecules ◽  
Protein Tyrosine Kinase ◽  
Neuroblastoma Cells ◽  
Cytoplasmic Domain ◽  
Lateral Mobility

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.

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