RESUMO
CD146 is an adhesion molecule expressed by both melanoma and endothelial cells and thus is well positioned to control melanoma extravasation. Nevertheless, during melanoma metastasis, the involvement of CD146 expressed within tumor microenvironment has never been analyzed. To investigate whether host CD146 mediates the extravasation of melanoma cells across the endothelium, we generated CD146 KO mice. We demonstrated that host CD146 did not affect melanoma growth or tumor angiogenesis but promoted hematogenous melanoma metastasis to the lung. Accordingly, the survival of CD146-deficient mice was markedly prolonged during melanoma metastasis. Interestingly, vascular endothelial growth factor-induced vascular permeability was significantly decreased in CD146 KO mice. We also provided evidence that VEGF-induced transendothelial migration of melanoma cells was significantly reduced across CD146 KO lung microvascular endothelial cells (LMEC). CD146 deficiency decreased the expression of VEGFR-2/Ve-cadherin and altered focal adhesion kinase (FAK) activation in response to VEGF. In addition, inhibition of FAK phosphorylation reduced transmigration of B16 melanoma cells across WT LMEC at the same level that across CD146 KO LMEC. Altogether, we propose a novel mechanism involving the VEGF/CD146/FAK/Ve-cadherin network in melanoma extravasation across the vessel barrier that identifies CD146-targeted therapy as a potential strategy for the treatment of melanoma metastasis.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Neoplasias Pulmonares/secundário , Pulmão/citologia , Melanoma Experimental/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Antígenos CD/metabolismo , Antígeno CD146/genética , Antígeno CD146/metabolismo , Caderinas/metabolismo , Endotélio Vascular/metabolismo , Regulação Neoplásica da Expressão Gênica , Pulmão/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Camundongos Knockout , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Células Tumorais CultivadasRESUMO
CD146 is a highly glycosylated junctional adhesion molecule, expressed on human vascular endothelial cells and involved in the control of vessel integrity. Galectin-1 is a lectin produced by vascular cells that can binds N- and O-linked oligosaccharides of cell membrane glycoproteins. Because both CD146 and Galectin-1 are involved in modulation of cell apoptosis, we hypothesized that Galectin-1 could interact with CD146, leading to functional consequences in endothelial cell apoptosis. We first characterized CD146 glycosylations and showed that it is mainly composed of N-glycans able to establish interactions with Galectin-1. We demonstrated a sugar-dependent binding of recombinant CD146 to Galectin-1 using both ELISA and Biacore assays. This interaction is direct, with a K(D) of 3.10(-7) M, and specific as CD146 binds to Galectin-1 and not to Galectin-2. Moreover, co-immunoprecipitation experiments showed that Galectin-1 interacts with endogenous CD146 that is highly expressed by HUVEC. We observed a Galectin-1-induced HUVEC apoptosis in a dose-dependent manner as demonstrated by Annexin-V/7AAD staining. Interestingly, both down-regulation of CD146 cell surface expression using siRNA and antibody-mediated blockade of CD146 increase this apoptosis. Altogether, our results identify Galectin-1 as a novel ligand for CD146 and this interaction protects, in vitro, endothelial cells against apoptosis induced by Galectin-1.
Assuntos
Apoptose , Antígeno CD146/química , Células Endoteliais/citologia , Galectina 1/metabolismo , Regulação da Expressão Gênica , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Fibroblastos/metabolismo , Galectina 2/metabolismo , Glicosilação , Células Endoteliais da Veia Umbilical Humana , Humanos , Cinética , Ligantes , Polissacarídeos/química , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
OBJECTIVES: During inflammation, cell adhesion molecules are modulated or redistributed for leukocyte transmigration. Among molecules at the interendothelial junction, CD146 is involved in cell-cell cohesion and permeability, but its role in monocyte transmigration is unknown. METHODS AND RESULTS: TNF enhanced CD146 expression at the junction and apical membrane of human umbilical veins endothelial cells (HUVECs) through CD146 synthesis and intracellular store redistribution. In addition, TNF increased the release of a soluble form (sCD146) through a metalloproteinase-dependent mechanism. The redistribution of CD146 to the junction led us to investigate its role in monocyte transmigration using THP1 and freshly isolated monocytes. Evidence that CD146 contributes to monocyte transmigration was provided by inhibition experiments using anti-CD146 antibodies and CD146 siRNA in HUVECs. In addition, sCD146 specifically bound both monocytes and HUVECs and dose-dependently increased monocyte transmigration. Assessment of sCD146 binding on immobilized CD146 failed to evidence any homophilic interaction. Together, our data suggest endothelial CD146 binds heterophilically with a yet unknown ligand on monocytes. CONCLUSIONS: Our results demonstrate that CD146 is regulated by the inflammatory cytokine TNF and that CD146 and sCD146 are both involved in monocyte transendothelial migration during inflammation.
Assuntos
Quimiotaxia/imunologia , Inflamação/imunologia , Monócitos/imunologia , Antígeno CD146/imunologia , Linhagem Celular Tumoral , Células Endoteliais/fisiologia , Humanos , Fator de Necrose Tumoral alfa/imunologia , Veias Umbilicais/citologiaRESUMO
BACKGROUND: Circulating CD34(+) cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN) could target progenitor cell injury by Natural Killer (NK) cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+)-derived Endothelial Colony Forming Cells (ECFC) can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+) progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+) cells expressing FKN was identified as an independent variable inversely correlated to CD34(+) progenitor cell count. We further showed that treatment of CD34(+) circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+) progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.
Assuntos
Quimiocina CX3CL1/metabolismo , Células Matadoras Naturais/citologia , Células-Tronco/citologia , Adesão Celular , Endotélio/citologia , Endotélio/metabolismo , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Células-Tronco/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
CD146/melanoma cell adhesion molecule is an adhesion molecule expressed by endothelial cells and by a small fraction of activated T and B lymphocytes in humans. In order to analyze the pattern of CD146 expression in mouse leukocytes at steady-state conditions, we generated a set of novel rat anti-mouse CD146 monoclonal antibodies. CD146 expression was undetectable on monocytes, dendritic cells, T cells or B cells, but was expressed on about 30% of neutrophils and 60% of NK cells. Within murine lymphocytes, CD146 was defined as a novel NK-specific surface molecule. An increased percentage of CD146+ cells was found in the most mature CD27(-)CD11b+ NK cell subpopulation, which also displays higher expression of Ly49C/I, Ly49D and KLRG1 and lower expression of NKG2A/C/E molecules. CD146+ NK cells were found to be less cytotoxic and produce less IFN-gamma than CD146(-) NK cells upon stimulation with target cells or activating antibodies. These findings define CD146 as a marker of mouse NK cell maturation that may be used as an alternative to the combined use of CD27 and CD11b staining to detect final stages of NK cell maturation.