Caracterización de la señalización inside-out implicada en la regulación de la adhesión de Linfocitos T dependiente de la integrina VLA-4

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular I, leída el 4-04-2016Sección Deptal. de Bioquímica y Biología Molecular (Biológicas)Fac. de Ciencias BiológicasTRUEunpu

VE-cadherin RGD motifs promote metastasis and constitute a potential therapeutic target in melanoma and breast cancers

13 p.-6 fig.We have investigated the role of vascular-endothelial (VE)-cadherin in melanoma and breast cancer metastasis. We found that VE-cadherin is expressed in highly aggressive melanoma and breast cancer cell lines. Remarkably, inactivation of VEcadherin triggered a significant loss of malignant traits (proliferation, adhesion, invasion and transendothelial migration) in melanoma and breast cancer cells. These effects, except transendothelial migration, were induced by the VE-cadherin RGD motifs. Co-immunoprecipitation experiments demonstrated an interaction between VE-cadherin and α2β1 integrin, with the RGD motifs found to directly affect β1 integrin activation. VE-cadherin-mediated integrin signaling occurred through specific activation of SRC, ERK and JNK, including AKT in melanoma. Knocking down VEcadherin suppressed lung colonization capacity of melanoma or breast cancer cells inoculated in mice, while pre-incubation with VE-cadherin RGD peptides promoted lung metastasis for both cancer types. Finally, an in silico study revealed the association of high VE-cadherin expression with poor survival in a subset of melanoma patients and breast cancer patients showing low CD34 expression. These findings support a general role for VE-cadherin and other RGD cadherins as critical regulators of lung and liver metastasis in multiple solid tumours. These results pave the way for cadherin-specific RGD targeted therapies to control disseminated metastasis in multiple cancers.BEP was an FPI fellow from Ministry of Economy and Competitiveness (MINECO). This research was supported by grants BIO2012-31023 and BIO2015-66849 from MINECO and PRB2 (IPT13/0001-ISCIII-SGEFI/FEDER) to JIC.Peer reviewe

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice

ICAP-1 regulates β1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4β1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1–/– spleen T and B cells displayed upregulation of α4β1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+- and CD19+-selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers

Effectiveness and safety of GLP-1 receptor analogues vs. Rapid-acting insulin added to basal insulin therapy in patients with type 2 diabetes mellitus: A living systematic review

Efectividad y seguridad de los análogos de los receptores de GLP-1 vs. insulina de acción rápida añadidos al tratamiento con insulina basal de pacientes con diabetes mellitus tipo 2 (protocolo

In vivo adhesion of malignant B cells to bone marrow microvasculature is regulated by α4β1 cytoplasmic-binding proteins

40 p.-7 fig. Martínez-Moreno, Mónica et al.Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4β1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are β1-integrin-binding partners that regulate β1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4β1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4β1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4β1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4β1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4β1 activity.This work was supported by the following grants from the Ministerio de Economía y Competitividad (Spain): SAF2011-24022 to J.T; SAF2012-31613 to A.G-P; SAF2012-31142 and SAF2013-49662-EXP to A.H; PI13/01454 to P.S-M; RD12/0036/0061 to J.T. and A.G-P; RD12/0036/0058 to N.C.G. The work was also funded by grant P2010/BMD-2314 from the Comunidad de Madrid to A.G-P., J.T., P. S-M and A.H., and partially supported by a CRIS foundation grant to fight cancer to J.M-L. N.C.G. is also funded by a grant from the Asociación Española Contra el Cáncer (GCB120981SAN), and from the Gerencia Regional de Salud, Junta de Castilla y León (GRS-702/A/11).Peer reviewe

Lateral mobility and nanoscale spatial arrangement of chemokine-activated α4β1 integrins on T cells

11p.-8 fig.Chemokine stimulation of integrin α4β1-dependent T lymphocyte adhesion is a key step during lymphocyte trafficking. A central question regarding α4β1 function is how its lateral mobility and organization influence its affinity and avidity following cell stimulation with chemokines and/or ligands. Using single particle tracking and super-resolution imaging approaches, we explored the lateral mobility and spatial arrangement of individual α4β1 integrins on T cells exposed to different activating stimuli. We show that CXCL12 stimulation leads to rapid and transient α4β1 activation, measured by induction of the activation epitope recognized by the HUTS-21 anti-β1 antibody and by increased talin-β1 association. CXCL12-dependent α4β1 activation directly correlated with restricted lateral diffusion and integrin immobilization. Moreover, co-stimulation by CXCL12 together with soluble VCAM-1 potentiated integrin immobilization with a five-fold increase in immobile integrins as compared to unstimulated conditions. Our data indicate that docking by talin of the chemokine-activated α4β1 to the actin cytoskeleton favors integrin immobilization, which likely facilitates ligand interaction and increased adhesiveness. Super-resolution imaging showed that the nanoscale organization of high-affinity α4β1 remains unaffected following chemokine and/or ligand addition. Instead, newly activated α4β1 integrins organize on the cell membrane as independent units without joining pre-established integrin sites to contribute to cluster formation. Altogether, our results provide a rationale to understand how the spatiotemporal organization of activated α4β1 integrins regulates T lymphocyte adhesion.This work was supported by Erasmus Mundus Doctorate Program Europhotonics Grant 159224-1-2009-1-FR-ERA MUNDUS-EMJD), Spanish Ministry of Economy and Competitiveness “Severo Ochoa” Programme for Centres of Excellence in R&D Grants SEV-2015–0522, FIS2014 –5617-R, SAF2014–53059-R, and RD12/0036/0061; Fundacio Privada CELLEX (Barcelona);HFSP Grant GA RGP0027/2012; and LaserLab Europe 4 Grant GA 654148.Peer reviewe

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1

Stimulation by chemokines of integrin α4β1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation.This work was supported by grants SAF2011-24022 from Ministerio de Economía y Competitividad, RD12/0036/0061, and S2010/BMD-2314 from Comunidad de Madrid to J.T.Peer Reviewe