Reyes Tejedor

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as α-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation.

Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes

Motile lymphocytes adopt a polarized morphology with different adhesion molecules (ICAM, CD43 and CD44) and ERM actin‐binding proteins concentrated on the uropod, a slender posterior appendage with important functions in cell‐cell interactions and lymphocyte recruitment. We have studied the role of Rho family of GTPases (Rho, Rac and Cdc42) in the control of lymphocyte polarity and migration by analyzing the effects of exogenously introduced Rho GTPase mutants. Transfection of T cell lines that constitutively display a polarized motile morphology with activated mutants of RhoA, Rac1 and Cdc42 impaired cell polarization. A guanosine nucleotide exchange factor for Rac, Tiam‐1, induced the same effect as activated Rac1. Conversely, dominant negative forms of the three GTP‐binding proteins induced a polarized phenotype in constitutively round‐shaped T cells with redistribution of ICAM‐3 and moesin to the uropod in an integrin‐dependent manner. On the other hand, overexpression of dominant negative Cdc42 and activated mutants of all three Rho GTPases significantly inhibited SDF‐1α‐induced T cell chemotaxis. Together, these data demonstrate that Rho GTPases regulate lymphocyte polarization and chemokine‐induced migration, and underscore the key role of Cdc42 in lymphocyte directional migration.

Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors.

CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-α synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-α was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcεRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.

TCR Engagement Induces Proline-Rich Tyrosine Kinase-2 (Pyk2) Translocation to the T Cell-APC Interface Independently of Pyk2 Activity and in an Immunoreceptor Tyrosine-Based Activation Motif-Mediated Fashion

The relocation of kinases in T lymphocytes during their cognate interaction with APCs is essential for lymphocyte activation. We found that the proline-rich tyrosine kinase-2 (Pyk2) is rapidly translocated to the T cell-APC contact area upon T cell-specific recognition of superantigen-pulsed APCs. Stimulation with anti-CD3-coated latex microspheres was sufficient for Pyk2 reorientation, and the coengagement of CD28 boosted Pyk2 redistribution. Nevertheless, Pyk2 translocation did not result in its recruitment to lipid rafts. Two results support that Pyk2 translocation was independent of its kinase activity. First, Lck activity was required for TCR-induced Pyk2 translocation, but not for TCR-induced Pyk2 activation. Second, a kinase-dead Pyk2 mutant was equally translocated upon TCR triggering. In addition, Lck activity alone was insufficient to induce Pyk2 reorientation and activation, requiring the presence of at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Despite the dependence on functional Lck and on phosphorylated ITAM for Pyk2 translocation, the ITAM-binding tyrosine kinase ζ-associated protein 70 (ZAP-70) was not essential. All these data suggest that, by translocating to the vicinity of the immune synapse, Pyk2 could play an essential role in T cell activation and polarized secretion of cytokines.

Photoprotective properties of a hydrophilic extract of the fern Polypodium leucotomos on human skin cells

The effect of a hydrophilic extract of the fern Polypodium leucotomos (PLE) has been investigated in terms of photoprotection against UV-induced cell damage. PLE efficiently preserved human fibroblast survival and restored their proliferative capability when the cells were exposed to UVA light. This effect was specific and dose-dependent. Photoprotection was not restricted to fibroblasts, as demonstrated by its effect on survival and proliferation of the human keratinocyte cell line HaCat. Finally, treatment of the cells with PLE prevented UV-induced morphological changes in human fibroblasts, namely disorganisation of F-actin-based cytoskeletal structures, coalescence of the tubulin cytoskeleton and mislocalization of adhesion molecules such as cadherins and integrins. Our in vitro results demonstrate the photoprotective effect of PLE on human cells and support its use in the preventive treatment of sunburning and skin pathologies associated with UV-mediated damage.

Involvement of α3 integrin/tetraspanin complexes in the angiogenic response induced by angiotensin II

The effect of angiotensin II (Ang II) on endothelial cell (EC) function has important potential implications, both under physiological conditions and in the pathogenesis of different cardiovascular diseases. We studied the effect of Ang II on EC junctions and its possible functional consequences. We found that Ang II induced a remarkable change on human umbilical vein endothelial cells (HUVEC) in the distribution of α3β1 integrin and CD151, CD9 tetraspanins on cell membrane, with an important decrease of these molecular complexes at intercellular contacts. However, the morphology of EC, the integrins located at cell‐substratum contact areas, the components of adherens and tight‐junctions, as well as the barrier functions of EC monolayers, remained unchanged after Ang II exposure. The Ang II effect on α3β1 1/tetraspanins complexes was associated with a significant induction of angiotube formation by HUVEC in an in vitro model of capillary formation on Matrigel. The effect of Ang II was mediated through the angiotensin II receptor 1 AT1 receptor and was inhibited with antibodies specific for the α3 integrin chain, which indicates the critical role of this integrin in the angiogenic effect of Ang II. These results show that Ang II exerts a direct and very specific effect on EC lateral junctions that is selectively associated with the induction of angiogenesis by this peptide.