Activation pathways of alpha4beta1 integrin leading to distinct T-cell cytoskeleton reorganization, Rac1 regulation and Pyk2 phosphorylation.
Alpha4beta1 integrin is highly expressed in lymphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. Alpha4beta1 can be activated by intracellular signals elicited upon T-cell activation by phorbol esters, CD3 crosslinking, or certain chemokine/receptor interactions (inside-out activation). Divalent cations or certain anti-beta1 mAbs (i.e., TS2/16) can also bind and activate integrins directly (outside-in activation). In both cases, activation results in increased adhesion and/or affinity for ligands. It is not known if these various stimuli produce the same or different post-adhesion events. To address this, we have studied the cytoskeleton organization and intracellular signaling following activation of 41 in Jurkat cells and in human T-lymphoblasts. Treatment with Mn2+, alpha-CD3 mAb or the chemokine SDF-1alpha followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM-1 (alpha4beta1 ligands), resulted in cell polarization and migration. In contrast, activation with PMA or TS2/16 induced cell spreading and strong adherence. Video microscopy and Transwell analyses confirmed these results, which correlated with different resistance to detachment under flow. Activation of the small GTPase RhoA or transfection with the constitutively active mutants V14RhoA or V12Rac1, abolished the alpha4beta1-induced cell polarization but did not affect cell spreading. Moreover, Rac1 activity was distinctly modulated by agents that induce a polarized or spread phenotype. The tyrosine kinase Pyk2 was highly phosphorylated upon induction of cell polarity but not during cell spreading. These results reveal novel properties of alpha4beta1 integrin, namely the ability to trigger two types of T-cell cytoskeletal response with different signaling requirements.
Maqueda
Alfredo
A
Departamento de Inmunología, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Moyano
José V
JV
Gutiérrez-López
M Dolores
MD
Ovalle
Susana
S
Rodríguez-Frade
José M
JM
Cabañas
Carlos
C
Garcia-Pardo
Angeles
A
D002448:Cell Adhesion; D002465:Cell Movement; D016764:Cell Polarity; D048430:Cell Shape; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D006801:Humans; D039041:Integrin alpha4beta1 / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D039041:Integrin alpha4beta1; D021122:Protein Subunits; D051418:Focal Adhesion Kinase 2; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein