Activation pathways of α4β1 integrin leading to distinct T-cell cytoskeleton reorganization, Rac1 regulation and Pyk2 phosphorylation

a4 beta 1 integrin is highly expressed in lyrnphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. alpha 4 beta 1 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-beta 1 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 alpha 4 beta 1 in Jurkat cells and in human T-lymphoblasts. Treatment with Mn2+, alpha-CD3 mAb or the chemokine SDF-1 alpha. followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM-1 (alpha 4 beta 1 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 alpha 4p1-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 alpha 4 beta 1 integrin, namely the ability to trigger two types of T-cell cytoskeletal response with different signaling requirements.