THROMBOPOIETIN AND ERYTHROPOIETIN ACTIVATE INSIDE-OUT SIGNALING OF INTEGRIN AND ENHANCE ADHESION TO IMMOBILIZED FIBRONECTIN IN HUMAN GROWTH-FACTOR-DEPENDENT HEMATOPOIETIC-CELLS

Erythropoietin (EPO) and thrombopoietin (c-MPL ligand; TPO) are struct urally similar cytokines and support respectively, the proliferation a nd differentiation for erythroid and megakaryocytic lineages, as well as more primitive progenitors. We studied the effect of these cytokine s on the induction of adhesion of human growth-factor-dependent hemato poietic cells to immobilized fibronectin, which is a main component of the extracellular matrix in the bone marrow. MO7ER cells that are gen etically engineered to express human EPO receptor and MO7e cells that express endogenous c-MPL were used. Stimulation with either TPO or EPO induced rapid increases in adhesion of M07ER cells to fibronectin wit hout apparent change of expression of integrins. Experiments with inhi bitory monoclonal antibodies (mAbs) demonstrated that CD41, which has been reported to be involved in TPO-induced adhesion of megakaryocytic cells, is not responsible for this enhanced adhesion. Anti-beta 1 int egrin mAb inhibited adhesion completely, while inhibition by anti-alph a 4 integrin mAb and anti-alpha 5 integrin mAb was partial. Combinatio n of anti-alpha 4 mAb plus anti-alpha 5 mAb completely abolished adhes ion, as did anti-beta 1 mAb, suggesting that the adhesion is mediated by both alpha 4 beta 1 and alpha 5 beta 1 integrins. Experiments using inhibitors suggested that ligand binding followed by activation of in tracellular tyrosine kinases along with PI3-kinase activation is requi red. After stimulation of M07ER cells with either TPO or EPO, fibronec tin-attached cells, but not cells in suspension, showed tyrosine phosp horylation of focal adhesion kinase, which plays a central role in int egrin-mediated signaling. These data suggest that TPO and EPO might be involved in homing/migration to the bone marrow microenvironment by h ematopoietic cells that express corresponding receptors.