REGULATION OF INTEGRIN-MEDIATED MYELOID CELL-ADHESION TO FIBRONECTIN - INFLUENCE OF DISULFIDE REDUCING AGENTS, DIVALENT-CATIONS AND PHORBOLESTER

Three different agents, dithiothreitol (DTT), Mn2+, and phorbol ester (TPA), were found to induce HL cell adhesion to fibronectin through di stinct mechanisms. The binding of HL-60 cells to fibronectin and a 120 -kDa fibronectin fragment is completely dependent on the alpha5beta1 i ntegrin, the adhesion activators, and appropriate divalent cations suc h as Mg2+. Mn2+ alone was able to induce maximal adhesion in the absen ce of these other activators. With any of the three activators, Ca2+ i nhibited adhesion to fibronectin substrates by inhibiting alpha5beta1- fibronectin binding. DTT and Mn2+ were both found to enhance the bindi ng of fibronectin to purified alpha5beta1, which suggests that both ag ents can directly stimulate the integrin-ligand binding reaction. TPA acts by inducing intracellular phosphorylation whereas neither DTT nor Mn2+ induced protein phosphorylation. TPA-treated HL-60 cells adhere and spread on fibronectin substrates, whereas DTT- and Mn2+-treated ce lls adhere but do not spread. The actin cytoskeletal inhibitor, cytoch alasin B, markedly blocks TPA-induced adhesion, has an intermediate ef fect on DTT-induced adhesion, and has a minimal effect on Mn2+-induced adhesion. Collectively, the data suggest that TPA seems to act by ind ucing phosphorylation events that lead to cytoskeletal changes and alp ha5beta1 integrin activation. In contrast, DTT and Mn2+ seem to act pr imarily by directly influencing the alpha5beta1 -fibronectin binding r eaction. These studies characterize in detail a regulatory system for studying leukocyte alpha5beta1-fibronectin adhesion and identify DTT a s a novel activator of alpha5beta1-fibronectin binding.