Mitogenic role for extracellular calmodulin-like activity in normal human umbilical vein endothelial cells

Normal human umbilical vein endothelial cells cultured on gelatin-coated plastic dishes were found to produce a protein in their media which had calmodulin-like immunoreactivity and biological activity. Further identification of the protein was achieved by examining the incorporation of 14C leucine into protein found in the conditioned medium. Cells produced 14C labelled protein in their medium which specifically bound to an affinity column for calmodulin. This latter material stimulated calmodulin dependent phosphodiesterase activity in vitro and this stimulation was inhibited by the addition of the calmodulin antagonist W7. The presence of calmodulin-like activity and immunoreactivity in the media varied as the cells grew from low to high density, a peak of extracellular calmodulin-like activity preceding an increase in cell number. Extracellular calmodulin-like activity did not correlate with the presence of lactate dehydrogenase in the medium. The addition of pure pig brain calmodulin affected the rate of cell proliferation; significant proliferation to pure calmodulin was only seen in cells at low density, at higher density calmodulin either had no effect or inhibited proliferation. Inhibition of extracellular calmodulin activity by a calmodulin antagonist immobilized on agarose beads, or by an antibody to calmodulin significantly decreased proliferation in all dividing cultures. Taken together this data suggests that, in vitro, calmodulin, or a very closely related protein, influences endothelial cell proliferation through an autocrine mechanism.