Expression and Modulation of Specific, High Affinity Binding Sites for Erythropoietin on the Human Erythroleukemic Cell Line K562

Abstract

AbstractErythroid differentiation is mediated by several interacting factors which include the glycoprotein hormone erythropoietin (Epo), interleukin-3 (IL-3) in the mouse, and erythroid-potentiating activity (EPA) in humans. Each of these factors binds to specific cell surface receptors on responsive target cells, but the way in which these factors interact to modulate erythropoiesis is unknown. In the present study, we used the human erythroleukemic cell line K562 to examine expression and regulation of the receptor for Epo using 126l-labeled, bioactive recombinant human Epo. K562 cells expressed low numbers of a single class of high-affinity Epo receptors corresponding to 4 to 6 receptors per K562 cell (KD = 270 to 290 pmol/L). Treatment of K562 cell cultures with medium conditioned by the EPA-secreting cell line U937 (U937CM) increased receptor expression 2.6 to 3.5-fold to 13 to 17 receptors/cell (KD = 260 to 300 pmol/L). That all of the Epo receptor-potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells. (kd = 260 to 300 pmol/L). That all of the Epo receptor-potentiating activity in U937CM was accounted for by EPA was shown by a similar increase in Epo receptor expression on K562 cells with recombinant EPA. The effect of U937CM on Epo receptors was reversed by culturing cells in inducer-free medium for 3 days. Medium conditioned by the 5637 cell line had no effect on Epo receptors on K562 cells. In methylcellulose culture, U937CM and Epo acted synergistically to increase erythroid differentiation of K562. Similarly, U937CM stimulated human cord blood CFU-E growth under conditions in which Epo was limiting or in excess. Increases in Epo receptor expression on K562 cells and on CFU-E in response to EPA may mediate the effects of Epo on these cells.