INHIBITION OF ERYTHROID-DIFFERENTIATION AND INDUCTION OF MEGAKARYOCYTIC DIFFERENTIATION BY THROMBOPOIETIN ARE REGULATED BY 2 DIFFERENT MECHANISMS IN TPO-DEPENDENT UT-7 C-MPL AND TF-1/C-MPL CELL-LINES/

Thrombopoietin (TPO) regulates megakaryocytic (MK) maturation and plat elet production. Molecular and cellular mechanisms of the TPO-induced MK differentiation are not totally understood. In order to develop cel lular models to study these mechanisms, we introduced c-mpl into UT-7 and TF-1 cells by means of a retroviral vector and compared the effect s of TPO on these two cell lines. UT-7 and TF-1 cell lines are two fac tor-dependent leukemic cell lines with an erythroid and MK phenotype. They proliferate in response to IL-3, GM-CSF and EPO, but not to TPO. The erythroid differentiation of both cell lines can be markedly incre ased by EPO. Several UT-7/c-mpl and TF-1/c-mpl cell clones which expre ss different levels of the c-mpl protein (Mpl) were obtained and ail b ecame TPO-dependent for their proliferation. The UT-7/c-mpl clones, bu t not the TF-l/c-mpl clones, were capable of undergoing MK differentia tion in response to TPO. This was demonstrated by the increase in MK m arkers (GPIIb, GPIIIa, GPIb alpha, GPIX and vWF), the appearance of cy toplasmic alpha-granules, intracellular membranes resembling demarcati on membranes which were immunologically labeled with an GPIIb/IIIa ant i-antibody, and a small percentage of polyploid cells (8N and 16N). In contrast, TPO inhibited the erythroid program of differentiation (gly cophorin A, beta-globin and EPO receptor) as well as the differentiati ve activity of EPO in both UT-7/c-mpl and TF-1/c-mpl crones. It is not eworthy that the differentiative effect of EPO in TF-1/c-mpl cells was associated with an increase in GATA-1 transcripts which was totally s uppressed by TPO. Overall the effects of TPO are the same as those of phorbol myristate acetate (PMA) which also induces MK differentiation and inhibits erythroid differentiation. These results suggest that: (1 ) Mpl expression is necessary but not sufficient for induction of MK d ifferentiation; and (2)induction of Mk differentiation and inhibition of erythroid differentiation by TPO involve different signaling pathwa ys; the pathway involved in the inhibition of erythroid differentiatio n might be related to a downregulation of GATA-1 expression in TF-1 ce lls.