Involvement of prolonged ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line

Thrombopoietin (TPO) is a hematopoietic growth factor that plays fundamental roles is both megakaryopoiesis and thrombopoiesis through binding to its receptor, c-mpl. Although, TPO has been shown to activate various types of intracellular signaling molecules, such as the Janus family of protein tyrosine kinases, signal transducers and activators of transcription (STATs), and ras, the precise mechanisms underlying TPOinduced proliferation and differentiation remain unknown. In an effort to clarify the mechanisms of TPOinduced proliferation and differentiation, c-mpl was introduced into F- 36P, a human interleukin-3 (IL-3)dependent erythroleukemia cell line, and the effects of TPO on the c-mpl-transfected F-36P (F-36P-mpl) cells were investigated. F-36P-mpl cells were found to proliferate an differentiate at a high rate into mature megakaryocytes in response to TPO. Dominant-negative (dn) forms of STAT1, STAT3, STAT5, and ras were inducibly expressed in F- 36P-mpl cells, and their effects on TPQ-induced proliferation and megakaryocytic differentiation were analyzed. Among these dn molecules, both dn ras and STAT5 reduced TPO- or IL-3-induced proliferation of F-36P-mpl cells by ~30%, and only dn ras could inhibit TPO-induced megakaryocytic differentiation. In accord with this result, overexpression of activated ras (H-ras(G12C)) for 5 days led to megakaryocytic differentiation of F-36P-mpl cells. In a time course analysis on H-ras(G12v)-induced differentiation, activation of the ras pathway for 24 to 28 h was required and sufficient to induce megakaryocytic differentiation. Consistent with this result, the treatment of F-36P-mpl cells-with TPO was able to induce prolonged activation of ras for more than 24 h, whereas IL-3 had only a transient effect. These results suggest that prolonged ras activation may be involved in TPO-induced megakaryocytic differentiation.