Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: Role in endomitosis

Thrombopoietin (TPO) plays a critical role in megakaryocyte proliferation a nd differentiation. Using various cultured cell lines, several recent studi es have implicated the mitogen-activated protein kinase (MAPK) pathway in m egakaryocyte differentiation, In the study reported here, we examined the r ole played by thrombopoietin-induced MAPK activity in a cytokine-dependent cell line (BAF3/Mpl) and in primary murine megakaryocytes, In both systems, extracellular signal-regulated protein kinase (ERK) 1 and 2 MAPK phosphory lation was rapidly induced by TPO stimulation. To identify the Mpl domain r esponsible for MAPK activation, BAF3 cells expressing truncated forms of th e Mpl receptor were studied. Phosphorylation of ERKs did not require elemen ts of the cytoplasmic signaling domain distal to Box 2 and was not dependen t on phosphorylation of the adapter protein Shc. ERK activation in murine m egakaryocytes was maximal at 10 minutes and was markedly decreased over the subsequent 3 hours. Next, the physiologic consequences of MAPK inhibition were studied. Using the MAPK kinase (MEK) inhibitor, PD 98059, blockade of MAPK activity substantially reduced TPO-dependent proliferation in BAFB/Mpl cells and markedly decreased mean megakaryocyte ploidy in cultures. To exc lude an indirect effect of MAPK inhibition on stromal cells in whole bone m arrow, CD41(+) cells were selected and then cultured in TPO. The number of polyploid megakaryocytes derived from the CD41-selected cells was also sign ificantly reduced by MEK inhibition, as was their geometric mean ploidy. Th ese studies show an important role for MAPK in TPO-induced endomitosis and underscore the value of primary cells when studying the physiologic effects of signaling pathways. (C) 1999 by The American Society of Hematology.