Thrombopoietin-Induced Activation of the Mitogen-Activated Protein Kinase (MAPK) Pathway in Normal Megakaryocytes: Role in Endomitosis

Abstract

Thrombopoietin (TPO) plays a critical role in megakaryocyte proliferation and differentiation. Using various cultured cell lines, several recent studies have implicated the mitogen-activated protein kinase (MAPK) pathway in megakaryocyte differentiation. In the study reported here, we examined the role 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 phosphorylation was rapidly induced by TPO stimulation. To identify the Mpl domain responsible for MAPK activation, BAF3 cells expressing truncated forms of the Mpl receptor were studied. Phosphorylation of ERKs did not require elements of the cytoplasmic signaling domain distal to Box 2 and was not dependent on phosphorylation of the adapter protein Shc. ERK activation in murine megakaryocytes 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 BAF3/Mpl cells and markedly decreased mean megakaryocyte ploidy in cultures. To exclude an indirect effect of MAPK inhibition on stromal cells in whole bone marrow, CD41+ cells were selected and then cultured in TPO. The number of polyploid megakaryocytes derived from the CD41-selected cells was also significantly reduced by MEK inhibition, as was their geometric mean ploidy. These 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.

2-(Trimethylammonium)ethyl (R)-3-methoxy-3-oxo-2-stearamidopropyl phosphate promotes megakaryocytic differentiation of myeloid leukaemia cells and primary human CD34+haematopoietic stem cells

“…It has been reported that activation of the MEK–ERK (Guerriero et al ., ; Majka et al ., ; Rojnuckarin et al ., ; Severin et al ., ) and PI3K‐AKT (Geddis et al ., ; Guerriero et al ., ) signalling pathways is important for TPO‐induced megakaryocyte differentiation. We thus sought to determine whether ( R )‐TEMOSPho activates the two signalling pathways, and whether such activation is required for megakaryocytic differentiation.…”

“…Importantly, however, the principal signalling pathway with a critical role appears to be the PI3K–AKT pathway, as blocking this pathway led to a far more visible inhibition of megakaryocytic differentiation. In sum, at least in our in vitro system, ( R )‐TEMOSPho appears to function as TPO does with respect to intracellular signalling (Guerriero et al ., ; Majka et al ., ; Rojnuckarin et al ., ). It should be noted that the function of ( R )‐TEMOSPho is independent of TPO, although we cannot rule out the possible role of serum‐derived auxiliary factors at this point.…”

“…Signalling is a complex issue requiring further work, particularly given the conflicting report made with regard to the role of MEK–ERK (Guerriero et al ., ). While some studies report the requirement of MEK–ERK activation for megakaryopoiesis (Majka et al ., ; Rojnuckarin et al ., ), others claim that blocking the pathway promotes certain aspects of the differentiation, such as polyploidization (Guerriero et al ., ). The situation possibly reflects diverse protocols, diverse cell types and diverse maturative stages used in different studies (Severin et al ., ).…”

“…Furthermore, at least for K562 cells, we show that functional platelets can be generated. Megakaryocytic differentiation induced by ( R )‐TEMOSPho appears to be mediated through the PI3K–AKT (Geddis et al ., ; Guerriero et al ., ) as well as the MEK–ERK (Guerriero et al ., ; Majka et al ., ; Rojnuckarin et al ., ; Severin et al ., ) pathways, as previous studies have shown for TPO‐induced megakaryopoiesis. These results indicate that ( R )‐TEMOSPho is a powerful novel tool for recapitulating and thereby dissecting the molecular nature of megakaryopoiesis and platelet genesis, and that ( R )‐TEMOSPho and its derivatives merit close examination as potential therapeutic agents for diseases originating from platelet dysfunction.…”

Functional expression of TRAIL and TRAIL‐R2 during human megakaryocytic development

“…Since the ERK1 and ERK2 subgroup of the MAPK family members has been involved in inducing megakaryocytic maturation (Rojnuckarin et al, 1999; Guerriero et al, 2001; Miyazaki et al, 2001; Rojnuckarin et al, 2001; Jiang et al, 2002), the last group of experiments was designed to investigate whether the ERK/MAPK pathway is engaged by the interaction between TRAIL and TRAIL receptors in primary megakaryocytic cultures. For this purpose, cells were exposed to recombinant TRAIL and activation of the ERK/MAPK pathway was investigated by Western Blot performed using antibodies specific for the residues that are phosphorylated upon kinase activation.…”