MAP Kinase Signaling Pathway Induced by TPO Is a Negative Regulator of Proplatelet Formation in Human Megakaryocytes.

Platelets are formed from mature megakaryocytes (MKs) and arise from the development of cytoplasmic extensions called proplatelets (PPT). Proliferation and full maturation of MKs require TPO, but it is dispensable for platelet shedding. To precisely define the role of different signaling pathway activated by TPO on proplatelet formation (PPF), chemical inhibitors of ERK (PD98059), p38 (SB 203580) and a pan PKC inhibitor (GF109203X) were added to MK derived from human CD34+ cells. As previously reported, the PKC inhibitor leads to a marked decrease in PPF and the p38 inhibitor has no effect on PPF. Addition at day 8 of an inhibitor of the ERK pathway (PD98059) to the purified CD34+CD41+ cultured cells leads to an early PPF associated to a 3-fold increase of PPT. As observed with PD98059, an overexpression of a dominant-negative (DN) form of MEK1 in primary MKs led to a 2-fold increase of PPT. Inhibition of the ERK pathway in the immature MKs surprisingly increased MK mean ploidy, (5.7N in the control versus 10.9N in presence of PD98059). Especially, the highest classes of ploidy (8N, 16N and 32N) were significantly increased. Altogether, those results suggest that the inhibition of MAP/ERK1/2 is an essential step for PPF in mature MKs but also that MAPK/ERK1/2 is a negative regulator of endomitosis. As PPF occurs in the presence of TPO, we investigated whether MAPK activation was down-regulated during MK differentiation. Western blot and flow cytometry analysis were performed and show that ERK activation induced by TPO was dramatically decreased at the late stages of megakaryocytopoiesis. In addition, inhibition of MEK prior to PPF was associated with an activation of caspase 3 in the absence of apoptosis suggesting a direct role of the ERK pathway in the regulation of proplatelet. Altogether, those results show a differential response of MAP kinase pathway to TPO depending on the maturation stage. ERK downregulation occurs when PPF begins and MEK inhibition is associated with increased PPF.