In the present study, we have compared the ability of BM- and FL-derived MKs to differentiate, migrate toward a gradient of SDF1α, and generate proplatelets. The role of MEK-ERK1/2 and p38MAPK pathways in these processes has been investigated. The results demonstrate a critical role for the ERK1/2 pathway, but not p38MAPK, in all of these processes, as well as several differences in the behavior of the two cell populations, of which the most notable was the failure of the FL-derived cells to migrate toward a gradient SDF1α due to a negligible level of CXCR4 receptor surface expression.
MK differentiation is characterized by expression of specific megakaryocytic surface glycoproteins and nuclear maturation, resulting in large polyploidy cells. In this study, the role of MEK-ERK1/2 pathway in the differentiation of BM- and FL-derived MK was investigated using relatively low concentrations of these two structurally distinct MEK inhibitors, UO126 and PD186141. Inhibition of MEK-ERK1/2 pathway is sufficient to induce an increase of expression of the immature marker CD34 and a decrease in the late MK commitment marker, GPIb, without affecting expression of the early megakaryocytic marker GPIIb. Furthermore, inhibition of the MEK-ERK1/2 pathway causes a leftward shift of the ploidy of the cells, showing a critical requirement of MEK-ERK1/2 pathway in late stage of MK differentiation. In contrast, the p38MAPK does not contribute to MK differentiation. Indeed, Miyazaki et al. has shown that, in cord blood primary MK, p38MAPK is required for TPO-induced erythroid differentiation and not for MK differentiation 
During megakaryopoiesis, differentiating MKs migrate within the complex BM stromal environment from the endosteal niche to the BM sinusoidal endothelial cells, the site of platelet formation. The chemokine SDF1α augments this motility and promotes the interaction of MKs with the BM vascular niche and, therefore, supports thrombopoiesis 
. Interestingly, we show that, contrary to BM-derived MKs, MKs derived from FL are not able to migrate in response to SDF1α because of a negligible level of CXCR4 cell surface expression, despite comparable level expression of CXCR4 gene in both models. This suggests a different regulation of megakaryopoiesis in these two models. One potential physiological explanation for this is that MKs in the liver do not need to migrate to release platelets because of a highly vascular environment. In contrast, within the complex BM microenvironment, MKs needs to migrate from the proliferative osteoblastic niche to the capillary-rich vascular niche to release platelets. Previous studies have revealed that MAPKs are important factors in the regulation of cell migration in numerous cell types in response to cell matrix proteins, growth factors, or cytokines 
. In this study, we demonstrate a critical role for ERK1/2, but not p38MAPK, in MK movement and chemotaxis in response to SDF1α. The defective migration might be explained by the altered polarization of the SDF1α receptor CXCR4, as shown previously 
. Moreover, ERK1/2 may govern cell movement by phosphorylating the myosin light-chain kinase, the protease calpain, or the focal adhesion kinase. These phosphorylations regulate the dynamics of focal adhesions and the reorganization of cytoskeleton, which are critical for cell migration [39−41]
. Further studies will focus upon elucidating the precise mechanisms by which ERK1/2 control cell migration.
The role of MEK-ERK1/2 and p38MAPK pathways in the terminal stage of megakaryopoiesis, proplatelet formation, was also investigated. An increased proportion of FL-derived MKs generate proplatelets compared to BM-derived MKs, perhaps reflecting the need to generate a large number of platelets in parallel to development of the vasculature in the fetus, whereas in contrast, the role of BM-derived MKs is to maintain a steady-state platelet production. Interestingly, in culture conditions, BM-derived MKs present a higher ploidy, but form fewer proplatelets than FL-derived MKs, suggesting that a higher threshold of MK maturation may be necessary to achieve proplatelet formation in BM. The present study also demonstrates that MEK-ERK1/2 pathway, but not p38MAPK, is required for proplatelet generation. It is now well-established that dynamic regulation of microtubules is essential for proplatelet formation [42−44]
and that microtubule-associated protein, which are well-characterized substrates of MAPKs [45,46]
, play a major role in microtubule dynamics 
. One of the potential mechanisms implicating ERK1/2 in proplatelet formation is therefore the regulation of the phosphorylation states of the microtubule-associated proteins and subsequent organization of the microtubule network.
In summary, we provide evidence for the critical role of the MEK-ERK1/2 pathway, but not p38MAPK, in MK differentiation, migration, and proplatelet formation, and describe several differences in the behavior of the two primary MK populations from BM and FL. Further experiments are required to identify and confirm the roles of the substrates of ERK1/2 that underlie these effects.