Shear flow vs. static culture of d9 and d12 megakaryocytes

d9 and d12 Mks were either cultured statically or subjected to shear flow for 30 min; at d9, half the Mks were placed back in culture for 30 min (60 min time point) Megakaryocytes (Mks) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation thus releasing platelets and pro/preplatelets directly into the blood stream. Shear forces have been now established as promoting Mk maturation and platelet biogenesis. In order to understand the underlying mechanisms that modulate the response of Mks to shear forces, we carried out transcriptional analysis on immature and mature stem cell-derived Mks that were exposed to physiologically-relevant shear (2.5 dyn/cm2). In immature (d9) Mks, shear exposure upregulated genes related to growth and Mk maturation, while in mature (d12) Mks, it upregulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, 6 AP-1 transcripts (ATF4, JUNB, JUN, FOSB, FOS, and JUND) were upregulated at d9 and two AP-1 proteins (JunD and c-Fos) were upregulated both at d9 and d12. Our data show that MAPK signaling is linked to both the shear-stress response and AP-1 upregulation. JNK phosphorylation increased significantly following shear stimulation, while JNK inhibition reduced shear-induced JunD protein expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos protein expression. JNK inhibition reduced fibrinogen binding of d9 and d12 platelet-like particle s (PLPs) and P-selectin expression at d12 PLPs, while p38 inhibition reduced fibrinogen binding of d12 PLPs. Here we show that mechanotransduction of shear forces in Mks results in JNK activation, AP-1 upregulation, and downstream transcriptional changes that promote maturation of immature Mks and platelet biogenesis in mature Mks. Two- and Three-condition experiment (flow vs. static culture condition, d9 vs. d12, and 30 min vs. 60 min at d9); Biological replicates: 3; Technical replicates: 1 (dye-swap)