Abstract
Development of human megakaryocytes is modulated at least in part by direct contact with stromal cells, however, the mechanisms that control this process have not been fully elucidated. Stromal contact is not needed for lineage commitment and direct contact of human progenitor cells with marrow stroma negatively influences megakaryocytic development. The K562 human erythroleukemia cell line has been extensively used as a model to study molecular aspects of megakaryocytic differentiation. Treatment of K562 with either phorbol-12 myristate-13 acetate (PMA), a PKC activator, or staurosporine (STSP) a PKC inhibitor, induces K562 cells to undergo differentiation to a megakaryocytic phenotype. Treated cells undergo growth arrest, changes in cellular morphology as well as increased expression of megakaryocytic/platelet-specific antigens, such as CD61. We have demonstrated that PMA-induced differentiation of K562 cells is inhibited by co-culture on the bone marrow stromal cell line, OP9. These cells remained small, continued to proliferate, and had less than 10% CD61 surface expression, similar to untreated K562. In contrast, K562 cells co-cultured on OP9 cells and treated with STSP retained the ability to differentiate. They were significantly larger than unstimulated K562 cells, exhibited megakaryocytic morphology, and had increased CD61 surface expression from 3.5±2.4% at baseline to 81±2.4%. These data suggest that while STSP and PMA recapitulate certain aspects of megakaryocytic differentiation, divergent signaling pathways may regulate the effects of stromal inhibition. Since stromal inhibition of differentiation plays an important role in the maintenance of hematopoietic progenitors in normal marrow, we investigated the molecular mechanisms involved in STSP induced differentiation. PMA induced megakaryocytic differentiation is dependent on sustained activation of ERK/MAPK, and stromal inhibition is associated with a loss of sustained Rap1 activation. The mechanism of action for STSP induced megakaryocytic differentiation has not been studied. Therefore we sought to investigate the ERK/MAPK signaling pathway during STSP induced differentiation. Using western blot analysis we found the small G protein c-RAF was phosphorylated at Ser338 when K562 cells were treated with either PMA or STSP, suggesting both agents activate c-RAF. In turn both led to a dramatic increase in ERK and p90RSK phosphorylation. We next examined the ERK signaling cascade during stromal inhibition. K562 cells co-cultured on OP9 cells and treated with PMA showed only a slight increase in c-RAF phosphorylation and no significant ERK or p90RSK phosphorylation, consistent with previous stromal inhibition data. However, STSP treatment of K562 co-cultured cells led to the robust phosphorylation of c-RAF and ERK. P90RSK was not significantly phosphorylated. Taken together these data suggest STSP treatment leads to activation of the ERK/MAPK pathway and that this activation may be Rap1 independent. Understanding the mechanisms involved in stromal inhibition and the divergent signaling pathways involved in megakaryocytic differentiation may bring to light novel functions of marrow stroma during normal and leukemic cell expansion.
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