OC 59.5 - Glucocorticoids Stimulate Thrombopoiesis in Murine Megakaryocytes

Background: Cushing's syndrome is characterized by elevated levels of endogenous glucocorticoids. Patients commonly present high platelet counts and increased thromboembolism, but the underlying mechanism remains unclear. Healthy men treated with dexamethasone evidenced a significant rise in the number of platelets. Platelet levels follow a circadian pattern like that of circulating glucocorticoids.

Aims: Determine a direct effect of glucocorticoids on platelet production by megakaryocytes.

Methods: Megakaryocytes were differentiated from C57BL/6 bone marrow and fetal livers. Expression and functionality of glucocorticoid receptors were addressed by immunofluorescence, western blot, and RT-qPCR. Megakaryocyte morphology, spreading and proplatelet formation, as well as in vitro platelet production, were analyzed by immunofluorescence, microscopic assays, and flow cytometry. In vivo effect was assessed by injecting dexamethasone to wild type C57BL/6 mice. Collected blood was examined using a hematology analyzer and a flow cytometer. Tail bleeding time was performed to explore any hemostatic outcome.

Results: Glucocorticoid receptor mRNA and protein were present in megakaryocytes. Glucocorticoid treatment induced nuclear translocation of the receptor and expression of Gilz and Fkbp5, two well-known glucocorticoid-responsive genes. A whole-genome expression microarray revealed 1325 genes differentially transcribed by dexamethasone (70% activated and 30% repressed). These genes were related to numerous cellular functions, particularly to the activation of cytoplasm and cytoskeleton organization and microtubule dynamics. Megakaryocytes incubated with dexamethasone on fibronectin displayed increased spreading and shape change associated, with augmented podosome formation and microtubule polymerization. Glucocorticoids increased the percentage of proplatelet-forming megakaryocytes and of platelet-like particles. Single dose of dexamethasone resulted in higher mature and young platelet counts and induced a more rapid hemostatic response.

Conclusion(s): We demonstrated that megakaryocytes express functional glucocorticoid receptors conferring glucocorticoids the ability to modulate megakaryocyte transcriptome and thus its functions. Furthermore, we present evidence that glucocorticoids stimulate thrombopoiesis in vitro and in vivo, providing a novel mechanism of action of glucocorticoids in health and disease.