The Bone Morphogenetic Protein (BMP)-4 Is Involved in the Regulation of Human Megakaryocytic Differentiation during Thrombopoietin Signaling.

It has been shown in the past that Activin A, BMP-2 and BMP-4, three members of the TGF-β family, are involved in the regulation of hematopoiesis and particularly erythropoiesis, in humans. In this study, we explored the role of these molecules in human megakaryopoiesis using an in vitro serum-free assay initiated with purified normal CD34⁺ human bone marrow (BM) cells (from allogeneic BM donors), that allows the analysis of the impact of such molecules on all stages of megakaryocytic differentiation. We could demonstrate for the first time, that in the absence of thrombopoietin (TPO), BMP-4 is able to induce CD34⁺ progenitor commitment and differentiation into megakaryocytes throughout all stages through means of cytology, flow cytometry, CFU and LTC-IC and ploidy assays, as well as in vitro platelet production. We analyzed as well the expression of megakaryocytic specific factors such as FOG-2, Fli-1 and PF4 by RQ-PCR, and PF4, BMP-4 secretion in culture supernatants. While we have previously shown that Activin A and BMP-2 are involved in the erythropoietic commitment even in the absence of erythropoietin, we were not able to demonstrate any effect of these molecules on megakaryopoietic commitment and differentiation. Using signaling pathways specific inhibitors such as AG490 (JAK-2 pathway inhibitor), PD98059 (ERK pathway inhibitor), LY294002 (PI3-K inhibitor) and Rapamycin (mTOR pathway inhibitor), we could show that BMP-4, as TPO, exerts its effects on human megakaryopoiesis involving specifically the JAK/STAT and mTOR signaling pathways. In addition, the specific inhibition of the BMP signaling pathway with blocking antibodies (CD34⁺ BM cells cultured in the presence of anti-TPO-R and mouse anti-BMP-4 Antibody), natural soluble inhibitors [such as FLRG (Follistatin related gene) protein or Follistatin], or soluble BMP-receptors (sBMPR-Ia, sBMPR-Ib) has revealed that TPO uses the BMP-4 pathway to induce the megakaryopoietic commitment of human BM CD34⁺ progenitors. Finally, we could demonstrate that TPO up-regulates a BMP-4 autocrine loop in megakaryocytic progenitors, by inducing their own production of BMP-4 associated to an up-regulation of BMP-receptor expression. In conclusion, this study illustrates that BMP-4 represents an important actor in the regulation of human megakaryopoiesis.