Identification of PDGF receptors on human megakaryocytes and megakaryocytic cell lines.
ABSTRACT Platelet-derived growth factor (PDGF) is a potent chemotactic and mitogenic factor implicated to play important roles in a variety of normal and pathophysiologic settings. We investigated PDGF receptor expression on human megakaryocytes and several megakaryocytic cell lines (CHRF, DAMI, Meg-01, M-07e) using enzyme-linked immunosorbent assay (ELISA), flow cytometry and immunocytochemical staining. Both PDGF receptor subtypes were identified on CHRF, DAMI, and Meg-01 cells by ELISA; PDGF beta-receptor levels exceeded alpha-receptor levels. Flow cytometry revealed that beta-receptor levels on CHRF and DAMI cells exceeded those on Meg-01 cells, and that M-07e expressed neither receptor. Immunocytochemical staining confirmed these findings and determined that bone marrow megakaryocytes also expressed PDGF receptors. Exposure of megakaryocytes to PDGF-BB dramatically induced the expression of the immediate-early gene, c-fos, within 30 min. Moreover, PDGF-BB significantly stimulated CHRF proliferation and colony formation. The present study demonstrates the presence of functional PDGF receptors on human megakaryocytes and their ability to mediate a mitogenic response.
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ABSTRACT: 5-Hydroxtryptamine (5-HT, serotonin) has been recognized not only as a neurotransmitter and vasoactive agent, but also as a growth factor. 5-HT mainly binds to 5-HT2 receptors or 5-HT1 receptors on cell surfaces to stimulate cell proliferation through Ras or MAPK (mitogen-activated protein kinase) pathways in many cell types. It has been reported that 5-HT stimulates megakaryocytopoiesis via 5-HT receptors (5-HTR). The possible mechanism by which 5-HT regulates the proliferation and differentiation of megakaryocytes (MK) is discussed in this review article. In early stages of megakaryocytopoiesis, 5-HT may bind to 5-HT2B receptors on MK to promote their proliferation and differentiation. In the late stages, 5-HT may be involved in platelet release by inducing nitric oxide (NO) synthesis via 5-HT2A receptors. 5-HT can also antagonize the apoptotic effect induced by thrombospondin-1 (TSP-1) which is a platelet alpha-granule protein and has synergic effects with platelet-derived growth factor (PDGF) to enhance MK proliferation. Therefore, 5-HT is likely to be an important substance in the feedback regulation of thrombopoiesis.Hematology 03/2006; 11(1):53-6. · 1.39 Impact Factor
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ABSTRACT: Platelet-derived growth factor is involved in the regulation of hematopoiesis. Imatinib mesylate, a platelet-derived growth factor receptor inhibitor, induces thrombocytopenia in a significant proportion of patients with chronic myeloid leukemia. Although our previous studies showed that platelet-derived growth factor enhances megakaryocytopoiesis in vitro, the in vivo effect of platelet-derived growth factor in a model of radiation-induced thrombocytopenia has not been reported. In this study, we investigated the effect of platelet-derived growth factor on hematopoietic stem/progenitor cells and platelet production using an irradiated-mouse model. We also explored the potential molecular mechanisms of platelet-derived growth factor on thrombopoiesis in M-07e cells. Platelet-derived growth factor, like thrombopoietin, significantly promoted the recovery of platelets and the formation of bone marrow colony-forming unit-megakaryocyte in irradiated mice. Histology confirmed the protective effect of platelet-derived growth factor, as shown by an increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis. In a megakaryocytic apoptotic model, platelet-derived growth factor had a similar anti-apoptotic effect as thrombopoietin on megakaryocytes. We also demonstrated that platelet-derived growth factor activated the PI3-k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings show that platelet-derived growth factor enhances platelet recovery in mice with radiation-induced thrombocytopenia. This radioprotective effect is likely to be mediated via platelet-derived growth factor receptors with subsequent activation of the PI3-k/Akt pathway. We also provide a possible explanation that blockage of platelet-derived growth factor receptors may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia.Haematologica 10/2010; 95(10):1745-53. · 5.94 Impact Factor
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ABSTRACT: We previously showed that the Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its receptor VPAC1 are negative regulators of megakaryopoiesis and platelet function, but their downstream signaling pathway that inhibits this process still remained unknown. A combined proteomic, transcriptomic, and bioinformatic approach was here used to elucidate the molecular mechanisms underlying PACAP signaling via VPAC1 in megakaryocytes. Two-dimensional difference gel electrophoresis and tandem MS were applied to detect differentially expressed proteins in megakaryocytic CHRF cells stimulated with PACAP. The majority of the 120 proteins modulated by PACAP belong to the class of "cell cycle and apoptosis" proteins. The up- or down-regulated expression of some proteins was confirmed by immunoblot and immunohistochemical analysis. A meta-analysis of our data and 12 other published studies was performed to evaluate signaling pathways involved in different cellular models of PACAP response. From 2384 differentially expressed genes/proteins, 83 were modulated by PACAP in at least three independent studies and Ingenuity Pathway Analysis further identified apoptosis as the highest scored network with NF-κB as a key-player. PACAP inhibited serum depletion-induced apoptosis of CHRF cells via VPAC1 stimulation. In addition, PACAP switched on NF-κB dependent gene expression since higher nuclear levels of the active NF-κB p50/p65 heterodimer were found in CHRF cells treated with PACAP. Finally, a quantitative real time PCR apoptosis array was used to study RNA from in vitro differentiated megakaryocytes from a PACAP overexpressing patient, leading to the identification of 15 apoptotic genes with a 4-fold change in expression and Ingenuity Pathway Analysis again revealed NF-κB as the central player. In conclusion, our findings suggest that PACAP interferes with the regulation of apoptosis in megakaryocytes, probably via stimulation of the NF-κB pathway.Molecular & Cellular Proteomics 01/2012; 11(1):M111.007625. · 7.25 Impact Factor