Identification of PDGF receptors on human megakaryocytes and megakaryocytic cell lines
The Centre for Thrombosis and Vascular Research, School of Pathology, The University of New South Wales and Department of Haematology, The Prince of Wales Hospital, Sydney, Australia. Thrombosis and Haemostasis
(Impact Factor: 4.98).
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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- "K562 cells were cultured in IMDM with 10% FCS as control cells. All cells were maintained with penicillin G (100 U/ml) and streptomycin (50 mg/ml) at 378C in 5% CO 2 and air in a humidified incubator (Yang et al, 1997). Primary megakaryocytic cells. "
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ABSTRACT: Megakaryocytopoiesis is regulated by thrombopoietin (TPO) and cytokines such as interleukin 3 (IL-3), IL-6 and IL-11. This study investigated the in vitro effects of IL-1β on megakaryocytopoiesis and the expression of IL-1 type I and type II receptors (IL-1 RI and RII) on megakaryocytic cell lines and primary cells. Our results demonstrated that IL-1β alone or in combination with TPO induced megakaryocyte colony forming units (CFU-MK) from murine and human haematopoietic cells. Using reverse-transcription polymerase chain reaction (RT-PCR) and Southern hybridization techniques, the mRNA of IL-1β, IL-1 RI, IL-1 RII and the transcription factor NF-E2 were detected in CD61+CD41+ cells cultured from cord blood and four megakaryocytic cell lines, Meg-01, DAMI, CHRF-288–11 and M-07e. The expression of IL-1 RI and RII proteins was confirmed by flow cytometry and immunofluorescence staining. In Meg-01 cells, the expression of NF-E2 was increased at both mRNA and protein levels after treatment with IL-1β for 4 h. This study demonstrated for the first time the presence of IL-1 receptors on megakaryocytic cells and the induction of NF-E2 by IL-1β. The mitogenic effect of IL-1β on this lineage could be mediated through IL-1 receptors and the activation of NF-E2.
Available from: Xiao-Bing Zhang
- "This family also encompasses macrophage colony-stimulating factor (M-CSF) receptor, stem cell factor (SCF) receptor and flk-2/flt3 (Lu et al, 2000). PDGF receptors are expressed on fibroblasts (Heldin et al, 1988), smooth muscle cells (Jin et al, 1990), T cells (Daynes et al, 1991), early B-lineage precursor cells (Tsai et al, 1994), myeloid cells (Alimandi et al, 1997), macrophages (Yan et al, 1993), platelets and megakaryocytes (Vassbotn et al, 1994; Yang et al, 1997). PDGF and its receptors play important roles in embryogenesis, as knock-out mice develop severe defects in major organs, including the kidney, lung, heart and central nervous system (Betsholtz & Raines, 1997; Heldin & Westermark, 1999; Kaminski et al, 2001). "
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ABSTRACT: Platelet-derived growth factor (PDGF) is a major mitogen for connective tissue cells. In this study, we investigated the effects and mechanism of PDGF on the ex vivo expansion of cord blood CD34+ cells. Our data demonstrated that among various cytokine combinations of thrombopoietin (TPO), interleukin 1 beta (IL-1beta), IL-3, IL-6 and Flt-3 ligand (Flt-3L), TPO + IL-6 + Flt-3L was most efficient in promoting the expansion of CD34+ cells, CD34+CD38- cells, mixed-lineage colony-forming units (CFU-GEMM) and long-term culture-initiating cells (LTC-IC) by 21.7 +/- 5.00-, 103 +/- 27.9-, 10.7 +/- 7.94- and 6.52 +/- 1.51-fold, respectively, after 12-14 d of culture. The addition of PDGF increased the yield of these early progenitors by 45.0%, 66.5%, 45.1% and 79.8% respectively. More significantly, PDGF enhanced the engraftment of human CD45+ cells and their myeloid subsets (CD33+, CD14+ cells) in non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) mice. The expression of PDGF receptor (PDGFR)-beta was not detectable in fresh CD34+ cells but was upregulated after culture for 3 d. PDGF also enhanced the development of adherent cells/clusters that expressed the endothelial markers VE-cadherin and CD31. These findings suggest that PDGF is an effective cytokine for the ex vivo expansion of early stem and progenitor cells. The mechanism could be mediated by PDGFR-beta on committed CD34+ progenitor cells and/or secondary to the stimulation of autologous, stromal feeder cells.
Available from: John Tennison Reilly
- "Its' ligand, PDGF AB or BB, is a potent stimulant of mesenchymal cell proliferation and migration, and plays a fundamental role in wound healing (Heldin & Westermark, 1999; Li et al, 2000). PDGFRb, however, has also been identified on a variety of haematopoietic cells, including B and T lymphocytes (Goustin et al, 1990; Tsai et al, 1994), NK cells (Gersuk et al, 1991), cultured monocytes (Inaba et al, 1993), HL60 myelomonocytic cells (Pantazis et al, 1990), platelets and megakaryocytes (Yang et al, 1997). Despite these observations, it is unclear whether PDGF directly affects haematopoietic function or indirectly stimulates haematopoietic precursors through the release of growth and differentiation factors from stromal cells (Kaminski et al, 2001). "
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