Regular ArticleThrombospondin-1 inhibits in vitro megakaryocytopoiesis via CD36
Introduction
Thrombospondin-1 (TSP-1) is a multimodular glycoprotein synthesized by megakaryocytes, endothelial cells, smooth muscle cells, fibroblasts and some tumor cells [1], [2]. Found in the platelet α-granules and extracellular matrix (ECM) of numerous tissues, it has a molecular weight of 450 kDa and is involved in the cell–cell, cell–growth factor and cell–ECM interactions [1]. Haematopoiesis is regulated by a complex set of events in which cells of the haematopoietic microenviroment interact with each other, with haematopoietic growth factors and with the surrounding ECM, where cytoadhesion molecules play a role [3]. As a multifunctional ECM protein, TSP-1 functions as a cytoadhesion protein for both haematopoietic pluripotent and committed progenitors, mediating the cell–cell and cell–matrix interactions [3]. It has been shown previously that ECM-bound TSP binds to haematopoietic progenitor cells and maturing megakaryocytes, and TSP may also be involved in the regulation of megakaryocytopoiesis as a negative modulator and this effect is at least partially mediated by the N-terminal heparin-binding domain [4].
CD36, known initially as glycoprotein IV, was first described in 1976 as an 88-kDa glycoprotein on the platelet membrane [5]. The cDNA was later sequenced from human platelets [6]. CD36 as an integral plasma membrane protein, is expressed on a variety of cell types including platelets, endothelial cells, monocytes, erythrocytes and various tumor cell lines [7], [8]. Since it can form multi-macromolecular complexes with TSP-1, it has been classified as a TSP-1 receptor [9], [10]. In addition, CD36 is known to be associated with collagen adhesion, malarial cytoadherence and signal transduction [7], [11]. Recently, it has been demonstrated that endothelial CD36, previously thought to be involved only in adhesion and scavenging activities, actually mediates the apoptosis-dependent inhibition of neovascularization by TSP-1 [12]. These findings have prompted us to investigate the role of CD36 in TSP-1-induced inhibition in vitro megakaryocytopoiesis.
Section snippets
Reagents
Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was a gift from AMGEM, USA. Recombinant human interleukin (IL)-3, IL-6 and GM-CSF (granulocyte monocyte-colony stimulating factor) were purchased from Genzyme (Boston, MA). Mouse anti-human CD36 antibody (FA6-152) was purchased from Immunotech (Miami, USA) and MOPC 21, the negative control mouse IgG1 antibody, from Sigma (MO, USA). Anti-transforming growth factor-beta (TGF-β) antibody was purchased from R&D
Comparison of TSP-1-induced suppression on murine CFU-MK and CFU-GM formation
In six separate experiments, murine bone marrow cells were cultured in the plasma clot system with increasing concentrations of TSP-1 (0, 1, 2.5, 5, 10 and 25 μg/ml) and the colony counts for CFU-MK and CFU-GM were scored. The results showed that TSP-1 significantly inhibited megakaryocyte colony formation in a dose-dependent manner (Fig. 1) and at a minimum dose of 2.5 μg/ml (p<0.05). By contrast, only a slight or moderate decrease in CFU-GM numbers was shown in the same dose range and only at
Discussion
As a widely expressed multimodular extracellular matrix (ECM) protein, TSP-1 participates in diverse biological processes through interaction with a specific receptor. Its involvement has extended far beyond the initial observations of their roles in platelet aggregation and coagulation [1]. The regulatory role of TSP-1 on endothelial cell adhesion, migration and proliferation has been intensely studied. It has been shown that human platelet TSP-1 specifically inhibits both the proliferation of
Acknowledgments
The authors thank Mrs. Leonie Gaudry for technical assistance with flow cytometry and Dr. B.N. Jimenez for her help in protein purification. The study was supported in part by Program support to the centre from the NH&MRC of Australia and the RGC grant of Hong Kong government.
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