The “feedback”mechanism of megakaryocytopoiesis via platelet molecules: a hypothesis Mo YANG PhD Department of Paediatrics, The Chinese University of Hong Kong. Contents Background and hypothesis: 		Platelet molecules may regulate 			megakaryocytopoiesis via direct effect on MK 	progenitors Part 1. PDGF and megakaryocytopoiesis 		I. PDGF on in vitro megakaryocytopoiesis 		II. PDGF receptors and signal pathway in 			MK cells 		III. To apply PDGF on ex vivo expansion of MK 		and in vivo megakaryocytopoiesis Part 2. 5-HT and megakaryocytopoiesis Part 3. TSP1 and megakaryocytopoiesis General conclusions Background The role of cytokines in megakaryocytopoiesis. Kaushansky. N Eng J Med 1998, 10; 746-754. Background Expression of IL-1 receptors on MK lineage. Yang & Li et al, Br J Haematol 2000,111:371-380 Background IL-1b enhances megakaryocytopoiesis and NF-E2 expression. Yang & Li et al, Br J Haematol 2000,111:371-380 Background TPO is a major growth factor for MK and platelets The existence of a humoral regulator for MK and platelet production, termed thrombopoietin (TPO) 		Kelemen et al. Acta Haematol 1958, 20;350) TPO was isolated and cloned. 		Bartley et al. Cell 1994, 77; 1117-1124. 		de Sauvage et al. Nature 1994, 369;533-538. 		Lok et al. Nature 1994, 369; 565-568. 		Kuter et al. PNAS 1994,91;11104-11108. 		Kato et al. J Biochem 1995, 118; 229-236. Background The structure of TPO gene. Background Cytokines in megakaryocytopoiesis Major Growth Factor: TPO Others: IL-1b, IL-3, IL-6, IL-11 SCF, FL G-CSF, EPO Background Transcriptional Regulation of Megakaryocytopoiesis: NF-E2 GATA-1 FOG-1 CBFA2 Shivdasani. Stem Cells 2001, 19: 397-407. Background TPO regulation by platelets. Kaushansky. N Eng J Med 1998, 10; 746-754. Hypothesis Alternatively, platelet molecules may regulate megakaryocytopoiesis by a direct effect on MK progenitors.				Yang et al. Blood 1999, 94; 41a. Objective Effect of PDGF, 5-HT and TSP1 on megakaryocytopoiesis? Possible mechanism? Part 1. PDGF and Megakaryocytopoiesis Introduction Platelet-derived growth factor (PDGF) is a peptide growth factor which was first isolated from human platelets. Rose et al, PNAS 1974.71;1207-10. Introduction Partially purified PDGF was inhibitory to CFU-MK growth? Komatsu et al, Br J Haematol 1986, 64: 241-252.  Part 1.1.To investigate the effect of PDGF on in vitro megakaryocytopoiesis Results 1. PDGF has a stimulating effect on megakaryocytopoiesis via stromal cells to produce IL-6 and GM-CSF. 		Yang et al Br J Haematol 1995, 91; 285-289. Results 2. PDGF stimulates TPO mRNA expression in human BM stromal cells. Sungaran (BH Chong) et al, Blood 2000, 95; 3094-3101. Results 3. PDGF promotes human CD34+ cells differentiation into stromal cells. Yang & Li et al Int J Hematol 2001, 73; 327-334. Part 1.1. Summary PDGF enhances megakaryocytopoiesis via BM stromal cells to produce TPO etc. 		 				 				Yang et al Br J Haematol 1995, 91; 285-289. 				 Yang et al Int J Hematol 2001, 73; 327-334. 				Sungaran (BH Chong) et al, Blood 2000, 95; 				3094-3101  Part 1.2.To identify PDGF receptors and signaling pathway on MK lineage Results 1. Identification of PDGF receptors on MK cells. 				 Results 2. PDGF receptors mediate the expression of immediate-early gene, c-fos, and CHRF cells proliferation. Results 3. PDGF also enhances the expression of transcription factor p45NF-E2 and GATA-1 on MK cells. 				 Chui, Li, Yang et al. Blood 2000, 96; 679a Part 1.2 Summary PDGF receptors were identified on human megakaryocytes and mediate mitogenic response via c-fos, NF-E2 and GATA-1. Yang et al, Thromb Haemostasis 1997, 78; 892-896. Chui, Li, Yang et al. Blood 2000, 96; 679a Part 3.3. To apply PDGF on ex vivo expansion of MK and in vivo megakaryocytopoiesis Results 1. PDGF enhances ex vivo expansion of MK progenitors and CD34+ cells from human cord blood. Su, Li, Yang et al. BMT 2001, 27; 1075-1080. Su, Li, Yang et al. Expt Hematol 2000, 28; 1500-1501. Results 2. PDGF and purified angelica polysaccharide (APS) also stimulate in vivo megakaryocytopoiesis. Part 1.3. Summary PDGF Part 2.5- HT and megakaryocytopoiesis Introduction 5- HT is a monoamine (176 dalton) and is stored in platelet dense granules. Effect of serotonin on haematopoiesis??? Results 1. Expression of 5-HT 2A, 2B and 2C receptors on MK cell lines by RT-PCR. Results 2. Identification of 5-HT 2A, 2B and 2C receptors on MK cell lines : Meg-01, Dami, CHRF and M-07e cells by western blot and FASC. Results 3. Identification of 5-HT 2A, 2B and 2C receptors on human bone marrow megakaryocytes by immunofluorescence staining. Results 4. 5- HT has mitogenic effect on megakaryocytopoiesis via 5-HT2B receptors. 			 	 Yang et al. Blood Coagul Fibrin 1996; 6:127-133. Results 5. 5- HT uptake ability is well established in megakaryoblasts via transporters. 					Yang et al. Int J Hematol 1996, 63: 137-142. Results 6. 5- HT can reduce TSP1-induced apoptosis on M-07e cells by annexin V assay. 	 	This effect may be mediated by caspase-3 signaling. Part 3.TSP1 and megakaryocytopoiesis Introduction Thrombospondin-1 (TSP1) is a major platelet alpha-granule protein. Our hypothesis is that: TSP-1 may affect megakaryocytopoiesis by binding to MK cells via CD36, thus inducing cell apoptosis. Results 1. Expression of CD36 (TSP receptor) on MK cells. Results 2. TSP1 and PF4 inhibited CFU-MK formation and MK cell growth via CD36. 	 Results 3. TSP-1 induced apoptosis in MK cells via CD36 and caspase-3 signaling pathway. 	 Results 4. TSP-1 did not show significant effect on the expression of bcl-2 and bax in MK cells. Part 3. Summary TSP-1 induces apoptosis in MK cells via CD36 and caspase-3 signaling pathway. 				Yang & Li et al. Thromb Haemostasis 2001,suppl, p316. 				 Yang et al. Blood 1999, 94; 41a. General Conclusions General Conclusions The studies provide evidences supporting the hypothesis: 		Negative regulator: 	TSP-1, PF4 		Positive regulator:		PDGF, 5-HT General Conclusions A New Link Between Nervous System and Hematopoiesis YANG, Mo ( 楊默) PhD Department of Paediatrics, 	The Chinese University of Hong Kong 	Alternatively, platelet molecules may regulate megakaryocytopoiesis by a feedback mechanism via direct effect on MK progenitors. 				Yang et al. Blood 1999, 94; 41a. Part 1:To investigate 5-HT effect and mechanism on MK cells 	5- HT uptake in Meg-01 cells via transporter. 				Yang et al. Int J Hematol 1996;63:137-142. 5- HT on CFU-MK formation via 5-HT2R 					Yang et al. Blood Coagul Fibrin 1996;6:127-133. 	5-HT 2R on MK cells by RT-PCR. 5-HT 2R on MK cell lines by western blot and FASC. 	5-HT 2R on human BM MK by immunofluorescence staining. Expression of 5-HT2A, 2B and 2C receptors on MK cells. 5- HT has growth effect on MK cells via 5-HT2R Ketanserin, 5-HT2BR antagonist, block the mitogenic effect on MK. Proposed transmembrane domain arrangements of BR-based models of human (a) and mouse (b) 5-HT2B receptors liganded with 5-HT.  	5- HT can reduce TSP1-induced apoptosis on M-07e cells by annexin V assay. Ablation of 5-HT2BR in mice leads to dilated cardiomyopathy 5-HT cyto-protective signaling pathways via 5-HT2BR and mitochondria. Percentage of B and C COMET produced by treatment with 10 μmol/L GSNO (NO) in the presence or absence of 100 ng/mL TPO (T), 100 μmol/L IL-11, or 100 μmol/L serotonin (S) pretreatment. Battinelli et al, Blood, 2000. 5- HT effect on CD34 cell expansion 5- HT effect on BM stromal cells Part 2:To investigate dopamine effect and mechanism on MK cells Dopamine D1 and D2 receptors on MK cells by RT-PCR Dopamine D1 and D2 R on Mk cells by western blot and FACS. DA (low dose) induces MK differentiation DA (high dose) induces MK apoptosis Dopamine induces differentiation and apoptosis in MK cells via its receptors. Hypothesis 	A new link between the nervous system and hematopoiesis: 5-HT acts as a m itogenic factor, dopamine as m aturation factor . Expression of neuronal marker MAP2, GFAP and Tau on MK cells. MK cells differentiate into neuron?TPO has neural effect? TPO in CNS TPO receptor c-mpl in CNS TPO has neural regeneration effect in a rat model Acknowledgements Dept. of Paediatrics, CUHK, HK Prof Karen Li