Promotion Effect And Mechanism Of Heparanase On Megakaryocytic Differentiation

Platelets are fragments of cytoplasm that are derived from the megakaryocytes of the bone marrow,which play critical roles in hemostasis and coagulation.The produce of platelets is divided into two process: megakaryopoiesis and thrombopoisis.Megakaryopoiesis is the process by which mature megakaryocytes develop from hematopoietic stem cells(HSCs),and the generation of platelets from megakaryocytes is termed thrombopoiesis.The cytokine thrombopoietin(TPO)is the major regulator of megakaryopoiesis and thrombopoiesis.It binds to its surface receptor,MPL,and acts through multiple downstream signaling pathways,including the PI3K-AKT,MAPK,and ERK1/ERK2 pathways.However,there are non-TPO pathways that influence megakaryopoiesis and thrombopoiesis.Certain molecular signals such as GM-CSF,IL-3,IL-6,IL-11,SDF-1,FGF-4 and EPO all stimulate the maturation of megakaryocytic progenitor cells.Other signals such as PF4,CXCL5,CXCL7 and CCL5 inhibit platelets formation.As the only endo-?-D-glucuronidase that cleaves heparan sulfate(HS)of heparan sulfate proteoglycans(HSPGs)in mammals,heparanase(HPA)facilitates degradation of the extracellular matrix.HPA expression is correlated with the metastatic potential of a variety of tumor-derived cell types owing to its enzymatic activity.Platelets express extremely high level of HPA under physiological conditions,suggesting a distinct expression process and potential function of HPA during megakaryopoiesis and thrombopoiesis.In this study,we investigated the effect of HPA on megakaryocytic differentiation both in vitro and in vivo.At the cellular level,leukemia cell lines(K562,HEL)and umbilical cord blood(UCB)CD34+ cells megakaryocytic differentiation models were used to investigate the expression pattern of HPA.Then,we proved that increasing HPA promotes the maturation of megakaryocytes.Subsequently,we investigated the mechanism of this observation.Finally,we verified the conclusions in HPA overexpressing mice(Hpa-tg).The main results and conclusions are summarized as follows: 1.HPA expression is increasing in the process of megakaryocytic differentiation.We first established megakaryocytic differentiation models with K562,HEL and UCB CD34+ cells.Then,q RT-PCR,Western blotting and immunofluorescence staining assays demonstrated that the expression of HPA dramatically increases along with differentiation,and the amounts of HPA developed from none to presence,from less to more.2.HPA modulates the cell surface HSPGs of megakaryocytes.K562 and HEL cell lines in which HPA overexpressed were used to investigate the enzymatic acticity of HPA in megakaryocytes.Flow cytometry analysis of cell surface HS revealed that HPA modulates megakaryocytes cell surface HSPGs along with differentiation.3.HPA promotes megakaryocytic differentiation.Flow cytometry and q RT-PCR analysis for megakaryocytes surface marker CD41 demonstrated that the overexpression of HPA improves megakaryocytic differentiation of K562 and HEL cells,whereas the absence of HPA led to a delayed development.Ploidy analysis as well as corroborated the positive impact of HPA on megakaryocytes maturation.4.HPA promotes megakaryocytic differentiation via upregulating IL-6 production,followed by enhanced STAT3 pathway.Megakaryocytic differentiation-related heparin binding proteins(HBPs)were screened with ELISA.The results showed that there was an exclusive positive correlation between the production of interleukin 6(IL-6)and the expression of HPA.Then,Western blot assays verified the enhanced IL-6/STAT3 activation induced by HPA,which explaining the positive effect of HPA on megakaryocytic differentiation.In addition,HPA promotes the megakaryocytic differentiation of primary cells as a same mechanism.5.HPA enhances the transcriptional activity of IL-6 via its endoglycosidase activity in megakaryocytes.K562 cells in which enzymatic activity deficiency HPA overexpressed was used to investigate the transcriptional activity and secretion amounts of IL-6.It was found that the expression of IL-6 return to normal levels,indicating the endoglycosidase activity of HPA played a key role on IL-6 upregulation.6.Megakaryocytes and platelets production are increased in mice overexpressing HPA.Based on the results of cellular level,we investigated the effect of HPA on megakaryocytic differentiation in mice overexpressing HPA(Hpa-tg).We first measured the numer of megakaryocytes,the results showed that the bone marrow,spleen and lung in Hpa-tg mice have more megakaryocytes than Ctr mice.Also,hematological analysis revealed a significantly higher number of platelets in Hpa-tg than in Ctr mice.7.HPA promotes TPO production in mice via upregulating IL-6.q RT-PCR assays revealed that overexpressing HPA also promotes the production of IL-6 in mice liver.Subsequent Western blot assays showed that the amounts of TPO in Hpa-tg mice liver is increased,and TPO ELISA in mive plasma confirmed the higher concentration of total TPO in the Hpa-tg liver samples.Conclusions: In this study,we revealed the expression pattern of HPA in megakaryocytic differentiation process.And we proved the positive effect of HPA on megakaryopoiesis and thrombopoiesis both in vivo and in vitro,thereby establishing a HPA/IL-6/STAT3 positive feedback loop that regulating megakaryocytes maturation.Our results implied that HPA holds a special place in the proteome of megakaryocytes and platelets,providing a new target for megakaryocytic regulation and platelets disorders.