Role of a serine/threonine kinase, Mst1, in megakaryocyte differentiation

Platelets, which play a central role in thrombosis and hemostasis, develop from megakaryocytes. Signal transduction originated from the megakaryocyte growth and development factor, the Mpl ligand, which leads to megakaryocyte differentiation, polyploidization, and maturation, has been gradually characterized. In this study, we report the inducibility of Mst1, a recently described serine/threonine kinase, by Mpl ligand and the effect of its induced expression on megakaryocyte differentiation. The steady-state level of mst1 message and Mst1-associated kinase activity increased in response to Mpl ligand. Ectopic expression of human mst1 in a mouse megakaryocytic cell line resulted in a drastic increase in DNA content per cell. Elevated expression of megakaryocyte differentiation markers, such as acetylcholine esterase, PF4, and GPIIb was also observed in hmst1-expressing cells. Activation of p38 MAPK, a known downstream effector of Mst1, was shown to be required for polyploidization, but not for enhanced expression of differentiation markers. Our study thus designates Mst1 as a Mpl ligand-responsive signaling molecule that promotes induction of lineage-specific cellular programming.     

Role of a serine/threonine kinase,Mst1, in megakaryocyte differentiation

Platelets, which play a central role in thrombosis and hemostasis, develop from megakaryocytes. Signal transduction originated from the megakaryocyte growth and development factor, the Mpl ligand, which leads to megakaryocyte differentiation, polyploidization, and maturation, has been gradually characterized. In this study, we report the inducibility of Mst1, a recently described serine/threonine kinase, by Mpl ligand and the effect of its induced expression on megakaryocyte differentiation. The steady‐state level of mst1 message and Mst1‐associated kinase activity increased in response to Mpl ligand. Ectopic expression of human mst1 in a mouse megakaryocytic cell line resulted in a drastic increase in DNA content per cell. Elevated expression of megakaryocyte differentiation markers, such as acetylcholine esterase, PF4, and GPIIb was also observed in hmst1‐expressing cells. Activation of p38 MAPK, a known downstream effector of Mst1, was shown to be required for polyploidization, but not for enhanced expression of differentiation markers. Our study thus designates Mst1 as a Mpl ligand‐responsive signaling molecule that promotes induction of lineage‐specific cellular programming. J. Cell. Biochem. 76:44–60, 1999. © 1999 Wiley‐Liss, Inc.     

A network map of thrombopoietin signaling

Thrombopoietin (THPO), also known as megakaryocyte growth and development factor (MGDF), is a cytokine involved in the production of platelets. THPO is a glycoprotein produced by liver and kidney. It regulates the production of platelets by stimulating the differentiation and maturation of megakaryocyte progenitors. It acts as a ligand for MPL receptor, a member of the hematopoietic cytokine receptor superfamily and is essential for megakaryocyte maturation. THPO binding induces homodimerization of the receptor which results in activation of JAKSTAT and MAPK signaling cascades that subsequently control cellular proliferation, differentiation and other signaling events. Despite the importance of THPO signaling in various diseases and biological processes, a detailed signaling network of THPO is not available in any publicly available database. Therefore, in this study, we present a resource of signaling events induced by THPO that was manually curated from published literature on THPO. Our manual curation of thrombopoietin pathway resulted in identification of 48 molecular associations, 66 catalytic reactions, 100 gene regulation events, 19 protein translocation events and 43 activation/inhibition reactions that occur upon activation of thrombopoietin receptor by THPO. THPO signaling pathway is made available on NetPath, a freely available human signaling pathway resource developed previously by our group. We believe this resource will provide a platform for scientific community to accelerate further research in this area on potential therapeutic interventions.     

Amphiregulin regulates the production of human chorionic gonadotropin in trophoblasts

AimsThe aim of this study was to investigate the significance of epidermal growth factor receptor (EGFR) ligands produced in syncytiotrophoblasts during normal pregnancy.Main methodsWe examined the expression of EGFR ligands in human pregnancy by real-time PCR, and analyzed the relationship between EGFR ligands and human chorionic gonadotropin (hCG) or human placental lactogen in amniotic fluid by ELISA. In addition, we also examined the EGFR ligands in syncytiotrophoblasts and the amount of hCG secretion in JAR, JEG3 and BeWo cells in the presence of each EGFR ligand.Key findingsIn order to identify possible candidates among the EGFR ligands, we examined the predominant expression of an EGFR ligand in the chorionic villi and amniotic fluid during normal pregnancy, and analyzed the relationship between EGFR ligands and hCG in trophoblastic model cells. Amphiregulin was primarily expressed throughout human pregnancy and stimulated the secretion of hCG, indicating that amphiregulin is a key molecule among EGFR ligands.SignificanceAmphiregulin may play a pivotal role in the development or maturation of placenta.     

Heat shock protein 27 expression in the human testis showing normal and abnormal spermatogenesis

Heat shock proteins (HSPs) are molecular chaperones involved in protein folding, assembly and transport, and which play critical roles in the regulation of cell growth, survival and differentiation. We set out to test the hypothesis that HSP27 protein is expressed in the human testes and its expression varies with the state of spermatogenesis. HSP27 expression was examined in 30 human testicular biopsy specimens (normal spermatogenesis, maturation arrest and Sertoli cell only syndrome, 10 cases each) using immunofluorescent methods. The biopsies were obtained from patients undergoing investigations for infertility. The seminiferous epithelium of the human testes showing normal spermatogenesis had a cell type-specific expression of HSP27. HSP27 expression was strong in the cytoplasm of the Sertoli cells, spermatogonia, and Leydig cells. Alternatively, the expression was moderate in the spermatocytes, weak in the spermatids and absent in the spermatozoa. In testes showing maturation arrest, HSP27 expression was strong in the Sertoli cells, weak in the spermatogonia, and spermatocytes. It was absent in the spermatids and Leydig cells. In Sertoli cell only syndrome, HSP27 expression was strong in the Sertoli cells and absent in the Leydig cells. We report for the first time the expression patterns of HSP27 in the human testes and show differential expression during normal spermatogenesis, indicating a possible role in this process. The altered expression of this protein in testes showing abnormal spermatogenesis may be related to the pathogenesis of male infertility.     

Microarray and proteomic analysis of brassinosteroid- and gibberellin-regulated gene and protein expression in rice

Brassinosteroid (BR) and gibberellin (GA) are two groups of plant growth regulators essential for normal plant growth and development. To gain insight into the molecular mechanism by which BR and GA regulate the growth and development of plants, especially the monocot plant rice, it is necessary to identify and analyze more genes and proteins that are regulated by them. With the availability of draft sequences of two major types, japonica and indica rice, it has become possible to analyze expression changes of genes and proteins at genome scale. In this review, we summarize rice functional genomic research by using microarray and proteomic approaches and our recent research results focusing on the comparison of cDNA microarray and proteomic analyses of BR- and GA-regulated gene and protein expression in rice. We believe our findings have important implications for understanding the mechanism by which BR and GA regulate the growth and development of rice.     

Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet-released glycoprotein

The suppressive role of platelets on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU-M growth, since marrow cell erythroid burst formation (BFU-E) and granulocytic-monocytic colony formation (CFU-GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP-stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl-m1. Partial purification of this activity was achieved by diethylaminoethyl (DEAE)-ion exchange and phytohemagglutinin (PHA)-E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12-17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU-M in vitro but acts on a day 6-8 CFU-M-derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU-M into megakaryocytes.     

Interference RNA (RNAi)-based silencing of endogenous thrombopoietin receptor (Mpl) in Dami cells resulted in decreased hNUDC-mediated megakaryocyte proliferation and differentiation

Recently our laboratory reported evidence showing that hNUDC acts as an additional cytokine for thrombopoietin receptor (Mpl). Previously known as the human homolog of a fungal nuclear migration protein, hNUDC plays a critical role in megakaryocyte differentiation and maturation. Here we sought to further clarify the hNUDC-Mpl ligand-receptor relationship by utilizing interference RNA (RNAi) to knockdown Mpl expression in a megakaryocyte cell line. We created U6 promoter driven constructs to express short hairpin RNAs (shRNA) with affinity for different sites on Mpl mRNA. By including Mpl-EGFP fusion protein in these constructs, we were able to effectively screen the shRNA that was most efficient in inhibiting Mpl mRNA expression. This shRNA was subsequently transferred into a lentivirus vector and transduced into Dami cells, a cell line which constitutively expresses endogenous Mpl. This lentiviral vector was also designed to simultaneously express EGFP to monitor transfection efficiency. Our results show that lentivirus can be used to effectively deliver shRNAs into Dami cells and cause specific inhibition of Mpl protein expression after transduction. Furthermore, we show the functional effects of shRNA-mediated Mpl silencing by demonstrating reduced hNUDC stimulated megakaryocyte proliferation and differentiation. Thus, the use of a RNAi knockdown strategy has allowed us to pinpoint the connection of hNUDC with Mpl in the regulation of megakaryocyte maturation.     

Recombinant gibbon interleukin-3 stimulates megakaryocyte colony growth in vitro from human peripheral blood progenitor cells

Gibbon interleukin-3 (rIL-3) has recently been cloned and found to have a high degree of homology with the human IL-3 molecule. In this investigation, we evaluated the effects of gibbon rIL-3 on normal human peripheral blood megakaryocyte progenitor cell growth in vitro. Gibbon rIL-3 exhibited substantial megakaryocyte colony stimulatory activity (Meg-CSA), supporting peak colony numbers at a concentration of 1 U/ml. Megakaryocyte colony growth induced by rIL-3 reached 58% of the maximum achieved with the active, Meg-CSA-containing protein fraction of aplastic canine serum. Increasing gibbon rIL-3 concentrations also stimulated a 4-5-fold increase in megakaryocyte colony size and resulted in a decrease in geometric mean megakaryocyte ploidy. Ploidy values fell from 8.5N +/- 1.4 (+/- SEM) at an rIL-3 concentration of 0.1 U/ml to a minimum of 2.9N +/- 0.3 at 10 U/ml. In the presence of rIL-3 at 1.0 U/ml, megakaryocyte colony growth was linear with cell plating density and the regression line passed approximately through the origin. The effects of rIL-3 on megakaryocyte colony growth were independent of the presence of T-lymphocytes in the cultures. Cross-species evaluation of murine and gibbon IL-3 indicated that its bioactivity is species restricted. Murine IL-3 did not support colony growth from human megakaryocyte progenitors and gibbon rIL-3 showed no activity in stimulating acetylcholinesterase production by murine bone marrow cells. Gibbon rIL-3 is a potent stimulator of the early events of human megakaryocyte progenitor cell development promoting predominantly mitosis and early megakaryocytic differentiation.     

Thrombopoietic-mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2

CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.     

Megakaryocyte development is normal in mice with targeted disruption of Tescalcin

BackgroundTescalcin is an EF-hand calcium-binding protein that interacts with the Na+/H + exchanger 1 (NHE1). Levay and Slepak recently proposed a role for tescalcin in megakaryopoiesis that was independent of NHE1 activity. Their studies using K562 and HEL cell lines, and human CD34 + hematopoietic stem cells suggested an essential role for tescalcin in megakaryocyte differentiation.ObjectiveTo study the role of tescalcin in megakaryocyte development using a murine model of megakaryopoiesis.MethodsWe generated a mouse with targeted disruption of tescalcin and investigated megakaryocyte development.ResultsTescalcin-deficient mice had a normal number of megakaryocytes and platelets. The morphology, polyploidization profile, and expression of Fli-1 in bone marrow-derived megakaryocytes were also normal.ConclusionTescalcin does not appear to be necessary for normal megakaryocyte development.     

人类胚胎期和成年期巨核细胞的分子特征比较

为探究人类不同发育时期巨核细胞的分子特征,基于人类胚胎期卵黄囊、胎肝和成年骨髓巨核细胞的单细胞转录组测序数据,从分子特征、基因调控网络等方面分别对其分子差异进行生物信息学分析。结果表明,胚胎期巨核细胞具有较强的增殖特征,高表达细胞增殖相关的转录因子;而成年期巨核细胞具有较强的血小板生成特征,高表达与巨核细胞分化成熟相关的转录因子。研究结果为研究不同发育阶段巨核细胞及其子代血小板的功能差异提供了理论依据。     

Functional characterization of hNUDC as a novel accumulator that specifically acts on in vitro megakaryocytopoiesis and in vivo platelet production

Human NUDC (hNUDC) has been previously described as a human homolog of a fungal nuclear migration protein. It is a multifunctional interactive protein that forms an association with the microtubule motor complex in a variety of cells. Our recent studies demonstrated that hNUDC could bind specifically to the thrombopoietin receptor (Mpl) and suggest a potential role for hNUDC in megakaryocytopoiesis and thrombopoiesis. The present study is designed to define its biological activity. We demonstrate that the recombinant hNUDC significantly increases megakaryocyte maturation in serum-free liquid-cultured human CD34(+) cells and stimulates colony formation in serum-free semi-solid cultures. Flow cytometry analyses also confirm the stimulatory effect of hNUDC on megakaryocyte polyploidization and in vitro platelet production. In vivo experiments further demonstrate that the administration of hNUDC substantially enhance the number of circulating platelets in normal mice.     

Hydrocortisone induces changes in gene expression and differentiation in immature human enterocytes

It is known that functional maturation of the small intestine occurring during the weaning period is facilitated by glucocorticoids (such as hydrocortisone, HC), including an increased expression of digestive hydrolases. However, the molecular mechanisms are not well understood, particularly in the human gut. Here we report a microarray analysis of HC-induced changes in gene expression in H4 cells (a well-characterized human fetal small intestinal epithelial cell line). This study identified a large number of HC-regulated genes, some involved in metabolism, cell cycle regulation, cell-cell or cell-extracellular matrix communication. HC also regulates the expression of genes important for cell maturation such as development of cell polarity, tight junction formation, and interactions with extracellular matrices. Using human small intestinal xenografts, we also show that HC can regulate the expression of genes important for intestinal epithelial cell maturation. Our dataset may serve as a useful resource for understanding and dissecting the molecular mechanisms of intestinal epithelial cell maturation.     

重组红细胞生成素对小鼠巨核细胞后期成熟及血小板生成的影响

本文应用血小板生成液体培养体系,检测了重组人红细胞生成素(r-EPO)对巨核细胞成熟及血小板生成的影响。r-EPO 能在1U 至6~U/ml 浓度范围内增加体系血小板数,r-EPO剂量与血小板数之间呈线性关系。r-EPO 还能促进巨核细胞 DNA 合成,并使 Ⅱ、Ⅳ 期巨核细胞比例增加,Ⅰ、Ⅱ 期巨核细胞比例减少。结果表明:r-EPO 可以促进巨核细胞成熟,并作为一种主要刺激因子,以增加血小板数的方式促进血小板生成。