| Summary: | 博士 === 長庚大學 === 生物醫學研究所 === 97 === Disabled-2 (DAB2) is up-regulated through platelet-derived growth factor autocrine signaling during megakaryocytic differentiation of human leukemic cell lines. In this study, human K562 leukemic cells, embryonic stem (ES) cell E14tg2a, and murine primary bone marrow cell were subjected to megakaryocytic differentiation to continue our efforts in understanding the functional role of DAB2 induction during megakaryopoiesis. Consistent with our previous observations, DAB2 expression was induced in all of these model systems during TPA or thrombopoietin treated megakaryocytic differentiation. Due to the properties of easier genetic manipulation, K562 leukemic cell was initially used for further characterization of DAB2 function during TPA-induced megakaryocytic differentiation. Upon K562 cells differentiating toward the megakaryocytic lineage, down-regulation of DAB2 by small interfering RNA siDAB2 augmented integrin alphaIIbbeta3 activation and resulted in the increase of cell adhesion to fibrinogen. Ectopic expression of DAB2 reversed the siDAB2 effect, or by itself, decreased fibrinogen adhesion of K562 cells. Mutational analysis revealed that a DAB2 Ser24 phosphorylation mutant S24A abrogated the inhibitory function of DAB2. Through cellular localization and co-immunoprecipiation analysis, we demonstrate for the first time that Ser24 phosphorylation promotes membrane translocation of DAB2 and its subsequent interaction with integrin beta3, thereby defining a mechanism for DAB2 in regulating integrin alphaIIbbeta3 activation and inside-out signaling during megakaryocytic differentiation. Based on these findings in K562 leukemic cells, the synchronized and mature differentiating ES/OP9 co-culture system was used for further characterization of DAB2 function in megakaryopoiesis. Accordingly, stable expression of siDAB2 in the E14tg2a cells abrogated mesodermal colony formation and the subsequent megakaryocytic differentiation. Morphological and cell growth rate analysis further revealed that DAB2 inhibition resulted in the reduced of cell-cell contact and induced cell death, suggesting that DAB2 may play an important role in the maintenance of cell survival for early stage of megakaryocytic differentiation. Molecular analysis demonstrated that DAB2 modulated Wnt signaling and the expression of apoptotic associated protein p53 leading to the blockage of differentiation programming. Microarray and miRNAs screening further unveiled an additional 17 genes and 26 miRNAs, mostly linked to cell death and metabolic pathways, were associated with DAB2-regulated signaling. Together with these effects, DAB2 is a key regulator in the control of cellular metabolism and cell survival that is required for mesodermal colony formation and the subsequent megakaryopoiesis. Based on these discoveries in cell based systems, DAB2 conditional knockout mice were established for advanced examining the in vivo roles of DAB2 in platelet biogenesis and function. However, DAB2 expression in platelets is extremely different from human, rat, to mice. Less DAB2 protein in murine platelets prompted us to investigate the role of DAB2 during platelet biogenesis, and we preliminarily found that DAB2 may regulate proplatelet formation. Abundant DAB2 expression in human platelets were worthy for us to characterize its role during platelet activation and aggregation. In washed human platelet, we found that DAB2 was distributed in the platelet alpha-granules and was released from the granular compartment upon platelet activation. The secreted DAB2 binds to the extracellular region of integrin alphaIIb on the platelet surface through the phosphotyrosine binding domain. The DAB2-platelet interactions result in the inhibition of agonist-induced platelet aggregation with the exception of thrombin which is a DAB2 protease that renders DAB2 inactive. These findings demonstrate for the first time that DAB2 is an integrin alphaIIb-binding protein that plays a novel role in the control of platelet-fibrinogen interactions and platelet aggregation. Together, this work contributes to our understanding of DAB2 in megakaryocytic differentiation and platelet function, and perhaps shed new light on platelet physiology.
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