Impaired thrombin generation in Reelin-deficient mice: a potential role of plasma Reelin in hemostasis. J Thromb Haemost 2014; 12: 2054-64.Summary. Background: Reelin is a large extracellular glycoprotein that is present in the peripheral blood. That Reelin interacts with the coagulation components and elicits a functional role in hemostasis has not yet been elucidated. Objectives: The hemostatic activity of Reelin is investigated and defined in this study. Methods: The interplay of Reelin with coagulation components was elucidated by far-Western and liposome/platelet binding assays. In vivo and ex vivo hemostasis-related analyses of Reelin-deficient mice and plasma were also performed. Results: Reelin interacted with the liposomes containing phosphatidylserine (PS) or phosphatidylcholine. Instead of interacting with known Reelin receptors (ApoE receptor 2, very low density lipoprotein receptor and integrin b1), Reelin interacted with PS of the activated platelets. The interaction between Reelin and the coagulation factors of thrombin and FXa was also demonstrated with the Kd of 11.7 and 21.2 nM, respectively. Reelin-deficient mice displayed a prolonged bleeding time and an increase in rebleeding rate. Despite the fact that Reelin deficiency had no significant effect on the clotting time of prothrombin and activated partial thromboplastin time, the fibrin clot formation was abnormal and the fibrin clot structure was relatively loosened with reduced clot strength. Abnormal fibrinogen expression did not account for the hemostatic defects associated with Reelin deficiency.Instead, thrombin generation was impaired concomitant with an altered prothrombin cleavage pattern. Conclusions: By interacting with platelet phospholipids and the coagulation factors, thrombin and FXa, Reelin plays a selective role in coagulation activation, leading to thrombin generation and formation of a normal fibrin clot.
a b s t r a c tReelin is an extracellular glycoprotein that is highly conserved in mammals. In addition to its expression in the nervous system, Reelin is present in erythroid cells but its function there is unknown. We report in this study that Reelin is up-regulated during erythroid differentiation of human erythroleukemic K562 cells and is expressed in the erythroid progenitors of murine bone marrow. Reelin deficiency promotes erythroid differentiation of K562 cells and augments erythroid production in murine bone marrow. In accordance with these findings, Reelin deficiency attenuates AKT phosphorylation of the Ter119 + CD71+ erythroid progenitors and alters the cell number and frequency of the progenitors at different erythroid differentiation stages. A regulatory role of Reelin in erythroid differentiation is thus defined.
Integrin-mediated signal transduction and membrane blebbing have been well studied to modulate cell adhesion, spreading and migration^1-6^. However, the relationship between membrane blebbing and integrin signaling has not been explored. Here we show that integrin-ligand interaction induces membrane blebbing and membrane permeability change. We found that sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are located in the membrane blebbing sites and inhibition of NHE1 disrupts membrane blebbing and decreases membrane permeability change. However, inhibition of NCX1 enhances cell blebbing to cause cell swelling which is correlated with an intracellular sodium accumulation induced by NHE17. These data suggest that sodium influx induced by NHE1 is a driving force for membrane blebbing growth, while sodium efflux induced by NCX1 in a reverse mode causes membrane blebbing retraction. Together, these data reveal a novel function of NHE1 and NCX1 in membrane permeability change and blebbing and provide the link for integrin signaling and membrane blebbing.
Prostate cancer is the leading cause of cancer-related death in men in the world. Previous studies have demonstrated that Disabled-2 (DAB2) is a tumor suppressor and down-regulation of DAB2 expression is accompanied with prostate cancer progression. On the other hands, protein kinase C epsilon (PKCε) functions as an oncogene and promotes human prostate cancer cell survival upon treatment of methyseleninic acid (MSA), an anti-cancer agent that induces prostate cancer cell apoptosis. However, little is known whether there is a crosstalk between DAB2 and PKCε. In this study, we first analyzed DAB2 and PKCε protein expression in three prostate cancer cell lines DU145, PC3 and C4-2. Western blot analysis revealed that DAB2 was relatively abundant in PC3 when compared with the DU145 and C4-2 cells. In contrast, the expression of PKCε was higher in DU145 and C4-2 cells when compared with the PC3 cells. Consistent with these findings, ectopic expression of DAB2 resulted in down-regulation of PKCε. These observations implicate a reverse association of DAB2 and PKCε and suggest that DAB2 is a regulator of PKCε expression. To further explore the interplay between DAB2 and PKCε, we determined the effect of DAB2 overexpression on DU145 cell susceptibility to MSA-induced cell death. Consistent with a previous report, overexpression of PKCε increased DU145 cell survival and knockdown of PKCε by small interfering RNA augmented cell death upon treatment of MSA but not Ibupofen. Accordingly, expression of DAB2 down-regulated PKCε and increased in cell susceptibility to MSA that resulted in a 40% decrease of cell survival. These findings suggest that DAB2 modulates PKCε expression and affects prostate cancer cell susceptibility to selective anti-cancer agents. This study represents the first report to unveil the interplay between DAB2, PKCε and prostate cancer cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5054.
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