Abstract

P1329 Demonstration of activation of integrin alphaIIbbeta3 by inside-out signaling in a megakaryocytic cell line, CMK NIA Exhibition Area 09:30 16th July, 2003 Session Type: Posters Subject area: Integrins and other adhesive proteins Session title: Megakarocytes Abstract: P1329 Authors: H. Kashiwagi, Y. Tomiyama, M. Shiraga, S. Honda, S. Kosugi, T. Kiyoi, H. Kato, Y. Kurata & Y. Matsuzawa Osaka University, Japan Affinity/avidity state of integrin alphaIIbbeta3 is tightly regulated by intracellular signaling, often referred to as inside-out signaling. Although several magakaryocytic cell lines have been established, activation of alphaIIbbeta3 by cellular agonists has not been observed in these cells, and that has made it difficult to use them for studying of inside-out signaling. Recent studies demonstrated that inside-out signals appear to be completed in the late-stage of megakaryocytic differentiation. Thus, we re-examined agonist-induced alphaIIbbeta3 activation on megakaryocytic cell lines with a marker of late-stage of megakaryocytic differentiation, GPIb. Activation of alphaIIbbeta3 was assessed by PAC1 and soluble fibrinogen binding to the cells. We studied Dami, HEL, K562 and CMK, and found that alphaIIbbeta3 expressed in CMK cells with high GPIb expression was activated with 200 nM of PMA. Although the population of PMA-reactive GPIb-high CMK cells was less than 0.5%, incubation with a nucleoside analogue, ribavirin, for 10-14 days efficiently increased the PMA-reactive GPIb-high cells. Not only PMA but also calcium ionophore, A23187, induced moderate alphaIIbbeta3 activation, whereas thrombin and ADP hardly activated alphaIIbbeta3. The alphaIIbbeta3 activation by PMA in GPIb-high CMK cells appeared to be comparable with that of platelets, because it was completely blocked by alphaIIbbeta3 specific antagonist, FK633, and also inhibited mildly by wortmannin and more efficiently blocked by cytochalasin-D as demonstrated in platelets. Although it is generally hard to transfect plasmids to CMK cells, we could get good transfection efficiency by adenovirus-mediated transfection or by electroporation of mRNA synthesized in vitro. These results indicate that GPIbhigh CMK cells combined with ribavirin-treatment is a useful model for studying the signaling events from PKC activation and intracellular calcium mobilization to alphaIIbbeta3 activation. Finally, we investigated expression of PKC isoforms in ribavirin-treated CMK cells. Platelets contained several PKC isoforms including PKCalpha, -beta, -delta, and -gamma, and ribavirin treatment increased the expression of PKC-beta, -delta, and -gamma in CMK cells, suggesting that these PKC isoforms may have direct or indirect roles in inside-out signaling. file:///E|/working/LAXMI-PRASAD/WileyML-3G/deepak/31-Jan/Wednesday/Abstract%20P1329.html (1 of 2) [1/31/2014 3:51:05 PM]