OBJECTIVE: Various cytokines and growth factors control the differentiation and maturation of megakaryocytes (MKs). However, the mechanism regulating platelet release from MKs is not well understood. Here, we investigated a role of Lnk/Sh2b3, an intracellular adaptor protein, in megakaryopoiesis. MATERIALS AND METHODS: Number of MK progenitor in bone marrow (BM) of wild-type or Lnk(-/-) mice and their sensitivity to thrombopoietin (TPO) were determined in colony-forming unit assay. Using BM-derived wild-type or Lnk(-/-) MKs stimulated with TPO, activation of the signaling molecules was biochemically analyzed and effect of integrin stimulation on TPO signals was studied by addition of vascular cell adhesion molecule (VCAM-1). Platelet production from MKs in the presence of VCAM-1 was counted by flow cytometry and their morphological change was observed by time-lapse microscopy. RESULTS: Lnk(-/-) mice showed elevated platelets and mature MKs due to enhanced sensitivity of progenitors to TPO. Erk1/2 phosphorylation induced by TPO was augmented and prolonged in Lnk(-/-) MKs while activation of signal transducers and activators of transcription (Stat)3, Stat5, and Akt was normal. Wild-type MKs, but not in Lnk(-/-) MKs on VCAM-1 showed reduced Stat5 phosphorylation and mitogen-activated protein kinases activation upon stimulation with TPO. Additionally, the presence of VCAM in culture accelerated spontaneous platelet release from mature wild-type MKs, but not from Lnk(-/-) MKs. CONCLUSIONS: Results suggest that contact of MKs with adhesion molecules via integrins might contribute to platelet release, which is under Lnk-mediated regulation of Stat-5 activation and show that Lnk functions in responses controlled by cell adhesion and in crosstalk between integrin- and cytokine-mediated signaling.
OBJECTIVE: Various cytokines and growth factors control the differentiation and maturation of megakaryocytes (MKs). However, the mechanism regulating platelet release from MKs is not well understood. Here, we investigated a role of Lnk/Sh2b3, an intracellular adaptor protein, in megakaryopoiesis. MATERIALS AND METHODS: Number of MK progenitor in bone marrow (BM) of wild-type or Lnk(-/-) mice and their sensitivity to thrombopoietin (TPO) were determined in colony-forming unit assay. Using BM-derived wild-type or Lnk(-/-) MKs stimulated with TPO, activation of the signaling molecules was biochemically analyzed and effect of integrin stimulation on TPO signals was studied by addition of vascular cell adhesion molecule (VCAM-1). Platelet production from MKs in the presence of VCAM-1 was counted by flow cytometry and their morphological change was observed by time-lapse microscopy. RESULTS:Lnk(-/-) mice showed elevated platelets and mature MKs due to enhanced sensitivity of progenitors to TPO. Erk1/2 phosphorylation induced by TPO was augmented and prolonged in Lnk(-/-) MKs while activation of signal transducers and activators of transcription (Stat)3, Stat5, and Akt was normal. Wild-type MKs, but not in Lnk(-/-) MKs on VCAM-1 showed reduced Stat5 phosphorylation and mitogen-activated protein kinases activation upon stimulation with TPO. Additionally, the presence of VCAM in culture accelerated spontaneous platelet release from mature wild-type MKs, but not from Lnk(-/-) MKs. CONCLUSIONS: Results suggest that contact of MKs with adhesion molecules via integrins might contribute to platelet release, which is under Lnk-mediated regulation of Stat-5 activation and show that Lnk functions in responses controlled by cell adhesion and in crosstalk between integrin- and cytokine-mediated signaling.
Authors: Nicole Soranzo; Tim D Spector; Massimo Mangino; Brigitte Kühnel; Augusto Rendon; Alexander Teumer; Christina Willenborg; Benjamin Wright; Li Chen; Mingyao Li; Perttu Salo; Benjamin F Voight; Philippa Burns; Roman A Laskowski; Yali Xue; Stephan Menzel; David Altshuler; John R Bradley; Suzannah Bumpstead; Mary-Susan Burnett; Joseph Devaney; Angela Döring; Roberto Elosua; Stephen E Epstein; Wendy Erber; Mario Falchi; Stephen F Garner; Mohammed J R Ghori; Alison H Goodall; Rhian Gwilliam; Hakon H Hakonarson; Alistair S Hall; Naomi Hammond; Christian Hengstenberg; Thomas Illig; Inke R König; Christopher W Knouff; Ruth McPherson; Olle Melander; Vincent Mooser; Matthias Nauck; Markku S Nieminen; Christopher J O'Donnell; Leena Peltonen; Simon C Potter; Holger Prokisch; Daniel J Rader; Catherine M Rice; Robert Roberts; Veikko Salomaa; Jennifer Sambrook; Stefan Schreiber; Heribert Schunkert; Stephen M Schwartz; Jovana Serbanovic-Canic; Juha Sinisalo; David S Siscovick; Klaus Stark; Ida Surakka; Jonathan Stephens; John R Thompson; Uwe Völker; Henry Völzke; Nicholas A Watkins; George A Wells; H-Erich Wichmann; David A Van Heel; Chris Tyler-Smith; Swee Lay Thein; Sekar Kathiresan; Markus Perola; Muredach P Reilly; Alexandre F R Stewart; Jeanette Erdmann; Nilesh J Samani; Christa Meisinger; Andreas Greinacher; Panos Deloukas; Willem H Ouwehand; Christian Gieger Journal: Nat Genet Date: 2009-10-11 Impact factor: 38.330