Growth and maturation of megakaryocytes is regulated by Lnk/Sh2b3 adaptor protein through crosstalk between cytokine- and integrin-mediated signals.
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.


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.
Takizawa
Hitoshi
H
Division of Immunology, Department of Microbiology and Immunology, The University of Tokyo, Tokyo, Japan.
Eto
Koji
K
Yoshikawa
Atsuko
A
Nakauchi
Hiromitsu
H
Takatsu
Kiyoshi
K
Takaki
Satoshi
S
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D018345:Mice, Knockout; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D013926:Thrombopoietin / Q000378:metabolism / Q000494:pharmacology*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D016023:Integrins; D047908:Intracellular Signaling Peptides and Proteins; C490777:Lnk protein, mouse; D008565:Membrane Proteins; D011506:Proteins; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; C494087:Stat3 protein, mouse; D019010:Vascular Cell Adhesion Molecule-1; D013926:Thrombopoietin; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3