|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Fibronectin promotes proplatelet formation in the human megakaryocytic cell line UT-7/TPO
Tatsuya Kawaguchi*1, Ryo Hatano†, Kyoji Yamaguchi‡, Katsuhiko Nawa*, Ryuji Hashimoto§ and Hiroshi Yokota†
*CardiovascularMetabolics Research Laboratories, DaiichiSankyo Co. Ltd, 1258 Hiromachi, Shinagawaku, Tokyo 1408710, Japan, †RD Planning Department, DaiichiSankyo Co. Ltd, 1258 Hiromachi, Shinagawaku, Tokyo 1408710, Japan, ‡Biological Research Laboratories, DaiichiSankyo Co. Ltd, 1258 Hiromachi, Shinagawaku, Tokyo 1408710, Japan, and §Biologics Research Laboratories, DaiichiSankyo Co. Ltd, 11613 Kitakasai, Edogawaku, Tokyo 1348630, Japan
We investigated PPF (proplatelet formation) in the human megakaryocytic cell line UT-7/TPO in vitro and signal transduction pathways responsible for PPF. The megakaryocytic cell lines are useful for studying megakaryocyte biology, although PPF is induced only in the presence of phorbol ester. TPO (thrombopoietin) stimulates megakaryocyte proliferation and differentiation; however, no PPF occurred in the megakaryocytic cell lines, even after the addition of TPO. Therefore, factors other than TPO may play an important role in the process of PPF. As PPF occurs in the bone marrow in vivo, we noted extracellular matrix proteins and found that soluble FN (fibronectin) induced potent PPF in UT-7/TPO without phorbol ester. A Western blot analysis showed that the expression of integrins was not increased by FN treatment. Anti-β1 antibody and the RGD (arginine-glycine-aspartate) peptide inhibited FN-induced PPF. This result indicates that the signal originated from integrin β1, which is essential to inducing PPF in UT-7/TPO. Results of the experiments using several inhibitors suggest that activation of the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]–ERK and PI3K (phosphoinositide 3-kinase) pathways are necessary for PPF. The phosphorylation of ERK gradually increased for 2 h after the addition of soluble FN, which suggests that activation of ERK is essential for the initial induction of FN-induced PPF in UT-7/TPO. UT-7/TPO is a useful cell line that enables us to study the signals of PPF without effects of chemical compounds.
Key words: extracellular-signal-regulated protein kinase (ERK), extracellular matrix (ECM), integrin, megakaryocyte, thrombopoietin (TPO)
Abbreviations: COX, cyclo-oxygenase, ECM, extracellular matrix, ERK, extracellular-signal-regulated kinase, FBS, fetal bovine serum; FN, fibronectin, GPVI, glycoprotein VI, IMDM, Iscove's modified Dulbecco's medium, MAPK, mitogen-activated protein kinase, MEK, MAPK/ERK kinase, PI3K, phosphoinositide 3-kinase, PF-4, platelet factor-4, PPF, proplatelet formation, RGD, arginine-glycine-aspartate, TPO, thrombopoietin, TXS, thromboxane synthase, vWF, von Willebrand factor
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Received 7 July 2011/22 August 2011; accepted 4 October 2011
Published as Cell Biology International Immediate Publication 4 October 2011, doi:10.1042/CBI20110383
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