Abstract
Fibronectin (FN) plays important roles in the proliferation, differentiation, and maintenance of megakaryocytic-lineage cells through FN receptors. However, substantial role of FN receptors and their functional assignment in proplatelet-like formation (PPF) of megakaryocytes are not yet fully understood. Herein, we investigated the effects of FN receptors on PPF using the CHRF-288 human megakaryoblastic cell line, which expresses VLA-4 and VLA-5 as FN receptors. FN and phorbol 12-myristate 13-acetate (PMA) were essential for inducing PPF in CHRF-288 cells. Blocking experiments using anti-β1-integrin monoclonal antibodies indicated that the adhesive interaction with FN via VLA-4 and VLA-5 were required for PPF. PPF induced by FN plus PMA was accelerated when CHRF-288 cells were enforced adhering to FN by TNIIIA2, a peptide derived from tenascin-C, which we recently found to induce β1-integrin activation. Adhesion to FN enhanced PMA-stimulated activation of extracellular signal-regulated protein kinase 1 (ERK1)/2 and enforced adhesion to FN via VLA-4 and VLA-5 by TNIIIA2-accelerated activation of ERK1/2 with FN plus PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38, and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN plus PMA, even with TNIIIA2. Thus, the enhanced activation of ERK1/2 by FN, PMA plus TNIIIA2 was responsible for acceleration of PPF with FN plus PMA.
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This work was supported by the Grants-in-Aid for Scientific Research grant to T.M, F.F, Y.K., and K.S.
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Supplementary Figure 1
Expression of fibronectin receptors and Syndecan-4 in CHRF-288 cells. (A) Flow cytometric (FACS) analyses of fibronectin receptor β1-integrins on CHRF-288 cells. FACS analyses were performed using the normal IgG or anti-β1-integrins mAb, (a) normal IgG (negative control), (b) anti-α3-integrin mAb, (c) anti-α4-integrin mAb, (d) anti-α5-integrin mAb, (e) anti-αV-integrin mAb, and (f) anti-β1-integrin mAb, as described in “Materials and methods”. Data shown are representative of three individual experiments. (B) FACS analyses of Syndecan-4 in CHRF-288 cells. FACS analyses were performed using the FITC-labeled normal IgG (┅┅┅) or FITC-labeled anti-Syndecan-4 mAb (━━━), as described in “Materials and methods”. Data shown are representative of three individual experiments. (JPEG 282 kb)
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Matsunaga, T., Fukai, F., Kameda, T. et al. Potentiated activation of VLA-4 and VLA-5 accelerates proplatelet-like formation. Ann Hematol 91, 1633–1643 (2012). https://doi.org/10.1007/s00277-012-1498-y
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DOI: https://doi.org/10.1007/s00277-012-1498-y