The induction of megakaryocyte differentiation is accompanied by selective Ser133 phosphorylation of the transcription factor CREB in both HEL cell line and primary CD34+ cells

Giorgio Zauli, Davide Gibellini, Marco Vitale, Paola Secchiero, Claudio Celeghini, Alessandra Bassini, Sabina Pierpaoli, Marco Marchisio, Lia Guidotti, Silvano Capitani

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Abstract

The addition of thrombopoietin (TPO) to HEL cells, cultured in a chemically defined serum-free medium, induced a rapid and dose-dependent phosphorylation of the transcription factor CREB on serine133 (PSer133), as detected by Western blot analysis. TPO also significantly increased the transactivation of CRE-dependent promoter, as determined in transient transfection experiments. On the other hand, neither erythropoietin (Epo; 1 to 10 U) nor hemin (10-7 mol/L) were able to significantly stimulate CREB-PSer133 or to activate CRE-promoter in HEL cells. Although pharmacological inhibitors of protein kinase C (chelerytrine and BIM) and protein kinase A (H-89) failed to block the TPO-mediated CREB phosphorylation, a specific inhibitor of the mitogen-activated protein kinases (PD98059) completely blocked the ability of TPO to stimulate CREB- PSer133. Moreover, PD98059 significantly decreased the ability of TPO to upregulate the surface expression of the α(IIb)β3 megakaryocytic marker in HEL cells. In parallel, primary CD34+ hematopoietic cells were seeded in liquid cultures supplemented with 100 ng/mL of TPO and examined by immunofluorescence for the coexpression of α(IIb)β3 and CREB-PSer133 at various time points. High levels of nuclear CREB-PSer133 were unequivocally demonstrated in α(IIb)β3+ cells, including morphologically recognizable megakaryocytes. Taken together, these data suggest that CREB plays a role in modulating the expression of genes critical for megakaryocyte differentiation and that the TPO-mediated CREB phosphorylation seems to be regulated via mitogen-activated protein kinases.

Original languageEnglish
Pages (from-to)472-480
Number of pages9
JournalBlood
Volume92
Issue number2
Publication statusPublished - Jul 15 1998

ASJC Scopus subject areas

  • Hematology

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    Zauli, G., Gibellini, D., Vitale, M., Secchiero, P., Celeghini, C., Bassini, A., Pierpaoli, S., Marchisio, M., Guidotti, L., & Capitani, S. (1998). The induction of megakaryocyte differentiation is accompanied by selective Ser133 phosphorylation of the transcription factor CREB in both HEL cell line and primary CD34+ cells. Blood, 92(2), 472-480.