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. 1998 Jul 15;92(2):472-80.


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 alphaIIIbbeta3 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 alphaIIIbbeta3 and CREB-PSer133 at various time points. High levels of nuclear CREB-PSer133 were unequivocally demonstrated in alphaIIIbbeta3+ 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD34
  • Blotting, Western
  • Cell Differentiation / physiology
  • Cell Line
  • Cyclic AMP Response Element-Binding Protein / physiology*
  • Enzyme Inhibitors / pharmacology
  • Flavonoids / pharmacology
  • Flow Cytometry
  • Hematopoietic Stem Cells / cytology*
  • Hematopoietic Stem Cells / physiology*
  • Humans
  • Megakaryocytes / cytology*
  • Megakaryocytes / physiology*
  • Phosphorylation
  • Protein Kinase Inhibitors
  • Protein Kinases / physiology
  • Serine
  • Signal Transduction / drug effects
  • Signal Transduction / physiology*
  • Thrombopoietin / pharmacology


  • Antigens, CD34
  • Cyclic AMP Response Element-Binding Protein
  • Enzyme Inhibitors
  • Flavonoids
  • Protein Kinase Inhibitors
  • Serine
  • Thrombopoietin
  • Protein Kinases
  • 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one