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.
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.
Zauli
G
G
Institute of Human Anatomy, University of Ferrara, Ferrara, Italy.
Gibellini
D
D
Vitale
M
M
Secchiero
P
P
Celeghini
C
C
Bassini
A
A
Pierpaoli
S
S
Marchisio
M
M
Guidotti
L
L
Capitani
S
S
D018952:Antigens, CD34; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000502:physiology*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology* / Q000502:physiology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000502:physiology; D012694:Serine; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013926:Thrombopoietin / Q000494:pharmacology
D018952:Antigens, CD34; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D005419:Flavonoids; D047428:Protein Kinase Inhibitors; D012694:Serine; D013926:Thrombopoietin; D011494:Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one