Anagrelide represses GATA-1 and FOG-1 expression without interfering with thrombopoietin receptor signal transduction.
BACKGROUND
Anagrelide is a selective inhibitor of megakaryocytopoiesis used to treat thrombocytosis in patients with chronic myeloproliferative disorders. The effectiveness of anagrelide in lowering platelet counts is firmly established, but its primary mechanism of action remains elusive.


OBJECTIVE
Here, we have evaluated whether anagrelide interferes with the major signal transduction cascades stimulated by thrombopoietin in the hematopoietic cell line UT-7/mpl and in cultured CD34(+) -derived human hematopoietic cells. In addition, we have used quantitative mRNA expression analysis to assess whether the drug affects the levels of known transcription factors that control megakaryocytopoiesis.


RESULTS
In UT-7/mpl cells, anagrelide (1μm) did not interfere with MPL-mediated signaling as monitored by its lack of effect on JAK2 phosphorylation. Similarly, the drug did not affect the phosphorylation of STAT3, ERK1/2 or AKT in either UT-7/mpl cells or primary hematopoietic cells. In contrast, during thrombopoietin-induced megakaryocytic differentiation of normal hematopoietic cultures, anagrelide (0.3μm) reduced the rise in the mRNA levels of the transcription factors GATA-1 and FOG-1 as well as those of the downstream genes encoding FLI-1, NF-E2, glycoprotein IIb and MPL. However, the drug showed no effect on GATA-2 or RUNX-1 mRNA expression. Furthermore, anagrelide did not diminish the rise in GATA-1 and FOG-1 expression during erythropoietin-stimulated erythroid differentiation. Cilostamide, an exclusive and equipotent phosphodiesterase III (PDEIII) inhibitor, did not alter the expression of these genes.


CONCLUSIONS
Anagrelide suppresses megakaryocytopoiesis by reducing the expression levels of GATA-1 and FOG-1 via a PDEIII-independent mechanism that is differentiation context-specific and does not involve inhibition of MPL-mediated early signal transduction events.
Ahluwalia
M
M
University of Wales Institute, Cardiff School of Health Sciences, Cardiff, UK.
Donovan
H
H
Singh
N
N
Butcher
L
L
Erusalimsky
J D
JD
D018952:Antigens, CD34 / Q000096:biosynthesis; D001792:Blood Platelets / Q000187:drug effects; D002460:Cell Line; D050982:GATA1 Transcription Factor / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D006801:Humans; D008533:Megakaryocytes / Q000187:drug effects; D009687:Nuclear Proteins / Q000096:biosynthesis*; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology*; D053628:Receptors, Thrombopoietin / Q000378:metabolism*; D015398:Signal Transduction; D014157:Transcription Factors / Q000096:biosynthesis*; D014158:Transcription, Genetic
D018952:Antigens, CD34; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D009687:Nuclear Proteins; D010975:Platelet Aggregation Inhibitors; D011799:Quinazolines; D053628:Receptors, Thrombopoietin; D014157:Transcription Factors; C498432:ZFPM1 protein, human; C021139:anagrelide