Transforming growth factor-beta1 interferes with thrombopoietin-induced signal transduction in megakaryoblastic and erythroleukemic cells.
OBJECTIVE
Thrombopoietin (TPO) and transforming growth factor-beta(1) (TGF-beta(1)) have been shown to exert opposite effects on proliferation and megakaryocytic differentiation of hematopoietic cells. To determine whether TGF-beta(1) interferes directly with TPO-induced signal transduction in hematopoietic cells, we compared the regulatory effects in the TPO-responsive cell lines Mo-7e and HEL.


METHODS
The cells were stimulated by 100 ng/mL TPO and/or 100 ng/mL TGF-beta1 and analyzed for proliferation (3H thymidine incorporation), viability (trypan blue exclusion), and protein expression and phosphorylation (Western blot).


RESULTS
TPO enhanced the proliferation of Mo-7e cells as determined by 3H-thymidine incorporation, whereas TGF-beta1 suppressed baseline cell growth and antagonized the proliferative effect of TPO. TPO-induced proliferation also was reduced by a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway (PD098059), which inhibits activation of the MAPK extracellular signal-regulated kinases (ERK) ERK1 and ERK2, and AG490, an inhibitor of Janus kinase-2, which completely blocked TPO-induced proliferation. As demonstrated by Western blotting, TGF-beta1 reduced the TPO-stimulated ERK1/ERK2 and STAT5 phosphorylation in Mo-7e and HEL cells. This effect was completely reversed by preincubation with a tyrosine phosphatase inhibitor (Na3VO4), which suggests that TGF-beta1 activated a phosphatase. Although STAT3 also was activated by TPO, STAT3 activation remained unaltered by TGF-beta1.


CONCLUSIONS
Taken together, these data suggest that TGF-beta1 modulates TPO-mediated effects on megakaryocytic proliferation by interfering with TPO-induced signal transduction, particularly by reducing the activities of MAPK ERK1/ERK2 and STAT5.
Kalina
U
U
Department of Hematology, Johann Wolfgang Goethe-University Hospital, Frankfurt/Main, Germany. Kalina@em.uni-frankfurt.de
Koschmieder
S
S
Hofmann
W K
WK
Wagner
S
S
Kauschat
D
D
Hoelzer
D
D
Ottmann
O G
OG
D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015672:Erythroid Precursor Cells / Q000187:drug effects*; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D053614:Janus Kinase 2; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology*; D007947:Leukemia, Megakaryoblastic, Acute / Q000473:pathology; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008533:Megakaryocytes / Q000187:drug effects*; D008894:Milk Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000187:drug effects*; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D013926:Thrombopoietin / Q000037:antagonists & inhibitors*; D015534:Trans-Activators / Q000037:antagonists & inhibitors / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D020032:Tyrphostins / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D008894:Milk Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D014638:Vanadates; D013926:Thrombopoietin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D017027:Protein Tyrosine Phosphatases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one