Thrombopoietin-induced differentiation of a human megakaryoblastic leukemia cell line, CMK, involves transcriptional activation of p21(WAF1/Cip1) by STAT5.
Although thrombopoietin (TPO) is known to play a fundamental role in both megakaryopoiesis and thrombopoiesis, the molecular mechanism of TPO-induced megakaryocytic differentiation is not known. In a human megakaryoblastic leukemia cell line, CMK, that showed some degree of megakaryocytic differentiation after culture with TPO, the cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/Cip1), but not p27(Kip1), p16(INK4A), p15(INK4B), or p18(INK4C), was found to be upregulated in an immediately early response to TPO. The expression of p21 was found to be sustained over a period of 5 days by treatment with TPO in large polyploid cells that developed in response to TPO, but not in small undifferentiated cells, indicating a close correlation between the ligand-induced differentiation and p21 induction in CMK cells. To examine potential roles of Cdk inhibitors in megakaryocytic differentiation, CMK cells were transfected with the p21, p27, or p16 gene, together with a marker gene, beta-galactosidase, and were cultured with medium alone for 5 days. The ectopic expression of p21 or p27 but not of p16 led to induction of megakaryocytic differentiation of CMK cells. Overexpression of the N-terminal domain (amino acids [aa] 1 to 75) of p21 was sufficient to induce megakaryocytic differentiation, whereas that of the C-terminal domain (aa 76 to 164) had little or no effect on morphological features. Furthermore, we found that although TPO induced tyrosine phosphorylation of both STAT3 and STAT5 in CMK cells, only STAT5 showed binding activities to potential STAT-binding sites that locate in the promoter region of p21 gene (p21-SIE sites), thereby leading to transactivation of p21. These results suggested that p21 induction, possibly mediated through activated STAT5, could play an important role in TPO-induced megakaryocytic differentiation.
Matsumura
I
I
Second Department of Internal Medicine, Osaka University Medical School, Suita, Japan.
Ishikawa
J
J
Nakajima
K
K
Oritani
K
K
Tomiyama
Y
Y
Miyagawa
J
J
Kato
T
T
Miyazaki
H
H
Matsuzawa
Y
Y
Kanakura
Y
Y
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000187:drug effects; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000378:metabolism*; D003561:Cytarabine / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism*; D016147:Genes, Tumor Suppressor; D006801:Humans; D007947:Leukemia, Megakaryoblastic, Acute / Q000473:pathology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008894:Milk Proteins; D047428:Protein Kinase Inhibitors; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013926:Thrombopoietin / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins
D000964:Antimetabolites, Antineoplastic; C493956:CDKN1A protein, human; C493966:CDKN2B protein, human; C493971:CDKN2C protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D008894:Milk Proteins; D047428:Protein Kinase Inhibitors; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D003561:Cytarabine; D050760:Cyclin-Dependent Kinase Inhibitor p27; D013926:Thrombopoietin; D013755:Tetradecanoylphorbol Acetate