THROMBOPOIETIN-INDUCED DIFFERENTIATION OF A HUMAN MEGAKARYOBLASTIC LEUKEMIA-CELL LINE, CMK, INVOLVES TRANSCRIPTIONAL ACTIVATION OF P21(WAF1CIP1) BY STAT5/

Although thrombopoietin (TPO) is known to play a fundamental role in b oth megakaryopoiesis and thrombopoiesis, the molecular mechanism of TP O-induced megakaryocytic differentiation is not known. In a human mega karyoblastic leukemia cell line, CMK, that showed some degree of megak aryocytic differentiation after culture with TPO, the cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/Cipl), but not p27(Kip1), p16(INK4A) p15(INK4B), Or p18(INK4C), found to be upregulated in an immediately e arly 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 diffe rentiation and p21 induction in CR IK cells. To examine potential role s of Cdk inhibitors in megakaryocytic differentiation, CMK cells were transfected with the p21, p27, or p16 gene, together with a marker gen e, beta-galactosidase, and were cultured with medium alone for i days. The ectopic expression of p21 or p27 hut not of p16 led to induction of megakaryocytic differentiation of CR IK cells, Overexpression of th e N-terminal domain (amino acids [aa] 1 to 75) of p21 was sufficient t o induce megakaryocytic differentiation, whereas that of the C-termina l domain (aa 76 to 164) had little or no effect on morphological featu res, Furthermore, we found that although TPO induced tyrosine phosphor ylation of both STAT3 and STAT5 in CR IK cells, only STAT5 showed bind ing activities to potential STAT-binding sites that locate in the prom oter region of p21 gene (p21-SIE sites), thereby leading to transactiv ation of p21. These results suggested that p21 induction, possibly med iated through activated STAT5, could play an important role in TPO-ind uced megakaryocytic differentiation.