| Research Abstract |
We recently reported that TGF-β is involved in negative feedback regulation of megakaryopoiesis in both normal volunteers (NV) and patients with Idiopathic Thrombocytopenic Purpura (Y.Hirayama et al. BLOOD,90,141,1998 ; S.Sakamaki et al. BLOOD,92,42,1999). In patients with Essential Thrombocythemia (ET), however, excess of megakaryopoiesis was observed despite the high concentration of TGF-β in BM, suggesting an escape mechanism for megakaryopoiesis from TGF-β action. To elucidate this mechanism, in this study, we analyzed TGF-β signal transduction pathways in megakaryocytes from ET and NV.Bone marrow mononuclear cells (BMMNC) (10^5 cells/dish) of ET and NV were cultured in α-MEM containing TGF-β (0, 0.1, 1.0 ng/mL), 100 ng/mL rh-TPO, 20% human fasting plasma and 0.8% methyl cellulose. After 14 days, CFU-Meg colony numbers were measured. RT-PCR of TGF-β receptor (type I, II) and Smad 2, 3, 4 and 5 were performed using total RNA prepared from CFU-Meg in liquid culture. TaqMan real-time RT-PCR were also employed to quantify the mRNA expression. The growth of CFU-Meg colonies derived from NV was completely suppressed by 1.0 ng/mL of TGF-β (mean inhibitory rate ; 92.7%) while those from ET patients (n=5) showed less suppression (mean inhibitory rate ; 24.1%) (p<0.05). Expressions of TGF-β receptor (type I, II) and Smad 2, 3 and 5 were equally detected in ET and NV.However, expression of Smad4 was significantly reduced in ET in comparison with those of NV (p<0.05). Transfection of Smad4 cDNA normalized the sensitivity of ET CFU-Meg to TGF-β. Thus, it was surmised that one of the mechanism for impaired sensitivity of CFU-Meg to TGF-β is the reduced expression of Smad4.
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