Thrombopoietin-Mediated Activation of the mTOR/S6K/4E-BP Signaling Pathway Is Required for Proliferation in Megakaryocyte Progenitors.

Translational regulation plays a central role in cell proliferation, survival and cell differentiation through activation of the target of rapamycin (mTOR) signaling pathway. mTOR controls the phosphorylation status of proteins involved in initiating translational control, including ribosomal S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Recently, the mTOR and phosphoinositide 3-kinase (PI3-K) pathways have been linked through the tumor suppressor complex TSC1/2. The PI3-K target PKB inactivates the TSC1/2 complex which acts as a GAP for the Ras homologue Rheb to suppress mTOR signaling. As the regulation of cell number and cell size are important factors during megakaryopoiesis, we investigated the role of mTOR signaling in thrombopoietin (TPO) induced proliferation and differentiation in primary human megakaryocyte progenitors and in megakaryoblastic MO7e cells. Preincubation of isolated CD34⁺ cells, primary cultured CD61⁺ cells and MO7e cells with the mTOR inhibitor rapamycin (10 nM) resulted in highly specific inhibition of TPO-mediated 4E-BP1 (S65) phosphorylation, S6K (T389) phosphorylation and its substrate S6 (S235/236), without affecting PKB (S473) phosphorylation. Activation of the mTOR signaling pathway by TPO was dependent on the PI3-K pathway as LY294002 (10 μM) inhibited phosphorylation of 4E-BP1, S6K and S6. Treatment of MO7e cells with rapamycin inhibited TPO-induced proliferation and cell cycling by reducing cells in S-phase and blocking cells in G₁. Rapamycin did not induce apoptosis as measured by cells in sub-G₀ phase and by Annexin V expression. Suspension cultures of CD34⁺ cells treated with rapamycin resulted in a 2.3-fold reduction in overall cell proliferation (p=0.01) and a reduction in the percentage of CD61⁺ megakaryocytic cells (55.2±7.93 vs 35.0±5.42, p=0.08) generated after 7 days. The mean fluorescence intensity of CD61 and CD42 expressing cells was however not decreased. In addition, polyploidisation levels of the CD61 expressing cells cultured in the presence of rapamycin were not decreased, indicating rapamycin inhibited megakaryocyte proliferation, not differentiation. Further analysis revealed that transforming growth factor β1 (TGF-β1), which inhibits proliferation of megakaryocyte progenitors, downregulated TPO-induced S6K/S6 phosphorylation. Concluding, we have shown that the mTOR pathway is activated by TPO and plays a role in regulating proliferation in megakaryocyte progenitors. Part of the effect of the PI3-K/PKB and TGF-β pathways in regulating proliferation may be mediated by the mTOR/S6K/4E-BP1 signaling pathway.