Inhibition of GSK-3β promotes survival and proliferation of megakaryocytic cells through a β-catenin-independent pathway

The interaction of thrombopoietin (TPO) with its receptor c-Mpl initiates intracellular signals that are critical for megakaryopoiesis. Previously we and others have shown that TPO activates PI3K and Akt and that this pathway is important for megakaryocyte growth. Here, we investigate the importance of the Akt substrate glycogen synthase kinase (GSK)-3β in TPO signaling. GSK-3β is phosphorylated and inhibited by Akt as part of the PI3K pathway. GSK-3β can also be inhibited by Wnt signaling through a distinct mechanism, leading to reduced phosphorylation and accumulation of the transcription factor β-catenin. Therefore, we asked if TPO and Wnt3a can both inhibit GSK-3β in megakaryocytic cells, and if they can act synergistically to promote cell growth. Although both TPO and specific chemical inhibitors of GSK-3β result in increased survival and proliferation in a megakaryocytic cell line model, treatment with Wnt3a failed to increase cell growth either in the absence or presence of TPO, despite inducing high levels of β-catenin. Similarly, expression of a constitutively active version of β-catenin did not increase cell growth either in the absence or presence of TPO, suggesting that the effects of GSK-3β inhibition downstream of TPO signaling are distinct from those induced by Wnt3a and independent of β-catenin. The growth promoting effects of TPO are not mediated by either of the two known GSK-3β targets, cyclin D or HIF-1α. We conclude that GSK-3β is phosphorylated and inhibited by TPO-induced Akt, promoting survival and proliferation in megakaryocytic cells through a pathway that does not involve β-catenin.