GSK-3β Negatively Regulates Megakaryocyte and Platelet Production in an In Vitro Human Bone Marrow Mononuclear Cell-Derived Differentiation System.

Glycogen synthase kinase (GSK)-3β, a serine/threonine kinase, reportedly regulates the canonical Wnt and Hedgehog pathways. Evidence indicates that thrombopoietin, a major regulator of thrombopoiesis, induces several signaling pathways, such as MAPK, PI3K, and JAK/STAT. Here, we present data showing a novel role of GSK-3β in megakaryocyte (MK) and platelet production in an in vitro hematopoietic cell culture system. Primary human low-density bone marrow mononuclear cells (BMMNCs) were grown in serum-free media containing thrombopoietin and several inhibitors: PD98059, SB202190, LY294002 or TWS119 was used to inhibit MAPK, p38MAPK, PI3K, or GSK-3β, respectively. The MK or platelet cell count was examined by flow cytometry on day 12 using the relative value of CD41 (+)/propidium iodide (+) cells or platelet size CD41 (+) cells, respectively, versus 10⁶ BMMNCs on day 0. When the cells were treated with PD98059, SB202190, or LY294002, the MKs and platelets decreased in number compared with the non-treated control cells. In contrast, MK and platelet production was increased by the presence of TWS119 in the cell culture [TWS119 (+)] as compared with its absence in the cell culture [TWS119 (−)]: MKs, 24965.3±5437.7 vs 5763.7±874.0, p=0.0038, platelets, 6645.8±792.0 vs 3276.0±298.1, p=0.0023. In addition, human CD34 (+) cells were obtained from BMMNCs and grown in the liquid culture system, and each small interference RNA (siRNA) for GSK-3β or negative control was transfected into the cells on day 3. The frequency of CD41 (+)/propidium iodide (+) cells in siRNA-GSK3β transfected-cells was 2-fold higher than that in siRNA-negative control transfected cells. The transcriptional profiling was then compared between BMMNCs in TWS119 (+/−) to study the mechanism underlying the effect of GSK-3β on MK and platelet production. This analysis using the Affymetrix U133 Plus 2.0 Gene Chip array, which covers approximately 50000 transcripts, demonstrated genes with significantly increased or decreased expression in TWS119 (+) relative to TWS119 (−) at day 8. Of those genes, some factors associated with cytoskeletal organization, such as Rho guanine nucleotide exchange factor and β tubulin cofactor A, had increased expression, while others, such as supervillin and α tubulin, had decreased expression. The BMMNC-derived MKs and platelets in TWS119 (+/−) were morphologically analyzed with electron microscopy. The size of MKs in TW119 (+) was larger in diameter than that of MKs in TWS119 (−). Also, there was abundant mature MKs in TWS119 (+) whereas most MKs in TWS119 (−) were immature at day 8. However, there was no difference in the ultrastructure of platelets obtained from the MKs in TWS119 (+) or TWS119 (−). The findings of the present study demonstrated that GSK-3β negatively regulates MK and platelet production. Alterations in the MK morphology and/or maturation time might contribute to this regulation.