The Role of Akt-Forkhead Signaling in the Hematopoietic Stem-Progenitor Cell Differentiation: Expansion of Hematopoietic and Endothelial Progenitors from Akt-1 Null Marrows and Peripheral Blood Involving FKHRL1-Activation.

Akt is an important regulator of cell survival, growth and glucose metabolism in many cell types. In the previous annual meeting, we first reported the role of Akt in the hematopoietic stem cells with Akt-gene knockout mouse models. We described that Akt-signaling modulates the side population (SP) cell by directly regulating the molecular site of Bcrp1, one of the ATP-binding cassette transporters. Akt regulates many downstream targets through phosphorylation of a number of cellular substrates. FKHRL1, one of the FOXO subclass members of the forkhead box transcription factors, has a role in not only regulation of cell-cycle progression and cell survival phosphorylated but also control of cell-differentiation and transformation. FKHRL1 transcription-activities are inhibited by Akt, following induction of a prompt and sustained nuclear exclusion through phosphorylation. In this meeting, we will exhibit how Akt and its downstream signaling, FKHRL1 act during hematopoietic stem-progenitor cell differentiation with Akt1-null mouse studies and endothelial progenitor cell (EPC) assays. In bone marrow cells, a significant increase in formation of macrophage colony-forming units (CFU-M) and granulocyte-macrophage colony-forming units (CFU-GM) was seen in Akt1-null mice. Multiple growth factor responsive progenitor cultures were also more from Akt1-deficient marrows. Moreover, flow cytometry analysis showed the higher ratio of the lineage-negative progenitor cell-population in these marrows. Previously, we reported that bone marrow cellularity and the number of hematopoietic stem cells is normal in Akt1-null mice. These results indicate that lacking of Akt1-signaling leads to the proliferation potential of progenitor cells. Next, we analyzed the number of EPCs in Akt1-deficit mice from peripheral blood mononuclear cells (PBMCs) cultured on mouse fibronectin (FN)-coated dishes. DiI-Ac-LDL/lectin stained EPCs were detected after 10 days. Interestingly, Akt1-defecit mouse-EPCs were quite an increase in number, whereas few EPCs are usually detected from wild-type PBMCs. To address why EPCs were expanded, we used human EPC assay for analyzing signal transduction at detail. After attached on FN, circulating stem cells in PBMCs differentiated into EPCs with four different steps; foci-formation, sprout from the foci, migration for cord-like structure and maturation. Western blot analysis clearly showed that Akt was gradually activated during EPC-differentiation following the inactivation at the first step of differentiation. On the contrary, Akt-downstream targets, FKHRL1 and GSK3-β were inactivated through phosphorylation during differentiation. Immunofluorescent staining showed FKHRL1 was located in nucleus at the foci-formation and translocated into cytosol at the time of sprout from foci formation. Finally, lentivirus-mediated overexpression of Akt and FKHRL1 gene into mouse PBMCs showed FKHRL1-triple mutant, which is not phosphorylatable because of replacing three phosphorylation sites by alanine residues, significantly increased the number of EPCs, while constitutive active-Akt and dominant negative-FKHRL1 failed to. These data suggest that Akt-signaling has an important modulator of the hematopoietic stem-progenitor cell differentiation. FKHRL1 is involved in this mechanism.