Influence of PI-3K/Akt Pathway on Wnt Signalling in Myeloid Progenitor Cells. Evidence for a Role of Autocrine/Paracrine Wnt Regulation in CFU-GM Proliferation

We have used a colony replating assay in conjunction with manipulation of the Wnt and PI-3K pathways to investigate regulation of myeloid progenitor cell proliferation/differentiation. We found that PI-3 kinase pathway via Akt acts as a proliferative brake and promotes differentiation in IL3-driven myelopoiesis, since inhibition of PI-3K/Akt pathway with LY294002 (PI-3 kinase inhibitor) and SH-5 (Akt inhibitor) increased proliferation (reduced differentiation). We investigated the involvement of Wnt signalling in CFU-GM proliferation by using exogenous recombinant canonical Wnt3a and non-canonical Wnt5a. We showed that both of the Wnt members cannot support colony growth alone, but when added to IL3 they increased proliferation potential compared with the IL3 control, indicating an involvement of canonical Wnt/β-catenin and non-canonical Wnt pathways in the myeloid progenitor cell proliferation. Immunoblotting analysis indicated that Wnt5a acts independently of β-catenin. Dkk-1 (Wnt-pathway inhibitor) alone did not affect IL-3 dependent proliferation but when combined with recombinant Wnts as expected it abrogated the effects of Wnt3a but not Wnt5a (acts as canonical-Wnt inhibitor). This was confirmed by β-catenin protein levels. Surprisingly, when Dkk-1 was added to LY294002 or SH-5, it blocked their proliferative effects. Dkk-1 acts as functional Wnt-receptor disabler and the finding that it blocks proliferation induced by PI-3K/Akt inhibitors’ indicates a link between the PI-3K and the Wnt signalling pathways. We hypothesised that increased proliferation induced by LY294002 or SH-5 was not mediated by downstream activation of the Wnt pathway but by induced endogenous expression of Wnt proteins and activation of the surface receptor. We conclude that there is a production of endogenous Wnt proteins that increases proliferation. Endogenous Wnt production has been reported in primitive haematopoietic cells so there is potential for a paracrine or autocrine role for these cell regulators. We tested this hypothesis in CD34⁺ cells and found the addition of SH-5 to IL3 creates an autocrine loop of endogenous Wnt production, which results in the phosphorylation and activation of the LRP6 receptor and the initiation of the canonical signalling pathway. Furthermore, the conditioned medium of cultured CD34⁺ cells was concentrated by using filtration devices (30kD cut-off) and added to IL3 to support the growth of progenitors of another sample in a CFU-GM assay. We indicated that Wnt production and secretion can act in a paracrine way as well, since IL3+SH-5 conditioned medium increased the proliferative index of CFU-GM cells whereas IL3 conditioned medium did not have significant effect. Dkk-1 abrogated the IL3+SH-5 conditioned medium’s induced proliferation, suggesting that the growth factors that had the proliferative effects were Wnt members. In conclusion, our data suggest that IL3 via PI-3K pathway promotes differentiation of progenitor myeloid cells through inhibition of endogenous Wnt production.