Opposing Roles for Class IA PI-3Kinase and Src Family Kinase in Regulating Myeloid Cell Growth and Lineage Commitment.

In adults, hematopoietic stem/progenitor (SC/P) cell expansion and lineage commitment is regulated by coordinated signals from cytokine receptors and transcription factors (TFs). Studies performed in cell lines using cytokine receptors lacking the binding sites for Src and PI-3Kinase family members have suggested a role for these signaling molecules in regulating growth and survival. However, the physiologic role(s) for these signaling molecules in primary SC/P cell growth and lineage commitment are poorly understood. Furthermore, the mechanism(s) via which these kinases regulate the expression of TFs in primary SC/Ps are not known. We demonstrate that Lyn Src family kinase and p85α subunit of class IA PI-3Kinase play opposing roles in regulating growth and lineage commitment in primary SC/Ps, in part by regulating the threshold of PI-3Kinase signaling, including the levels of phosphatidylinositol-3,4,5,-trisphosphate (PIP3) and Akt. Loss of Lyn in SC/P cells results in elevated PIP3 levels and hyperactivation of Akt. In contrast, loss of p85α subunit of class IA PI-3Kinase results in reduced levels of PIP3 and Akt activation. Functionally, Lyn−/− or Lyn+/− SC/P cells show enhanced growth and commitment towards myeloid lineage, including granulocytes, monocytes and mast cells, which is associated with enhanced and accelerated induction of myeloid lineage markers including c-Kit and the high affinity receptor for IgE. The enhanced myeloid lineage commitment in Lyn−/− SC/P cells is also associated with hyper induction of PU.1, CEB/Pα as well as members of the GATA family. In spite of high degree of homology between various Src family kinases, reconstitution of Lyn−/− SC/Ps with Lyn, but not with other related family members such as Hck or Fgr resulted in the rescue of Lyn−/− SC/P cell phenotype, suggesting that Lyn functions with specificity in regulating SC/P cell growth and survival, including lineage commitment. Enhanced lineage commitment, growth and survival as well as PIP3 levels seen in Lyn−/− SC/Ps were in part due to impaired activation of SHIP in these cells. Remarkably, in spite of continued reduction in the activation of SHIP and hyperactivation of Stat5 and Erk MAP kinase in Lyn−/− SC/P cells, haplo-insufficiency or complete deficiency of p85α subunit of class IA PI-3Kinase in the setting of Lyn deficiency or Lyn-haplo-insufficiency not only corrected the elevated PIP3 levels, cytokine induced hypersensitivity and lineage commitment to near wildtype levels, but in some cases made it worst than that seen in wildtype controls. Taken together, utilizing a genetic and a biochemical approach, our results illustrate a complex network of interactions between cytokine receptors and TFs involving Lyn, p85α and SHIP in positive and negative regulation of growth and lineage commitment in primary SC/P cells. Furthermore, our results demonstrate that the level of PIP3 in SC/P cells plays a critical role in determining the fate and lineage commitment in these cells. In vivo consequences on the long-term repopulating ability of stem cells lacking both Lyn and p85α subunit of class IA PI-3Kinase will be discussed.