Project Goal:  To define the role of PI3-kinase and Akt in the CSF-1 growthand survival program in myeloid cells

Theprimary aim of this project is to develop an understanding of how the ColonyStimulating Factor-1 (CSF-1) supports the proliferation and survival ofmyelomonocytic cells. To do this, we genetically complemented a CSF-1 receptor(CSF-1R) mutant defective in mitogenic and survival signaling with downstreamsignaling molecules. DeltaKI is a CSF-1R mutant that lacks thereceptor binding site for PI3-kinase but can still activate this pathway via analternate route involving Src family kinases and Gab2. Unlike cells expressingthe WT receptor, CSF-1 cannot support the proliferation and survival of DeltaKI cells. CSF-1 induction of the PI3-kinase/Aktpathway in DeltaKI cells was significantly impairedbut stable expression of a constitutively active p110* restored their abilityto function mitogenically in response to CSF-1. These results establish thatdirect recruitment of PI3-kinase to the receptor is essential for optimalactivation and normal proliferative and survival functions require a thresholdlevel of PI3-kinase activity.

BothAkt-dependent and independent pathways downstream of PI3-kinase played a roleas stable expression of a constitutively active Akt (E40K) was unable tocompletely substitute for p110* in restoring the mitogenic response of DeltaKI cells. DeltaKIcells in the presence of CSF-1 undergo a protracted form of cell deathdisplaying several features characteristic of apoptosis (Fig. 1). Cell deathwas not accompanied by early changes in Delta Psi-mor by an early release of cytochrome c into the cytosol. Atypical also ofapoptosis was the relative lack of detectable caspase activation associatedwith dying DeltaKI cells (Fig. 2). Overexpression ofthe pro-survival molecule Bcl-xL delayed but did not prevent death of DeltaKI cells in the presence or absence of CSF-1. Theseresults are consistent with a form of cell death that is independent ofmitochondrial dysfunction and caspase activation.

  See our latest publication on the role of PI3K and Akt in cell survivalpdf.

Fig. 1   Electronmicrographs.  Starved: deprived of growth factors –typical marginated and condensed chromatin of apoptotic cells. IL-3: cells grown in IL-3 are healthy andnuclei show the fine lacy pattern. WT CSF-1:cells expressing the WT CSF-1R are healthy when grown in CSF-1.  DeltaKI CSF-1:cells expressing DeltaKI CSF-1R are dying in the presenceof CSF-1: a mix of late apoptotic cells with heavy vacuolization (left) andcells with some early changes (right).


Fig. 2  PARP cleavageshown by immunoblotting.  PARP is asubstrate of several executioner caspases and is cleaved into a large fragment(p85) and a small fragment (not shown). Starved cells are included as a positive control for PARP cleavage.  Aliquots of cells are processed at theindicated times after CSF-1 treatment.

Src kinases