Differential Role forpP85α and p85β Regulatory Subunits of Class IA PI-3Kinase in Mast Cell Growth and Maturation.

Abstract

While it has been established that c-Kit/stem cell factor (SCF) interactions are indispensable for normal mast cell development and functions, the intracellular signals that regulate the development and function of these cells downstream from c-Kit are not fully understood. Here, we demonstrate that p85 regulatory subunits of class IA PI-3Kinase (p85α and p85β) play an essential role in regulating mast cell growth and maturation. We show that deficiency of p85α in mast cells results in reduced growth and survival in response to SCF stimulation, which is associated with impaired activation of Akt, Rac and JNK MAP kinase. Furthermore, we demonstrate that in addition to regulating growth and survival, loss of p85α also impairs the maturation of mast cells as seen by significantly reduced expression of c-Kit and the IgE receptor on p85α−/− mast cells compared to wildtype controls. To determine the extent to which Akt contributes to the growth, survival and maturation of p85α−/− mast cells, we reconstituted p85α−/− mast cells with a retrovirus encoding an activated form of Akt. Remarkably, p85α−/− mast cells expressing an activated Akt completely rescued the proliferative defect associated with p85α deficient mast cells in response to IL-3 as well as IL-3 plus SCF, and a 70% correction in response to SCF alone. The phenotypic abnormalities seen in p85α−/− mast cells were surprising in light of the sequence homology and continued expression in these cells of p85β subunit of class IA PI-3Kinase, and suggested that p85α might function with specificity in mast cells in regulating c-Kit functions. To examine the relationship between p85α and p85β in regulating mast cell functions, we generated mast cells from p85β−/− mice and compared them with p85α−/− mast cells. We found that p85α and p85β perform qualitatively distinct functions in regulating mast cell growth and differentiation. We found that although deficiency of p85α in mast cells consistently results in reduced growth and impaired maturation, deficiency of p85β in these same cells results in enhanced growth and no significant defect in maturation. To further demonstrate that p85α and p85β play qualitatively distinct functions in regulating mast cell growth and differentiation, a genetic intercross between p85α+/− and p85β+/− mice was performed. Since mice completely deficient in both the isoforms of p85 (α & β) die early in embryonic development, we analyzed mast cell growth and differentiation in mice that were deficient in the expression of p85α(p85α−/−) and heterozygous at the p85β (p85β+/−) locus, along with the appropriate controls. We found that heterozygosity of p85β (p85β+/−) in the setting of p85α deficiency (p85α−/−) significantly corrected the mast cell defect associated with p85α deficiency (p85α−/−) alone. Finally, we reconstituted p85α−/− mast cells with a bicistronic retrovirus encoding EGFP and a cDNA for p85β, and found that p85β could not rescue the mast cell defect associated with p85α deficiency. In contrast, reconstituting p85α−/− mast cells with a retrovirus encoding the p85α cDNA completely rescued the mast cell growth defect associated with p85α deficiency. The level of expression of p85α and p85β in p85α−/− reconstituted mast cells was similar as determined by western blot analysis. Taken together, our results demonstrate qualitatively distinct functions for two closely related regulatory subunits of class IA PI-3Kinase in controlling mast cell growth and maturation.