Protein kinase Calpha is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the (A)gamma globin-promoter in cellular models of hemoglobin switching.

PKCa was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the g/gþb globin expression ratio represented the major difference between neonatal and adult erythroblasts (5812 vs. 73, respectively), we tested the hypothesis that PKCa might affect g-globin expression by measuring the levels of Ag- or b-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual mLCRbprRlucAgpr- Fluc reporter in the presence of transient expression of either the constitutively active (sPKCa) or catalytically inactive (iPKCa) PKCa. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 mM). In all the cells analyzed, sPKCa significantly increased (by two- to sixfold) the levels of luciferase activity driven by the Ag-promoter and the Ag-F/(Ag-Fþ2b-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the Ag-driven luciferase activity and the Ag-F/(Ag-Fþ2b-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCa might affect the activity of Ag-promoter-driven reporters