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.
PKCalpha 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 gamma/gamma + beta globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 +/- 12 vs. 7 +/- 3, respectively), we tested the hypothesis that PKCalpha might affect gamma-globin expression by measuring the levels of (A)gamma- or beta-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual microLCRbetaprRluc(A)gammaprFluc reporter in the presence of transient expression of either the constitutively active (sPKCalpha) or catalytically inactive (iPKCalpha) PKCalpha. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 microM). In all the cells analyzed, sPKCalpha significantly increased (by two- to sixfold) the levels of luciferase activity driven by the (A)gamma-promoter and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the (A)gamma-driven luciferase activity and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCalpha might affect the activity of (A)gamma-promoter-driven reporters.
Di Baldassarre
Angela
A
Department of Biomorphology, University of Chieti-Pescara, Chieti, Italy.
Di Rico
Mariacristina
M
Di Noia
Antonella
A
Bonfini
Tiziana
T
Iacone
Antonio
A
Marchisio
Marco
M
Miscia
Sebastiano
S
Alfani
Elena
E
Migliaccio
Anna Rita
AR
Stamatoyannopoulos
George
G
Migliaccio
Giovanni
G
D000098:Acetophenones / Q000378:metabolism; D000328:Adult; D001578:Benzopyrans / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D004900:Erythroblasts / Q000166:cytology / Q000502:physiology; D004920:Erythropoiesis / Q000502:physiology*; D017930:Genes, Reporter; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D006454:Hemoglobins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D011401:Promoter Regions, Genetic; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism
D000098:Acetophenones; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D006454:Hemoglobins; D007527:Isoenzymes; D005914:Globins; C085746:rottlerin; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta