Protein kinase Cα is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the Aγ globin-promoter in cellular models of hemoglobin switching
✍ Scribed by Angela Di Baldassarre; Mariacristina Di Rico; Antonella Di Noia; Tiziana Bonfini; Antonio Iacone; Marco Marchisio; Sebastiano Miscia; Elena Alfani; Anna Rita Migliaccio; George Stamatoyannopoulos; Giovanni Migliaccio
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 369 KB
- Volume
- 101
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
Abstract
PKCα 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 γ/γ + β globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 ± 12 vs. 7 ± 3, respectively), we tested the hypothesis that PKCα might affect γ‐globin expression by measuring the levels of ^A^γ‐ or β‐promoter‐driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual µLCRβprRluc^A^γprFluc reporter in the presence of transient expression of either the constitutively active (sPKCα) or catalytically inactive (iPKCα) PKCα. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 µM). In all the cells analyzed, sPKCα significantly increased (by two‐ to sixfold) the levels of luciferase activity driven by the ^A^γ‐promoter and the ^A^γ‐F/(^A^γ‐F + 2β‐R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the ^A^γ‐driven luciferase activity and the ^A^γ‐F/(^A^γ‐F + 2β‐R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic‐specific functions in erythropoiesis and that modulation of PKCα might affect the activity of ^A^γ‐promoter‐driven reporters. J. Cell. Biochem. 101: 411–424, 2007. © 2007 Wiley‐Liss, Inc.