PKCs are serine-threonine kinases that play an important role in many cellular functions. Almost all the 12 PKC isoforms described so far are expressed throughout erythroid differentiation of human CD34pos cells. Besides the downmodulation of PKCε expression (

Bassini et al,
), little is known on the role exerted by each PKC on erythroid differentiation. PKCα and PKCδ are two isoforms expressed at comparable levels (as mRNA and protein) throughout differentiation of CD36highCD235alow pro-erythroblasts into orthochromatic erythroblasts. For both proteins, the ratio between phosphorylated and total form remains constant during the differentiation of adult cells. In contrast, the ratio P-PKCα/total PKCα (but not that of P-PKCδ/total PKCδ) increases by 3-fold (with slight donor-to-donor variability) during the differentiation of CD36highCD235alow cells from cord blood. Furthermore, the protein becomes prominently localized in the nucleus of these cells. Since the most stricking difference in the differentiation of adult vs. neonatal erythroblasts is the γ/γ+β globin expression ratio (0.02–0.08 vs. 0.20–0.40 by Taqman, respectively), we hypothesized that the levels of PKCα activity might affect γ-globin expression in erythroblasts. To test this hypothesis, we used two in vitro models of HbF switching. The first model is represented by GM979 cells stably transfected with a dual luciferase (Renilla, R - Firefly, F) reporter driven by either the human γ- or β-globin promoter. The second model is represented by human erythroblasts obtained in HEMA culture. GM979 cells were transiently transfected with expression constructs encoding either the catalytic subunit (sPKCα) or the catalytic inactive (iPKCα) PKCα. The levels of expression of the γ-driven and β-driven luciferase reporters were then measured 24 hrs after transfection. Alternatively, GM979 cells were incubated with concentrations of rottlerin (30 μM) that specifically inhibit PKCα activity. sPKCα increased by 2–3-fold the expression of the luciferase driven by the γ-promoter but did not affect expression from the β promoter. Therefore, the ratio Aγ-F/(Aγ-F+2β-R) was also increased by 2–3 fold. Conversely, rottlerin inhibited (by 50%) both expression of the γ-driven luciferase and the Aγ-F/(Aγ-F+2β-R) ratio. On the other hand, transfection of the double luciferase reporter gene into adult and neonatal CD36highCD235alow cells (30–50% transfection efficiency) resulted in high levels of expression of both reporters, in a ratio consistent with the ontogenic stage of the cells [high Aγ-F/(Aγ-F+2β-R) in neonatal erythroblasts; low Aγ-F/(Aγ-F+2β-R) in adult erythroblasts]. Co-trasfection of sPKCα with this luciferase reporter in CD36highCD235alow cells (both adult and neonatal) increased by 5–6-fold the expression of the luciferase driven from the γ-promoter. Co-trasfection of iPKCα, sPKCδ and iPKCδ had no effect on the expression of the reporters in these cells. In conclusion, using several models of erythroid differentiation, we observed that increased levels of PKCα activity results in increased activity of the γ-promoter, suggesting that element(s) of the haemoglobin switching machinery may represent a specific PKCα substrate in erythroid cells.

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