Parallel regulation of PKC-alpha and PKC-delta characterizes the occurrence of erythroid differentiation from human primary hematopoietic progenitors.
OBJECTIVE
Erythroid differentiation is a process characterized by modulation of different proteins including phosphoinositide-related enzymes such as protein kinase C (PKC) isoforms. Because in different cell lines PKC-alpha and PKC-delta have been reported to be involved in the mechanisms controlling proliferation and differentiation, the aim of this study was to examine the relative involvement of these PKC isoforms in the development of CD235a+ erythroid cells from human healthy hematopoietic progenitors.


METHODS
Erythroid differentiation from human primary hematopoietic progenitor cells was achieved by adopting the human erythroblasts mass amplification culture. Expression and activity of PKC isoforms and their relationship with proliferation and differentiation were investigated by morphologic analysis, reverse-transcriptase polymerase chain reaction, Western blotting, multiparametric flow cytometry, and transfection experiments.


RESULTS
PKC-alpha was found expressed and phosphorylated in cells undergoing both proliferation and differentiation, although PKC-delta, largely expressed and activated during proliferation, was evidently downregulated during differentiation. Overexpression of PKC-delta-CAT scarcely influenced the development of glycophorin-A (CD235a)+ erythroid cells from hematopoietic progenitors, although overexpression of PKC-alpha-CAT strongly induced the development of CD235a+ erythroid cells. On the other hand, in PKC-alpha-CAT-transfected cells, pharmacologic inhibition of PKC-delta further increased the number of CD235a+ cells, although inhibition of PKC-alpha resulted in an evident impairment of the development of CD235a+ erythroid cells.


CONCLUSIONS
Our results indicate that the suppression or at least a strong downregulation of PKC-delta, concomitant to PKC-alpha expression and activity, might be a cofactor to be further investigated and might be involved in the events regulating erythropoietin-induced erythroid differentiation from human primary hematopoietic progenitor cells.
Lanuti
Paola
P
Cell Signalling Unit, Section of Human Anatomy, Department of Biomorphology, University G. d'Annunzio of Chieti-Pescara, Chieti, Italy.
Bertagnolo
Valeria
V
Gaspari
Anna Rita
AR
Ciccocioppo
Fausta
F
Pierdomenico
Laura
L
Bascelli
Adriana
A
Sabatino
Giuseppe
G
Miscia
Sebastiano
S
Marchisio
Marco
M
D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D041905:Erythroid Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004921:Erythropoietin / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D016195:G2 Phase / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D010641:Phenotype; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D012333:RNA, Messenger; D004921:Erythropoietin; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta