Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 114-126
Erythropoietin- and Stem Cell Factor-Induced DNA Synthesis in Normal
Human Erythroid Progenitor Cells Requires Activation of Protein Kinase
C
and Is Strongly Inhibited by Thrombin
Michael Haslauer,
Kurt Baltensperger, and
Hartmut Porzig
From the Department of Pharmacology, University of Bern, Bern,
Switzerland.
Proliferation, differentiation, and survival of erythroid progenitor
cells are mainly regulated by stem cell factor (SCF) and erythropoietin
(Epo). Using normal human progenitors, we analyzed the role of
Ca2+-sensitive protein kinase C (PKC) subtypes and of
G-protein-coupled receptor ligands on growth factor-dependent DNA
synthesis. We show that stimulation of DNA synthesis by the two growth
factors requires activation of PKC
. Inhibitors of
Ca2+-activated PKC subtypes blocked the growth
factor-induced 3H-thymidine incorporation. SCF and Epo
caused no significant translocation of PKC into the membrane, but
treatment of intact cells with either of the two cytokines resulted in
enhanced activity of immunoprecipitated cytosolic PKC. Stimulation
of PKC with the phorbol ester PMA mimicked the cytokine effect on DNA
synthesis. Epo-, SCF-, and PMA-induced thymidine incorporation was
potently inhibited by thrombin (half-maximal inhibition with 0.1 U/mL).
This effect was mediated via the G-protein-coupled thrombin receptor
and the Rho guanosine triphosphatase. Adenosine
diphosphate caused a modest Ca2+-dependent
stimulation of DNA synthesis in the absence of cytokines and
specifically enhanced the effect of SCF. Cyclic 3',5'-adenosine monophosphate exerted a selective inhibitory effect on Epo-stimulated thymidine incorporation. Our results define PKC as major
intermediate effector of cytokine signaling and suggest a role for
thrombin in controlling erythroid progenitor proliferation.
