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Protein Kinase C Mediates the Mitogenic Action of Thrombopoietin
in c-Mpl-Expressing UT-7 Cells
Ying Hong,
Dominique Dumènil,
Bernd van der Loo,
Frédérique Goncalves,
William Vainchenker, and
Jorge D. Erusalimsky
From the Cruciform Project and Department of Medicine, University
College London, London, UK; and INSERM U362, Institut Gustave Roussy,
Villejuif Cedex, France.
Protein kinase C (PKC) has been implicated in signal transduction
events elicited by several hematopoietic growth factors. Thrombopoietin
(TPO) is the major regulator of megakaryocytic lineage development, and
its receptor, c-Mpl, transduces signals for the proliferation and
differentiation of hematopoietic progenitors. In this study we have
examined the effect of TPO on the subcellular distribution of PKC (a
measure of enzyme activation) in a growth factor-dependent pluripotent
hematopoietic cell line that was engineered to express the c-Mpl
receptor (UT-7/mpl). In addition, we have assessed the significance of
this activation for the induction of both mitogenesis and
differentiation. Using a PKC translocation assay, TPO was found to
stimulate a time- and dose-dependent increase in the total content of
PKC activity present in the membrane fraction of UT-7/mpl cells
(maximum increase = 2.3-fold above basal level after 15 minutes
with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a
decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that
TPO treatment led to a marked increase of the
Ca2+/diacylglycerol-sensitive PKC isoforms  and  found in the membrane fraction. In contrast, the subcellular
distribution of these isoforms did not change after treatment with
granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of
UT-7/mpl cells to the selective PKC inhibitor GF109203X completely
inhibited the PKC activity associated to the membrane fraction after
TPO treatment, and blocked the mitogenic effect of TPO. In contrast,
GF109203X had no effect on the TPO-induced expression of GpIIb, a
megakaryocytic differentiation antigen. Downregulation of PKC isoforms
and to less than 25% of their initial level by treatment with
phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic
response, but had no significant effect when this response was induced
by GM-CSF. Taken together, these findings suggest that (1) TPO
stimulates the activation of PKC, (2) PKC activation mediates the
mitogenic action of TPO, and (3) PKC activation is not required for
TPO-induced expression of megakaryocytic surface markers.
Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 813-822
© 1998 by The American Society of Hematology.
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