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Prepublished online as a Blood First Edition Paper on September 5, 2002; DOI 10.1182/blood-2002-04-1278.
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Blood, 1 March 2003, Vol. 101, No. 5, pp. 1744-1751
HEMATOPOIESIS
Stromal inhibition of megakaryocytic differentiation is
associated with blockade of sustained Rap1 activation
Lorrie L. Delehanty,
Michael Mogass,
Sara L. Gonias,
Frederick K. Racke,
Brian Johnstone, and
Adam N. Goldfarb
From the Department of Pathology, University of
Virginia Health Sciences Center, Charlottesville; Department of
Pathology, Johns Hopkins Medical Institutions, Baltimore, MD; and
Department of Orthopedics, Case Western Reserve University and
University Hospitals Research Institute, Cleveland, OH.
Coculture with stromal cells tends to maintain normal hematopoietic
progenitors and their leukemic counterparts in an undifferentiated, proliferative state. An example of this effect is seen with
megakaryocytic differentiation, wherein stromal contact renders
many cell types refractory to potent induction stimuli. This inhibitory
effect of stroma on megakaryocytic differentiation correlates with a blockade within hematopoietic cells of protein kinase C- (PKC-) up-regulation and of extracellular signal-regulated
kinase/mitogen-activated protein (ERK/MAP) kinase activation, both of
which have been implicated in promoting megakaryocytic differentiation.
In this study K562 RafER.5 cells, expressing an estradiol-responsive
mutant of the protein kinase Raf-1, were used to determine the
relevance and stage of ERK/MAPK pathway blockade by stromal contact.
Activation of RafER by estradiol overrode stromal blockade of
megakaryocytic differentiation, implicating the proximal stage of the
ERK/MAPK pathway as a relevant control point. Because stromal contact
blocked delayed but not early ERK activation, the small guanosine
triphosphatase (GTPase) Rap1 was considered as a candidate inhibitory
target. Activation assays confirmed that Rap1 underwent sustained
activation as a result of megakaryocytic induction, as previously
described. As with ERK activation, stromal contact selectively blocked
delayed but not early Rap1 activation, having no effect on Ras
activation. Enforced expression of either wild-type Rap1 or the GTPase
(GAP) resistant mutant Rap1 V12 failed to override stromal inhibition, suggesting that the inhibitory mechanism does not involve GAP up-regulation but rather may target upstream guanine nucleotide exchange factor (GEF) complexes. Accordingly,
coimmunoprecipitation demonstrated stromally induced alterations in a
protein complex associated with c-Cbl, a scaffolding factor for
Rap1-GEF complexes.
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