5 research outputs found
Differential fMet-Leu-Phe- and Platelet-activating Factor-induced Signaling Toward Ral Activation in Primary Human Neutrophils
We have measured the activation of the small GTPase
Ral in human neutrophils after stimulation with fMet-
Leu-Phe (fMLP), platelet activating factor (PAF), and
granulocyte macrophage-colony stimulating factor and
compared it with the activation of two other small
GTPases, Ras and Rap1. We found that fMLP and PAF,
but not granulocyte macrophage-colony stimulating factor,
induce Ral activation. All three stimuli induce the
activation of both Ras and Rap1. Utilizing specific inhibitors
we demonstrate that fMLP-induced Ral activation
is mediated by pertussis toxin-sensitive G-proteins and
partially by Src-like kinases, whereas fMLP-induced
Ras activation is independent of Src-like kinases. PAFinduced
Ral activation is mediated by pertussis toxininsensitive
proteins, Src-like kinases and phosphatidylinositol
3-kinase. Phosphatidylinositol 3-kinase is not
involved in PAF-induced Ras activation. The calcium
ionophore ionomycin activates Ral, but calcium depletion
partially inhibits fMLP- and PAF-induced Ral activation,
whereas Ras activation was not affected. In addition,
12-O-tetradecanoylphorbol-13-acetate-induced
activation of Ral is completely abolished by inhibitors of
protein kinase C, whereas 12-O-tetradecanoylphorbol-
13-acetate-induced Ras activation is largely insensitive.
We conclude that in neutrophils Ral activation is mediated
by multiple pathways, and that fMLP and PAF induce
Ral activation differently
Mammalian target of rapamycin activity is required for expansion of CD34+ hematopoietic progenitor cells
Background
The mammalian target of rapamycin is a conserved protein kinase known to regulate protein
synthesis, cell size and proliferation. Aberrant regulation of mammalian target of
rapamycin activity has been observed in hematopoietic malignancies, including acute
leukemias and myelodysplastic syndromes, suggesting that correct regulation of mammalian
target of rapamycin is critical for normal hematopoiesis.
Design and Methods
An ex vivo granulocyte differentiation system was utilized to investigate the role of mammalian
target of rapamycin in the regulation of myelopoiesis.
Results
Inhibition of mammalian target of rapamycin activity, with the pharmacological inhibitor
rapamycin, dramatically reduced hematopoietic progenitor expansion, without altering
levels of apoptosis or maturation. Moreover, analysis of distinct hematopoietic progenitor
populations revealed that rapamycin treatment inhibited the expansion potential of committed
CD34+ lineage-positive progenitors, but did not affect early hematopoietic progenitors.
Further examinations showed that these effects of rapamycin on progenitor expansion
might involve differential regulation of protein kinase B and mammalian target of
rapamycin signaling.
Conclusions
Together, these results indicate that mammalian target of rapamycin activity is essential
for expansion of CD34+ hematopoietic progenitor cells during myelopoiesis. Modulation
of the mammalian target of rapamycin pathway may be of benefit in the design of new
therapies to control hematologic malignancie
Activation of the Small GTPase Rap1 in Human Neutrophils
The small GTPase Rap1 is highly expressed in human neutrophils,
but its function is largely unknown. Using the Rap1-
binding domain of RalGDS (RalGDS-RBD) as an activationspecific
probe for Rap1, we have investigated the regulation
of Rap1 activity in primary human neutrophils. We found
that a variety of stimuli involved in neutrophil activation,
including fMet-Leu-Phe (fMLP), platelet-activating factor
(PAF), granulocyte-macrophage colony-stimulating factor
(GM-CSF), and IgG-coated particles, induce a rapid and
transient Rap1 activation. In addition, we found that Rap1 is
normally activated in neutrophils from chronic granulomatous
disease patients that lack cytochrome b558 or p47phox
and have a defective NADPH oxidase system. From these
results we conclude that in neutrophils Rap1 is activated
independently of respiratory burst induction. Finally, we
found that Rap1 is activated by both the Ca21 ionophore
ionomycin and the phorbol ester 12-O-tetradecanoylphorbol
13-acetate (TPA), indicating that phospholipase C (PLC)
activation leading to elevated levels of intracellular free Ca21
and diacylglycerol (DAG) can mediate Rap1 activation. However,
inhibition of PLC and Ca21 depletion only marginally
affected fMLP-induced Rap1 activation, suggesting that additional
pathways may control Rap1 activation
Activation of FoxO transcription factors contributes to the antiproliferative effect of cAMP
cAMP is a potent inhibitor of cell proliferation in a
variety of cell lines. Downregulation of cyclin D1 and
upregulation of the cell cycle inhibitor p27Kip1 are two
mechanisms by which cAMP may induce a G1-arrest.
Here we show that cAMP inhibits proliferation of cells
that constitutively express cyclin D1 or are deficient for
Rb, demonstrating that changes in these cell cycle
regulators do not account for the cAMP-induced growth
effects in mouse embryo fibroblasts (MEFs). Interestingly,
the antiproliferative effect of cAMP mimics the
effect previously observed for FoxO transcription factors.
These transcription factors are under negative control of
protein kinase B (PKB). We show that in MEFs cAMP
strongly induces transcriptional activation of FoxO4
through the inhibition of PKB. Accordingly, not only
p27Kip1 but also the FoxO target MnSOD is upregulated
by cAMP. Importantly, introduction of dominant-negative
FoxO partially rescues cAMP-induced inhibition of
proliferation. From these results we conclude that
inhibition of PKB and subsequent activation of FoxO
transcription factors mediates an antiproliferative effect
of cAMP
The ENOD12 gene product is involved in the infection process during the pea-rhizobium interaction
The pea cDNA clone pPsENOD12 represents a gene involved in the infection process during Pisum sativum L.-Rhizobium leguminosarum bv. viciae symbiosis. The ENOD12 protein is composed of pentapeptides containing two hydroxyprolines. The expression of the ENOD12 gene is induced in cells through which the infection thread is migrating, but also in cells that do not yet contain an infection thread. Soluble compounds from Rhizobium are involved in eliciting ENOD12 gene expression. Rhizobium common and host-specific nodulation genes are essential for the production of these compounds. Two ENOD12 genes are expressed in nodules and in stem tissue of uninoculated plants. The gene represented by the cloned ENOD12 mRNA is also expressed in flowers, but a different transcription start may be used