From the Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084
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ABSTRACT |
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Lysophosphatidic acid (LPA) is a growth factor
that exerts a number of well characterized biological actions on
fibroblasts and other cells. In the present study, we investigated the
possibility that LPA activates the transcription factor NF- Lysophosphatidic acid
(LPA1;
1-acyl-2-lyso-sn-glycero-3-phosphate) is a naturally
occurring, water-soluble glycerophospholipid that exhibits striking
hormone- and growth factor-like activities (1, 2). Synthesized and
released by platelets, LPA represents a major bioactive constituent of
serum, and its actions on fibroblasts, endothelial cells, and smooth
muscle cells in particular suggest roles in wound healing among other
events. Indeed, LPA acts on a large number of cells to achieve a broad
range of immediate and long lasting effects. Specific responses to LPA
include changes in cell shape and tension, chemotaxis, proliferation,
and differentiation.
The molecular actions of LPA have been characterized best in rodent
fibroblasts, where at low concentrations (i.e. 10-100 nM) LPA stimulates phosphoinositide hydrolysis (3) and
promotes the Rho-dependent formation of stress fibers and
focal adhesions (4). The stimulation of phosphoinositide hydrolysis is
thought to occur through the GTP-binding regulatory protein (G protein) Gq. The formation of stress fibers and focal adhesions is
consistent with activation of Rho through G12 or
G13 (5). One or a combination of these G proteins is also
responsible for the protein tyrosine phosphorylation elicited by LPA
(6-8). LPA additionally inhibits adenylyl cyclase, an action achieved
through the pertussis toxin (PTX)-sensitive G protein Gi
(9). LPA uses Gi, moreover, to activate Ras, Raf, and the
extracellular signal-regulated kinases (ERKs) ERK1 and ERK2 (10, 11).
The activation of ERKs (11), and presumably the inhibition of adenylyl
cyclase (3), occurs at concentrations of LPA comparable with those
stimulating phosphoinositide hydrolysis and cytoskeletal changes. At
higher concentrations (i.e. 5-70 µM), LPA
promotes reinitiation of DNA synthesis in quiescent fibroblasts (9).
Whether G proteins are sufficient for this action is unclear, but the
sensitivity of the phenomenon to PTX implies that Gi
represents at least one necessary input. The need for high
concentrations of LPA in this context may relate to a requirement for
more persistent signaling and/or engagement of other receptors or
pathways. Micromolar concentrations of LPA also promote arachidonic
acid formation, a second phase of inositol phosphate accumulation
(PTX-insensitive), and activation of serum response factor (9, 12, 13).
Receptors that recognize LPA are poorly characterized; however, several
have been identified that conform to the seven-transmembrane domain
motif characteristic of G protein-coupled receptors (GPCRs)
(14-17).
NF- The binding of agonists to certain GPCRs promotes activation of
NF- Reagents--
L- Cell Culture--
Swiss 3T3 mouse embryo fibroblasts (a gift
from Dr. E. Rozengurt, Imperial Cancer Research Fund, London, UK) were
maintained at 37 °C under a humidified atmosphere of 10%
CO2 in Dulbecco's modified Eagle's medium containing 10%
(v/v) fetal calf serum, supplemented with penicillin (100 units/ml) and
streptomycin (100 µg/ml). For all experiments, 1 × 106 cells were subcultured into 10-cm tissue culture plates
(Nunc). After 48 h, the medium was replaced with 6 ml of
Dulbecco's modified Eagle's medium containing 1% fetal calf serum
and antibiotics, and the cells were incubated for an additional 18 h. LPA (prepared as a stock of 1 mg/ml in phosphate-buffered saline
containing 10 mg/ml essentially fatty acid-free bovine serum albumin
(Sigma)) and/or other reagents or vehicles were added to achieve the
concentrations specified.
Nuclear Extract Preparation--
Nuclear extracts were prepared
by the method of Dignam et al. (24) with minor
modifications. Following incubation with LPA or other reagents, cells
were washed twice in ice-cold phosphate-buffered saline, harvested, and
resuspended in 400 µl of hypotonic buffer (10 mM HEPES
(pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride). After 10 min on ice, 30 µl of Nonidet P-40 (10% (v/v)) was added with mixing for 2 s.
The nuclei were pelleted by centrifugation at 20,000 × g for 10 s. The supernatant was removed, and the nuclei were resuspended in hypertonic buffer (20 mM HEPES (pH
7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM
EGTA, 0.1 mM phenylmethylsulfonyl fluoride) and shaken for
45 min at 4 °C. The samples were centrifuged at 20,000 × g for 30 s, and the supernatants (nuclear extracts)
were saved. Protein concentrations were determined using the Bradford method (Bio-Rad).
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assays were performed using a double-stranded
oligonucleotide containing a consensus Western Blot Analysis--
Following exposure to LPA with or
without cycloheximide as specified, cells were washed with ice-cold
phosphate-buffered saline and lysed in 10 mM Tris-HCl (pH
7.6), 5 mM EDTA, 50 mM NaCl, 30 mM
sodium pyrophosphate, 50 mM NaF, 100 mM
Na3VO4, 0.5% Triton X-100, 10 mg/ml leupeptin,
10 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride.
Lysates were clarified by centrifugation at 20,000 × g
for 15 min at 4 °C. Supernatants were collected and subjected to
SDS-polyacrylamide gel electrophoresis (11% acrylamide). Protein was
transferred to nitrocellulose membrane and probed with polyclonal rabbit anti-human I The possibility that LPA activates the transcription factor
NF-B.
NF-
B is a target of cytokines, but its activation by other classes
of agonists has raised considerable interest in the control of
processes such as inflammation and wound healing through varied
mechanisms. We find that LPA causes a marked activation of NF-
B in
Swiss 3T3 fibroblasts as determined by the degradation of I
B-
in
the cytosol and the emergence of
B binding activity in nuclear
extracts. The EC50 for activation of NF-
B is 1-5
µM, a range similar to that reported for reinitiation of
DNA synthesis and activation of the serum response element. Activation
of NF-
B is attenuated by pertussis toxin and inhibitors of protein
kinase C, and it is completely blocked by the Ca2+ chelator
BAPTA-AM. The combination of phorbol ester and thapsigargin promotes an
activation comparable with that of LPA. Activation by LPA is
additionally inhibited by tyrphostin A25 but not genistein or AG1478,
indicating a selective utilization of protein-tyrosine kinases, and by
certain antioxidants, implying a role for reactive oxygen species. The
activation is also inhibited by tricyclodecan-9-yl-xanthogenate (D609),
implying a requirement for hydrolysis of phosphatidylcholine. The data
demonstrate the utilization of multiple pathways in the activation of
NF-
B by LPA, not inconsistent with the relevance of several families
of GTP-binding regulatory proteins.
INTRODUCTION
Top
Abstract
Introduction
References
B (nuclear factor-
B) is the prototype of a family of dimers
whose constituents are members of the Rel family of
transcription factors (18). In most types of cells, NF-
B is present
as a heterodimer comprising p50 (NF-
B1) and p65 (RelA). NF-
B is
normally retained in the cytosol in an inactive form through
interaction with I
B inhibitory proteins. Release of NF-
B for
translocation to the nucleus and interaction with cognate DNA sequences
is accomplished through a signal-induced phosphorylation and subsequent
degradation of I
B. Originally described as a necessary element for
expression of the immunoglobulin
gene in mature B cells, NF-
B is
now recognized to be an important transcriptional regulatory protein in
a variety of cell types (19).
B. Agonists include serotonin (working through the
5-HT1A receptor) (20), platelet-activating factor (21),
thrombin (22), and bradykinin (23). That GPCRs are linked to NF-
B is
particularly significant, since these receptors are widely distributed,
the actions of NF-
B are notable, and the coincident activation of
NF-
B and other GPCR-regulated transcription factors can provide
unique forms of transcriptional regulation. Because LPA exerts a wide
range of actions in part or entirely through GPCRs, and because NF-
B
is especially relevant to inflammation and wound healing, we instituted
efforts here to understand whether LPA promotes the activation of
NF-
B. We explored the possible relationship between LPA and NF-
B
in fibroblasts and the mechanisms by which this relationship is established.
EXPERIMENTAL PROCEDURES
-Lysophosphatidic acid
(C18:1,[cis]-9), cycloheximide, ascorbic acid,
pyrrolidinedithiocarbamate, and dimethyl sulfoxide were obtained from
Sigma. Phorbol-12-myristate-13-acetate (PMA), calphostin C, Ro 31-8220, bisindolylmaleimide I, tyrphostins A25 and AG1478,
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), thapsigargin,
N-acetylcysteine, and diphenyleneiodonium were obtained from
Calbiochem. Dithiothreitol was obtained from Boehringer Mannheim.
Tricyclodecan-9-yl xanthogenate (D609) was obtained from Biomol
Research Laboratories (Plymouth Meeting, PA) or Sigma. TNF
was
obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Potassium
ethylxanthate was obtained from Aldrich. The double-stranded
oligonucleotide conforming to 5'-AGTTGAGGGGACTTTCCCAGGC-3' was obtained
from Promega Corp. (Madison, WI), and those conforming to
5'-AGTTGAGGCGACTTTCCCAGGC-3' and 5'-ATTCGATCGGGGCGGGGCGAGC-3' and the
antibody toward p65 (RelA) were obtained from Santa Cruz Biotechnology,
Inc. (Santa Cruz, CA). [
-32P]ATP was obtained from NEN
Life Science Products. Electrophoretic reagents were obtained from
Bio-Rad.
B binding site
(5'-AGTTGAGGGGACTTTCCCAGGC-3'; the underlined sequence
represents the consensus
B region), which was end-labeled with
[
-32P]ATP and T4 polynucleotide kinase. Nuclear
extracts (2.5 µg of protein) were incubated in 10 mM
Tris-HCl (pH 7.9), 50 mM NaCl, 1 mM EDTA, 10%
glycerol, 0.15 mg/ml poly(dI-dC), and 20-30 fmol of
32P-labeled oligonucleotide (50,000-100,000 cpm) in a
total volume of 15 µl at room temperature for 10 min. The reaction
mixture was then subjected to electrophoresis in a 5% polyacrylamide
slab gel containing 50 mM Tris, 380 mM glycine,
and 2 mM EDTA, pH 8. The gels were dried under vacuum for
analysis by autoradiography (overnight exposure) or PhosphorImager
analysis. For competition studies, nuclear extracts were incubated
prior to the addition of labeled oligonucleotide for 10 min at room
temperature with unlabeled oligonucleotide, unlabeled oligonucleotide
containing a G
C substitution in the
B binding motif
(5'-AGTTGAGGCGACTTTCCCAGGC-3'), or an unlabeled
oligonucleotide containing the consensus binding site for Sp1
(5'-ATTCGATCGGGGCGGGGCGAGC-3'). For supershift analysis, nuclear
extracts were incubated with approximately 2 µg of antibodies specific for p65 (RelA) or nonimmune goat IgG for 30 min at 4 °C in
the presence of radiolabeled oligonucleotide prior to electrophoresis. The results shown in all figures are representative of at least three experiments.
B-
(0.4 mg/ml) detected subsequently by
chemiluminescence using a donkey anti-rabbit IgG conjugated with
horseradish peroxidase and luminol as recommended by the manufacturer
(ECL Western; Amersham Pharmacia Biotech).
RESULTS
B was investigated in Swiss 3T3 fibroblasts, which have been used
extensively in studies of agonists, including LPA, linked to changes in
cell morphology and reinitiation of DNA synthesis (4, 25, 26). We
examined first the extent to which LPA promotes degradation of
I
B-
, an inhibitory protein whose proteolysis would precede the
translocation of NF-
B to the nucleus. As shown in Fig.
1 (left panel), LPA
caused a transient degradation of this protein. Levels of I
B-
decreased slowly following introduction of LPA, reaching a minimum at
40-60 min, and increased thereafter to near control values. The
time-dependent resynthesis of I
B-
is a common finding
in cytokine action (27) and appears to reflect activation of the
I
B-
gene by NF-
B as part of a feedback loop (28). To
circumvent resynthesis of I
B-
, we also evaluated degradation of
this protein in the presence of cycloheximide. As expected, degradation
of I
B-
under this condition, where protein synthesis is blocked,
was complete.
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Fig. 1.
LPA-induced degradation of
I B-
. Swiss 3T3
fibroblasts were preincubated without (left) or with
(right) 50 µg/ml cycloheximide for 60 min and then with 40 µM LPA for the times indicated. Cell extracts were
prepared and analyzed for I
B-
by Western blotting. This
experiment is representative of several similar experiments.
A more direct evaluation of NF-B activation was carried out by
electrophoretic mobility shift assays. The data in Fig.
2 demonstrate that LPA promotes a time-
and concentration-dependent appearance of a factor(s)
within nuclear extracts that binds to an oligonucleotide probe
containing an NF-
B binding site. The proinflammatory cytokine TNF
also promotes the appearance of this factor. The relevant protein-DNA
complex was evident as a band of radioactivity (denoted by an
arrow) positioned above two less prominent bands. This band,
but not the other two, was supershifted with a p65 (RelA)-directed
antibody (Fig. 3, top
panel), confirming the identity of the induced factor as
NF-
B. The nature of the protein-DNA interaction was evaluated
further in competition experiments (Fig. 3, bottom
panel), where the 32P-labeled oligonucleotide
was found to be displaced by unlabeled oligonucleotide. The same
unlabeled oligonucleotide, but containing a mutation in the
B site,
and an altogether unrelated oligonucleotide (containing an Sp1 binding
site) did not displace the 32P-labeled oligonucleotide. The
EC50 for LPA based on the intensity of the shifted band was
1-5 µM, and the time required for full development of
the response was 40-60 min (Fig. 2). The response was transient, as
the level of shifted oligonucleotide began to decrease by 3 h (not
shown).
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The extent to which the G protein Gi might contribute to
the activation of NF-B was assessed with PTX, which suppresses
activation of Gi by GPCRs. Pretreatment of cells with PTX
attenuated LPA-induced activation of NF-
B by approximately 60%
(Fig. 4, top
panel). Efforts to enhance the attenuation by manipulating
pretreatment time and concentration of PTX were unsuccessful. That the
actions of LPA typically assigned to Gi are not completely
suppressed by PTX, for example ERK activation and reinitiation of DNA
synthesis, is not without precedent (9-11).
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A downstream target for both Gi and Gq is the
phosphoinositide-specific phospholipase C-, whose activation
results in recruitment of PKC and mobilization of Ca2+.
Overnight treatment of cells with a high concentration of PMA (1 µM) to induce down-regulation of classical and novel
forms of PKC suppressed activation of NF-
B by 70-80% (not shown),
as did Ro-31-8220 (Fig. 4, middle panel), which
inhibits all forms of PKC. Other inhibitors of PKC, including
bisindolylmaleimide I (not shown) and calphostin C, inhibited
activation of NF-
B nearly as well. Activation of NF-
B was
completely suppressed by pretreatment of cells with the cell-permeable
Ca2+ chelator BAPTA-AM (Fig. 4, bottom
panel).
Given the apparent requirements for PKC and intracellular
Ca2+, we tested whether the activation of PKC and/or
mobilization of Ca2+ might be sufficient to activate
NF-B. Some degree of activation was achieved with PMA, but not the
extent observed with LPA (Fig. 5). Only a
small degree of activation was achieved with thapsigargin, moreover, an
inhibitor of the endoplasmic reticular Ca2+-ATPase that
causes a time-dependent increase in cytosolic
Ca2+. However, the combination of thapsigargin and PMA
activated NF-
B ultimately to an extent somewhat greater than that
achieved with LPA.
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Because considerable attention has been devoted to the utilization of
the EGF receptor and other tyrosine kinases by GPCRs, including the one
or more receptors that mediate the actions of LPA (29, 30), we tested
inhibitors of different tyrosine kinases for their effect on
LPA-induced activation of NF-B. Genistein, which inhibits a wide
range of tyrosine kinases, was without effect (Fig.
6). Tyrphostin AG1478, a relatively
specific inhibitor of the EGF receptor tyrosine kinase, caused some
degree of inhibition but only at very high concentrations (16 µM is shown; concentrations normally used to inhibit the
EGF receptor are 0.125-1 µM (29, 31)). At concentrations
less than 5 µM, AG1478 had no effect. In contrast,
tyrphostin A25, like genistein regarded as a general inhibitor of
tyrosine kinases, achieved a significant degree of inhibition at 25 and
50 µM and complete inhibition by 150 µM
(not shown; 150 µM is a commonly employed concentration
(31, 32)).
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Chen et al. (33) demonstrated that LPA stimulates reactive
oxygen species production in HeLa cells and that antioxidants inhibit
LPA-stimulated MAP kinase kinase activity. We therefore evaluated the
effects of antioxidants on the activation of NF-B. As shown in Fig.
7, N-acetylcysteine completely
inhibited LPA-induced activation of NF-
B. Pyrrolidinedithiocarbamate
was similarly effective. Dimethyl sulfoxide achieved a less extensive,
but still notable, inhibition. Ascorbic acid and dithiothreitol were
without effect. The activation of NF-
B was also highly sensitive to
diphenyleneiodonium, an inhibitor of flavanoid-containing enzymes such
as NADPH oxidase.
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The tricyclodecan xanthogenate D609 inhibits the hydrolysis of
phosphatidylcholine at the level of a phosphatidylcholine-specific phospholipase C-like enzyme and/or phospholipase D (34-36), among perhaps other actions (37), and has been used to explore signaling pathways utilized by growth factors, TNF, and GTPase-deficient G
protein subunits. Schütze et al. (35), for example,
found that D609 inhibits the activation of NF-B by TNF
, and
Wadsworth et al. (38) demonstrated that this compound
inhibits Na+/H+ exchange stimulated by
12. We found here that D609 inhibits quite effectively
the activation of NF-
B by LPA (Fig.
8). The EC50 was about 3 µg/ml, and the maximum degree of inhibition was greater than 90%.
Potassium ethylxanthate had no effect. D609 was not nearly as potent an
inhibitor of TNF
's activation of NF-
B as it was of LPA's.
Inhibition in the case of TNF
occurred only at concentrations of
D609 exceeding 50 µg/ml. The activation of NF-
B by LPA was thus
selectively inhibited by D609.
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DISCUSSION |
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That proinflammatory cytokines such as interleukin-1 and TNF
activate NF-
B has long been appreciated, and the sequence of events
by which the activation occurs is now emerging (39, 40). Of perhaps
equal significance, however, is the fact that agonists working through
GPCRs can also activate NF-
B (20-23). In the work described here,
we have focused on LPA. LPA has a rich and important biology; it
induces a number of cells to proliferate and others to differentiate
and, at a molecular level, works through one or more G proteins to
stimulate MAP kinases, phospholipid metabolism, and cytoskeletal
rearrangement. Activation of NF-
B clearly constitutes an additional
action of LPA, and one to be reconciled in any setting of transcription
relevant to this agonist.
LPA triggers a pronounced activation of NF-B, as ascertained by the
degradation of I
B-
and the emergence of
B binding activity in
nuclear extracts. Based on supershift experiments, the activated form
of NF-
B contains the p65 (RelA) subunit. The other component may be
p50 (NF-
B1), given the widespread occurrence of NF-
B as a p50-p65
heterodimer, but this remains to be determined. Dimeric complexes of
NF-
B that contain p65 function as strong activators of gene
expression (41). The transience of NF-
B activation suggests that
I
B-
alone of the inhibitory proteins is the target of LPA's
action (28).
The concentration of LPA supporting activation of NF-B
(EC50 = 1-5 µM) is higher than that reported
to inhibit adenylyl cyclase (3), activate ERKs (10), elevate
IP3 and Ca2+ (3), and stimulate formation of
stress fibers and focal adhesions (4) but is similar to that needed for
activation of serum response factor (12), initiation of a "second"
phase of inositol phosphate production (9), and reinitiation of DNA
synthesis (9). It is tempting to speculate that activation of NF-
B
by LPA, like that of serum response factor, may function as a
counterpart to proliferative signaling. Mayo et al. (42)
have reported that activation of NF-
B by oncogenic Ras is required
for progress toward cell transformation and, in particular, that
NF-
B prevents a Ras-induced apoptosis that would otherwise abrogate
transformation. It is not implausible that mitogens such as LPA, as
they drive replication, similarly use NF-
B to foil any tendency of
the cell to enter into an apoptotic program. Alternatively, the
activation of NF-
B may play a more direct role in proliferative
signaling, for example through activation of genes immediately relevant
to DNA synthesis.
Because the roles of G proteins in the phenomena induced by LPA are not
completely established, a basic question is whether LPA utilizes G
proteins to achieve its activation of NF-B. That PTX attenuates
activation of NF-
B would suggest a role for Gi. The
participation of this G protein would not be surprising; NF-
B can be
activated by oncogenic Ras (43), and both Ras and ERKs are activated by
LPA through a pathway(s) at least partly sensitive to PTX (10, 11, 44).
pp90rsk1 lies downstream of ERKs and has been implicated in the
phosphorylation of I
B-
(45, 46). Yet, the activation of
Gi would seem to occur at concentrations of LPA lower than
those required for activation of NF-
B (3, 10, 11). Thus, while
Gi may contribute to the activation of NF-
B, it may not
provide a sufficient stimulus. The actions of PTX, moreover, must be
qualified, since PTX conceivably affects the activation of NF-
B by
LPA indirectly, for example through elevations in cAMP or through
actions not related specifically to ADP-ribosylation.
Gq may well provide an additional and/or independent input.
As implied above, the stimulation of inositol phosphate production by
LPA occurs in two phases (3, 9). Some production of inositol phosphates
is observed at low concentrations of agonist, but the preponderance is
achieved at micromolar concentrations. The latter (at least) is
insensitive to PTX (9). It is conceivable that low concentrations of
LPA activate Gi and that higher concentrations activate
Gq. Gq may be responsible for the activation of
protein kinase C and mobilization of Ca2+ of sufficient
magnitude and/or duration to bring, together with signals from
Gi, activation of NF-B to fruition. That the combination of PMA and thapsigargin achieves activation of NF-
B supports the
notion that Gq might even be sufficient. However, PMA and thapsigargin would exert more potent and long lasting actions than
those achieved by agonist-activated Gq. With respect to the involvement of PKC, we have used both down-regulation and several pharmacological inhibitors to implicate a role for this enzyme(s). That
Ro-31-8220 can activate JNK is a potentially confounding issue, but
bisindolylmaleimide I lacks this attribute (47).
The possibility that Rho is engaged by G12 or
G13 in the activation of NF-B by LPA is most intriguing.
LPA appears to activate Rho (4), and Rho when overexpressed as a
wild-type or constitutively active protein activates NF-
B (48). The
activation of Rho by LPA no doubt proceeds through G12
and/or G13. The GTPase-deficient forms of
12
and
13, for example, cause a Rho-dependent
formation of stress fibers and focal adhesions, as does LPA (5).
Labeling of G proteins with [32P]GTP azidoanilide,
moreover, reveals activation of G12 and G13, together with Gi and Gq, by LPA (31).
Tyrphostin A25 has been demonstrated previously to prevent the
induction of stress fiber and focal adhesion assembly by the
GTPase-deficient form of
13 (31), as it does that by LPA
at a step upstream from Rho (32). Of interest, we find here that A25
prevents LPA-induced activation of NF-
B. Thus, the activation of
NF-
B by LPA may well involve an A25-sensitive activation of Rho. Rho
has recently been demonstrated to be required in the activation of
NF-
B by bradykinin in human epithelial cells (49). How a
Rho-dependent pathway integrates with those pathways
sensitive to inhibitors of PKC and intracellular Ca2+
fluxes, D609, and PTX remains to be determined. In a departure from the
results presented for cytoskeletal changes (31), we do not have firm
evidence for the utilization of the EGF receptor by LPA. AG1478, an
inhibitor of EGF receptor autophosphorylation, had only a modest impact
at high concentrations on the activation of NF-
B. Genistein, which
would block phosphorylation of the receptor by Src among other kinases,
had no effect.
The relevance of reactive oxygen intermediates to the activation of MAP
kinases and NF-B has generated considerable interest (33, 50-52).
N-Acetylcysteine has been used extensively in this regard
and was found here to effectively inhibit the activation of NF-
B by
LPA, as did pyrrolidinedithiocarbamate and dimethyl sulfoxide. The
nature and source of the reactive oxygen species is unclear, although
the inhibition achieved with diphenyleneiodonium might suggest the
involvement of NADPH oxidase. Of interest, several reports find
reactive oxygen species to be involved in activating components of the
ERK activation cascade, ostensibly through phosphorylation of the EGF
receptor (50, 53). For reasons outlined above, we suspect that the EGF
receptor is not a participant in the activation of NF-
B, and we know
that ERK is activated at concentrations of LPA well below those
required for activation of NF-
B. It is nevertheless intriguing that
the reactive oxygen species-dependent phosphorylation of
the EGF receptor reported in HeLa cells is implied to occur at only
higher (i.e. micromolar) concentrations of LPA (53).
Perhaps, then, the formation of reactive oxygen species will emerge as
the most closely correlated requirement for NF-
B activation, SRF
activation, and reinitiation of DNA synthesis.
The activation of NF-B by LPA is exceptionally sensitive to
inhibition by the tricyclodecan xanthogenate D609. Sensitivity to this
compound has been used in other contexts to suggest the relevance of a
phosphatidylcholine-specific phospholipase C (34, 35). More recent
reports suggest that phospholipase D, too, can be inhibited by D609
(36, 54), perhaps at higher concentrations. Our use of D609 was
prompted by the observation that D609 inhibits the activation of
NF-
B by TNF
(35), as it does that of sphingomyelinase and of Raf
by oncogenic Ras (34). We found that D609 indeed inhibited the
activation of NF-
B by TNF
but only at high concentrations (>50
µg/ml), consistent with the observation of Schütze et
al. (35). In contrast, the amount of D609 required to inhibit
activation of NF-
B by LPA was considerably lower; almost complete
suppression could be achieved at concentrations less than 10 µg/ml.
D609 at these lower concentrations has been argued to inhibit a
phosphatidylcholine-specific phospholipase C-like activity selectively
(36). The most obvious contribution of a phosphatidylcholine-specific
phospholipase C would be the activation of Raf (34) and hence ERKs and
pp90rsk by LPA-activated Ras. However, low concentrations of
D609, perhaps through inhibition of a phosphatidylcholine-specific
phospholipase C but conceivably other means, can cause
hyperphosphorylation and concomitant release of Raf from membrane as a
form of negative modulation (37). We therefore cannot rule out the
possibility that D609 has actions beyond phosphatidylcholine
hydrolysis. It is of considerable interest that
12,
which can activate Rho, utilizes a D609-sensitive step in the
regulation of Na+/H+ exchange (38). The
possibility therefore exists that
12 utilizes the same
step in the activation of Rho, Raf, and/or PKCs (55), leading to
activation of NF-
B.
Whether any or all of the receptors for LPA so far cloned mediate the
activation of NF-B represents a subject of interest. Receptors
include PSP24, Edg2 (Vzg-1), and Edg4 (14-17). Edg2 and Edg4, at
least, are linked to the activation of the serum response element (16,
17), and the activation through Edg4 was demonstrated to be partly
sensitive to PTX and to the inhibition of Rho. Edg2 and Edg4 therefore
represent reasonable candidates for linking LPA to NF-
B. Regardless,
the activation of NF-
B, together with the means by which the
activation is coordinated with other signals elicited by LPA
(e.g. activation of ERK and serum response factor), must now
be considered in any response to LPA of significant duration and/or
explicitly involving gene expression.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM51196.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology,
University of Pennsylvania School of Medicine, 3620 Hamilton Walk,
Philadelphia, PA 19104-6084. Tel.: 215-898-1775; Fax: 215-573-2236; E-mail: manning{at}pharm.med.upenn.edu.
The abbreviations used are:
LPA, lysophosphatidic acid; ERK, extracellular signal-regulated kinase; G protein, GTP-binding regulatory protein; GPCR, G protein-coupled
receptor; PKC, protein kinase C; PMA, phorbol-12-myristate-13-acetate; PTX, pertussis toxin; TNF, tumor necrosis factor-
; EGF, epidermal
growth factor; D609, tricyclodecan-9-yl-xanthogenate; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetra(acetoxymethyl) ester.
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