(Received for publication, August 1, 1995; and in revised form, October 16, 1995)
From the
The abilities of platelet-derived growth factor (PDGF) and
insulin-like growth factor (IGF-I) to regulate cAMP metabolism and
mitogen-activated protein kinase (MAP kinase) activity were compared in
human arterial smooth muscle cells (hSMC). PDGF-BB stimulated cAMP
accumulation up to 150-fold in a concentration-dependent manner
(EC
0.7 nM). The peak of cAMP formation and
cAMP-dependent protein kinase (PKA) activity occurred approximately 5
min after the addition of PDGF and rapidly declined thereafter.
Incubating cells with PDGF and 3-isobutyl-1-methylxanthine (IBMX, a
phosphodiesterase inhibitor) enhanced the accumulation of cAMP and PKA
activity by an additional 2.5-3-fold, whereas IBMX alone was
essentially without effect. The PDGF-stimulated increase in cAMP was
prevented by addition of the cyclooxygenase inhibitor indomethacin,
consistent with release of prostaglandins stimulating cAMP. PDGF, but
not IGF-I, stimulated MAPK activity, cytosolic phospholipase
A
(cPLA
) phosphorylation, and cAMP synthesis
which indicated a key role for MAP kinase in the activation of
cPLA
. Further, PDGF stimulated the rapid release of
arachidonic acid and synthesis of prostaglandin E
(PGE
) which could be inhibited by a cPLA
inhibitor (AACOCF
). Calcium mobilization was required
for PDGF-induced arachidonic acid release and PGE
synthesis
but not for MAPK activation, whereas PKC was required for
PGE
-mediated activation of PKA. In summary, these results
demonstrate that PDGF increases cAMP formation and PKA activity through
a MAP kinase-mediated activation of cPLA
, arachidonic acid
release, and PGE
synthesis in human arterial smooth muscle
cells.
The proliferation of arterial smooth muscle cells is a key event
in the formation and progression of lesions of atherosclerosis and in
restenosis following angioplasty. This proliferation is most likely
initiated and regulated by growth factors, such as the platelet-derived
growth factors, PDGF-AA, ()PDGF-BB, and PDGF-AB. In
atherosclerotic lesions, a major source of PDGF-BB is activated
macrophages, although smooth muscle, endothelial cells, and other cells
can also express and secrete PDGF dimers (reviewed in (1) ).
Many growth factor receptors, including PDGF receptors, activate a
signal transduction pathway that includes conversion of inactive
RasGDP to active Ras
GTP, activation of Raf, MAP kinase
kinase (MAPKK or MEK(2) ), and MAP kinase (MAPK) (for review,
see (3) ). Activation of the MAPK cascade results in the
stimulation of DNA synthesis and cell proliferation, which can be
inhibited by expression of a dominant-negative
MAPKK(4, 5) . Conversely, expression of a
constitutively active form of MAPKK can stimulate cell proliferation
and transformation(4, 6) . A number of nuclear and
non-nuclear proteins have been identified as substrates for MAPK. Among
the latter is phospholipase A
, thereby providing a
potential link to arachidonic acid metabolism (reviewed in (7) ).
Activation and translocation of the cytosolic 85-kDa
phospholipase (cPLA), which catalyzes the release of
arachidonic acid from the sn-2 position of phospholipids in
the plasma membrane, is an important signal leading to prostaglandin
synthesis (reviewed in (8) and (9) ). This enzyme can
be distinguished from the low molecular weight forms of PLA
by insensitivity to disulfide-reducing agents or inhibition by
arachidonic acid analogues (for reviews, see (10) and (11) ). Regulation of cPLA
appears to be critically
dependent on the integration of multiple signals, including
intracellular calcium, protein kinase C (PKC), and phosphorylation by
MAP kinase(12, 13, 14, 15) .
However, in some cells, calcium-independent forms of cPLA
(16) and PKC-independent mechanisms of cPLA
activation have also been observed (15) .
Although the coupling of hormonal and neurotransmitter receptors to cAMP synthesis is well established (reviewed in (17) ), the means by which growth factor receptor tyrosine kinases (e.g. the PDGF receptor) regulate cAMP metabolism is less well understood. At present, there are few examples of growth factors stimulating cAMP accumulation. Before elucidation of the MAP kinase cascade, it was reported that PDGF (in the presence of phosphodiesterase inhibitors) increased cAMP synthesis 6-8-fold in Swiss 3T3 cells(18) . In perfused rat hearts, epidermal growth factor (EGF) was found to stimulate cAMP accumulation (19) . In epithelial cells overexpressing the EGF receptor (A431 cells), EGF alone did not affect cAMP accumulation, but did potentiate an increase in cAMP in response to cAMP-elevating agents(20) .
Sustained elevation of cAMP inhibits the proliferation of many cell types, including smooth muscle cells(21) . This phenomena may in part be explained by the fact that in many cell types, including human arterial SMC (hSMC), MAP kinase activation is inhibited by the cyclic AMP-dependent protein kinase (PKA)(22, 23, 24, 25, 26) . The target for inhibition by PKA in the MAP kinase pathway may be Raf-1(23, 27) , although other unidentified targets are also likely to play an important role(28) .
To further
evaluate how growth regulatory molecules may regulate cAMP metabolism
in hSMC, we examined the effect of two major factors known to influence
hSMC (i.e. PDGF and IGF-I). We report here that PDGF rapidly
stimulates cAMP synthesis through a mechanism requiring intracellular
calcium, PKC activity, MAPK-dependent phosphorylation of
cPLA, and activation of prostaglandin synthesis, an effect
which culminates in increased PKA activity.
Figure 1:
PDGF-BB and PDGF-AA, but not IGF-I,
increase cAMP and activate PKA. Human SMC were incubated in 10-cm
plates containing DMEM plus 1% PDS for 48 h. A shows the time
course of cAMP formation by 1 nM PDGF-BB () and 1
nM IGF-I (
) and PKA activation by 1 nM PDGF-BB
(
). Samples were rapidly harvested, and the cAMP concentration
was measured in triplicate by radioimmunoassay whereas PKA activity was
measured as phosphorylation of Kemptide in the presence or absence of
the PKA inhibitor PKI. B shows dose-response curves for
PDGF-BB (
), PDGF-AA (
), and IGF-I (
) on cAMP
formation. The results are shown as mean ± S.E. of triplicate or
duplicate samples. These experiments were repeated 3 times with similar
results.
The
stimulation of cAMP accumulation was dose-dependent, with up to a
150-fold increase over basal levels (10-50 pmol/ml) seen at the
highest concentration (10 nM) of PDGF-BB (Fig. 1B). The PDGF isoform, PDGF-AA, also increased
cAMP levels in hSMC, although not to the same extent as PDGF-BB. This
is likely a reflection of the 10-fold lower number of PDGF receptor
subunits compared to PDGF receptor
-subunits in these
cells(32) . Interestingly, despite the fact that hSMC also
contain functional IGF-I receptors (similar in number to PDGF-
receptors), IGF-I, at concentrations as high as 10 nM, did not
increase cAMP synthesis, even in the presence of the phosphodiesterase
inhibitor IBMX (Fig. 1, A and B, and data not
shown).
Figure 2: PDGF-BB-induced cAMP formation, but not MAPK activation is dependent on calcium mobilization and PKC. Human SMC in 10-cm plates were incubated for 5 min with 1 nM PDGF-BB, 1 nM IGF-I or for 20 min with 100 nM PMA. In some cells, PKC was down-regulated by a 20-h preincubation with 1 µM PMA, or intracellular calcium stores were depleted by a 1-h preincubation with 300 nM thapsigargin. Levels of cAMP (A) were measured by the cAMP immunoassay as in Fig. 1, and MAPK activity (B) was measured as phosphorylation of MBP for 15 min at 30 °C. These results are shown as mean ± S.E. of duplicate samples. These experiments were repeated twice with similar results.
Since calcium is a key
second messenger in the regulation of cAMP metabolism in many cell
types (reviewed in (35) and (36) ), we investigated
whether calcium was required for the stimulation of cAMP synthesis by
exposing cells to the tumor promoter, thapsigargin. Thapsigargin
stimulates the release of calcium from intracellular stores, resulting
in an initial increase of intracellular calcium and later (at
approximately 1 h), as the calcium is transferred to the extracellular
space, a depletion of calcium from intracellular stores (37) .
Addition of PDGF-BB to hSMC rapidly increases intracellular calcium, an
event that can be inhibited by prior incubation with 300 nM thapsigargin for 1 h. ()Preincubation with thapsigargin
(300 nM, 1 h) eliminated the synthesis of cAMP stimulated by
PDGF-BB, demonstrating a requirement for intracellular calcium in this
process (Fig. 2A). The effect of this compound on the
activation of MAPK by PDGF was examined. Neither an extended (1-h) (Fig. 2B) nor a brief (5-min) (data not shown) exposure
of cells to thapsigargin affected the basal or PDGF-stimulated levels
of MAP kinase activity in these cells (Fig. 2B).
However, a brief incubation with thapsigargin (5 min) did increase PKA
activity above basal levels (thapsigargin, 404 ± 16 pmol/min/ml;
basal, 80 ± 2 pmol/min/ml) and cAMP accumulation (data not
shown).
Protein kinase C (PKC) has also been implicated in the regulation of cAMP formation in many cell types (reviewed in (36) ). Therefore, the requirement for protein kinase C (PKC) activity in PDGF-stimulated cAMP synthesis was investigated. Incubating hSMC with PMA (1 µM, 20 h) resulted in the complete loss of PMA-stimulated MAPK and MAPKK activity and in keeping with a known effect of this procedure for down-regulation of PKC activity (data not shown). Down-regulation of PMA-sensitive PKC activity inhibited the PDGF-stimulated increase in cAMP by >90% (Fig. 2A), without a significant influence on the activation of MAPK by the growth factor (Fig. 2B). Similarly, addition of the PKC inhibitor (1 µM), bisindoylmaleimide (GF109203X) prevented the formation of cAMP by PDGF without inhibiting that stimulated by forskolin (data not shown). Interestingly, PMA alone did not increase cAMP accumulation, although it did activate MAP kinase as expected (Fig. 2B).
Figure 3:
PDGF stimulates arachidonic acid- and
PGE release via a cPLA
and calcium-dependent
mechanism. Human SMC in 6-well plates were incubated in DMEM with 1%
PDS for 48 h. In A, the cells were labeled with 1 µCi/ml
[
H]arachidonic acid for the last 24 h. The cells
were washed three times with fresh DMEM without
[
H]arachidonic acid, and release of
[
H]arachidonic acid to the medium during a 5-min
stimulation with 1 nM PDGF-BB, 1 nM PDGF-AA, 1 nM IGF-I was measured. In some instances, intracellular calcium
stores were depleted by preincubation with 300 nM thapsigargin, or cPLA2 activity was inhibited by a 30-min
preincubation with 30 µM AACOCF
. The
AACOCF
analogue AACOCH
(30 µM) was
used as a control. In B, the cells were washed 3 times with
DMEM and then stimulated with 1 nM PDGF-BB (
) or
vehicle (
, 10 mM acetic acid, 0.25% bovine serum albumin)
for the indicated periods of time. PGE
release into the
medium was measured using a PGE
enzyme immunoassay
(Amersham). The results are expressed as mean ± S.D. of
triplicate samples. The experiment was repeated twice with similar
results.
One of the major products of arachidonic
acid metabolism in hSMC is prostaglandin E2
(PGE)(38) , which is formed by the action of
cyclooxygenases on arachidonyl precursors. To investigate the potential
role of prostaglandin release in cAMP formation, hSMC were incubated
with the cyclooxygenase inhibitor, indomethacin, prior to the addition
of PDGF-BB. As seen in Table 1, indomethacin (10 µM,
30 min) completely inhibited the PDGF-stimulated increase in cAMP and
PKA activity and slightly inhibited the basal levels of cAMP.
Incubation with indomethacin did not affect the activation of MAPK,
suggesting that the effects of this compound were specific to
inhibition of cyclooxygenase activity. These results demonstrated that
prostaglandin synthesis was required for PDGF-stimulated cAMP
synthesis.
To specifically investigate whether PGE was
involved in stimulating the increase in cAMP, PGE
formation
was measured in response to PDGF. Within 1 min of addition, PDGF
increased PGE
synthesis in hSMC, which continued to
accumulate with extended exposure to PDGF (Fig. 3B).
PGE
increased PKA activity by 330% as early as 1 min after
addition (187.6 ± 0.6 to 622.6 ± 12.0 pmol/min/ml). The
peak of PKA activity occurred within 5 min of PGE
addition,
in good agreement with the ability of PDGF to rapidly increase PKA
activity through PGE
release (Table 2). PDGF-induced
PGE
release was dependent on intracellular calcium. Thus,
both arachidonic acid release and PGE
release stimulated by
PDGF were inhibited by depletion of intracellular calcium stores using
thapsigargin (Fig. 3A and data not shown).
Down-regulation of PKC inhibited the ability of PGE to
stimulate PKA activity from 6.5-fold in vehicle-treated cells to
1.4-fold in cells subjected to PKC down-regulation (Table 2). In
contrast, neither down-regulation of PKC nor addition of the PKC
inhibitor, bisindolylmaleimide, inhibited the formation of cAMP
stimulated directly by forskolin (data not shown). PKC down-regulation
did not inhibit PDGF-induced PGE
release during a 5-min
stimulation (11.1 ± 0.6 to 30.6 ± 0.7 ng of
PGE
released/10
cells subjected to PKC
down-regulation compared to 3.3 ± 0.4 to 25.7 ± 1.4 ng of
PGE
released/10
vehicle-treated cells).
Figure 4:
PDGF-induced MAPK activity phosphorylates
cPLAin vitro. Human SMC in 10-cm plates were
incubated without addition (
), with 1 nM PDGF-BB
(
) or 1 nM IGF-I (
) for 5 min. The samples were
separated on a Mono Q column, and the fractions were measured for MAP
kinase activity (A). Fractions 22, 30, 32, 34, 36, and 44 from
each sample were immunoblotted for the presence of MAPK by using an
anti-ERK antibody (B). The same fractions were incubated with
1 µg of recombinant cPLA
, and the phosphorylation of
cPLA
(C) was measured as described under
``Experimental Procedures.'' Lane C contains active
Erk2 (B) or cPLA
phosphorylated by recombinant
Erk2 (C). This experiment was repeated twice with similar
results.
We further
investigated the phosphorylation of endogenous cPLA under
conditions that led to the activation of cAMP synthesis in hSMC.
Phosphorylation of cPLA
by MAPK results in a mobility shift
on SDS-PAGE that correlates with the activation of this
enzyme(13) . Extracts of hSMC stimulated with PDGF or IGF-I
were examined by immunoblotting for cPLA
. As seen in Fig. 5, the cPLA
from untreated hSMC was a doublet
similar to the baculovirus-expressed human recombinant cPLA
standard which is partially phosphorylated in the SF9
cells(31) . Samples from PDGF-treated cells showed that
cPLA
mobility was shifted completely relative to the
untreated or IGF-I-treated samples. The lack of effect of IGF-I is
consistent with the finding that IGF-I did not activate MAPK in these
cells and that cPLA
-dependent release of arachidonic acid
was not stimulated by IGF-I.
Figure 5:
PDGF-induced phosphorylation of cPLA in hSMC. Lysates (50 µg) from hSMC treated with vehicle (lane 2), 1 nM PDGF-BB (lane 3), or 10
nM IGF-I (lane 4) for 5 min were immunoblotted using
an antibody to cPLA
as described under ``Experimental
Procedures.'' The standard human cPLA
(5 ng) was
purified from baculovirus-infected Sf9 cells (lane 1). The upper band represents the phosphorylated form of
cPLA
. The experiment was repeated twice with similar
results.
PDGF initiates a multitude of biological effects through the
activation of intracellular signal transduction pathways such as the
MAP kinase cascade, phosphatidylinositol turnover, and calcium
mobilization (reviewed in (41) ), and these effects are
believed to contribute to smooth muscle cell proliferation and directed
migration (32) . Further, changes in eicosanoid metabolism can
regulate smooth muscle cell growth and contraction through alterations
in cAMP metabolism and calcium homeostasis (reviewed in (38) ).
How these key signal transduction pathways are integrated is not
presently well understood. Because of our interest in the cross-talk
between the MAPK cascade and PKA, we investigated the effects of growth
factors on cAMP metabolism in hSMC. We found that PDGF induces a strong
and rapid formation of cAMP through a mechanism that includes MAP
kinase-mediated activation of cPLA, release of arachidonic
acid, prostaglandin PGE
, and the subsequent activation of
adenylyl cyclase. Although parts of these signaling pathways have been
described previously in other cell types, we report here the complete
conversion of a growth factor signal (i.e. PDGF) to cAMP in
primary cultures of normal diploid cells, specifically human arterial
SMC. In addition, our studies demonstrate that at least three
independent signals, i.e. calcium, PKC, and MAP kinase
activity are necessary for this event to occur. Several observations
support these concepts.
Prostaglandins, such as PGE are
produced from arachidonyl precursors and potently stimulate cAMP
formation and PKA activity in human and other smooth muscle
cells(22, 38, 42) . In the experiments
described here, the PDGF-dependent formation of cAMP required
prostaglandin synthesis, as it was completely inhibited by the
cyclooxygenase inhibitor indomethacin. Our results demonstrate that
both arachidonic acid release and PGE
formation were
stimulated by PDGF and that the rapid increase in PGE
synthesis could account for the formation of cAMP. Addition of
PGE
was sufficient to trigger PKA activation in hSMC, and
the time course of PGE
formation was consistent with the
activation of PKA elicited by PDGF. In support of this model, Rozengurt et al. (18) reported that addition of PDGF to Swiss
3T3 cells stimulated a slow, sustained formation of cAMP that was
prevented by the addition of indomethacin. However, in Swiss 3T3 cells,
addition of phosphodiesterase inhibitors was required to observe this
effect. In contrast, in hSMC, PDGF stimulates a potent increase in cAMP
and PKA activity in the absence of phosphodiesterase inhibitors.
Interestingly, cyclic AMP is a mitogen for Swiss 3T3
cells(43) , whereas this nucleotide inhibits the proliferation
of smooth muscle cells(21) .
We examined the possibility
that, in addition to effects of PDGF on prostaglandin metabolism,
alternative mechanisms could facilitate the coupling of growth factor
signals to cAMP synthesis in hSMC. We were unable to find a direct
effect of PDGF on adenylyl cyclase activity in membranes obtained from
hSMC or on the phosphorylation of the -subunit of the G-protein
G
, (
)in contrast to results obtained in
epidermal growth factor (EGF)-stimulated
cells(44, 45, 46) . Instead, our results
suggest that increased prostaglandin metabolism through the activation
of cPLA
can account for the majority of PDGF-stimulated
cAMP synthesis observed in hSMC.
Both PDGF-BB and PDGF-AA potently
stimulated MAPK activity, cPLA phosphorylation, arachidonic
acid release, and cAMP synthesis in this study. IGF-I did not influence
any of these events, despite the fact that in hSMC the number of IGF-I
receptors is equivalent to the number of PDGF-
receptors, and that
the IGF-I receptor is coupled to phosphatidylinositol turnover, calcium
mobilization, and chemotaxis(32) . In the present study, the
inability of IGF-I to elevate cAMP correlates with an absence of effect
of this growth factor on MAPK activity and cPLA
phosphorylation in hSMC. These experiments confirm the findings of
others(13, 14, 15) , demonstrating that MAPK
phosphorylation is an essential signal in the activation of
cPLA
. Recently, Sa et al. (47) reported
that, in endothelial cells, basic fibroblast growth factor stimulates a
MAPK-dependent activation of cPLA
, supporting the role for
MAPK in cPLA
regulation in other cell types.
In addition
to MAP kinase activation, PKC activity and intracellular calcium
mobilization were critical for the activation of PKA by PDGF in SMC.
Since both cPLA(13, 14, 15) and
adenylate cyclase (48, 49, 50) can be
regulated by calcium and PKC in other cell types, we investigated some
of the mechanisms responsible for this calcium and PKC dependence. PMA
did not significantly stimulate arachidonic acid release, PGE
release, or cAMP synthesis, although PMA increased both MAPK and
PKC activities as expected. Thus, activation of MAPK or PKC alone is
insufficient for stimulation of cAMP synthesis in hSMC. PKC
down-regulation blocked both PDGF-induced and PGE
-induced
PKA activation, without affecting the ability of forskolin to activate
the adenylate cyclase or the ability of PDGF to stimulate PGE
release. Together, these results suggest that PKC is required for
PGE
receptor signaling to PKA activation. Depletion of
calcium from intracellular stores blocked PDGF-induced arachidonic acid
release, PGE
release, and the subsequent PKA activation in
hSMC without inhibiting MAPK activation. Further, no effect on
PGE
-stimulated PKA activation was seen, suggesting that
inhibition of PDGF-induced PKA activation by intracellular calcium
depletion is due to inhibition of cPLA
activation. In
addition, transient increases in cAMP synthesis were observed in hSMC
when intracellular calcium levels were increased dramatically by a
short stimulation with thapsigargin or by
sphingosine-1-phosphate(51) , for example. This effect was
independent of MAPK activation, and the concentrations of intracellular
calcium required for direct cAMP stimulation were higher than those
obtained following stimulation with PDGF or IGF-I (51) . (
)Possibly, high intracellular concentrations of calcium may
directly stimulate the adenylate cyclase types I and III (reviewed in (35) ).
Previously, we reported that PKA can inhibit PDGF-stimulated MAPK signaling in hSMC(22) . Therefore, we examined whether the increase in cAMP and PKA activity in response to PDGF could inhibit the MAP kinase cascade in a ``feedback'' manner. Such a negative feedback mechanism on MAPK (Erk2) activity has recently been demonstrated by Pyne and co-workers following bradykinin-induced cAMP accumulation(52) . We were unable to find an effect of the PDGF-stimulated PKA activity on the time course of MAPKK or MAPK activation in response to PDGF, nor was the PDGF-stimulated increase in PKA activity sufficient to limit the magnitude of MAPK activation by this growth factor. At this time, we can only speculate that the PDGF-stimulated increase in PKA activity occurs too transiently to sufficiently impede the activation of MAPK in hSMC. Alternatively, the PDGF-stimulated increase in cAMP and PKA activity may be involved in PKA-mediated transcription or cytoskeletal remodeling events such as actin filament reorganization which is known to occur in response to PDGF(51) .