From the Howard Hughes Medical Institute and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050
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ABSTRACT |
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The fibroblast, a cell central to effective wound
remodeling, not only contains various growth factor receptors but also
high activities of a guanylyl cyclase receptor (GC-B). Here we
demonstrate that marked elevations of cyclic GMP induced by C-type
natriuretic peptide (CNP), the ligand of GC-B, blocks activation of the
mitogen-activated protein kinase cascade in fibroblasts. We also show
that platelet-derived growth factor, fibroblast growth factor, serum,
or Na3VO4 rapidly (within 5 min) and
extensively (up to 85% inhibition) disrupt CNP-dependent
elevations of cyclic GMP. In addition, the mitogens also lower cyclic
GMP concentrations (50% decrease) in cells not treated with CNP.
Cytoplasmic forms of guanylyl cyclase, in contrast to the
CNP-stimulated pathway, are not antagonized by the various mitogens.
The effects of the mitogens on cellular cyclic GMP are fully explained
by a direct and stable inactivation of GC-B. Homogenates obtained from
fibroblasts treated with or without the various mitogens contain
equivalent amounts of GC-B protein, but both ligand-dependent and ligand-independent activity are
markedly (up to 90% inhibition of CNP-dependent activity)
decreased after mitogen addition. The stable inactivation is correlated
with the dephosphorylation of phosphoserine and phosphothreonine
residues of the cyclase receptor. These results not only establish a
specific and reciprocal antagonistic relationship between
mitogen-activated and GC-B-regulated signaling pathways in the
fibroblast but also suggest that one of the earliest events following
mitogen activation of a fibroblast is an interruption of cyclic GMP
production from this receptor.
Wound healing and tissue remodeling require exquisite spatial and
temporal coordination of chemotactic, proliferative, and secretory
responses in multiple cells (1). The fibroblast, a cell central to the
above processes, is tightly regulated by a host of growth and
chemotactic factors that govern its migration, proliferation, and
extracellular matrix remodeling (2, 3). A number of years ago we
demonstrated that fibroblast cell lines contain particularly high
activities of a guanylyl cyclase receptor, GC-B,1 that binds C-type
natriuretic peptide (CNP) with high affinity (4). CNP, the most highly
conserved of the natriuretic peptides (5, 6), is synthesized in various
regions throughout the body including endothelial cells but is not
found in appreciable quantities in blood, suggesting it acts in an
autocrine or paracrine manner. Aside from GC-B, some fibroblast cell
lines also appear to contain a soluble form of guanylyl cyclase
responsive to nitric oxide (7, 8) and low activities of GC-A, the
atrial natriuretic peptide receptor (4).
Substantial evidence exists that cyclic GMP is an antagonist of mitogen
action in many cell types. Whether elevated by stimulation of
cell-surface receptor-linked guanylyl cyclases, by stimulation of
cytosolic guanylyl cyclases, or by direct addition of cell-permeant analogs, cyclic GMP slows the onset of DNA synthesis, decreases cell
proliferation, and inhibits chemotaxis (8-14). Thus, significant antagonistic interplay may occur between growth factor-regulated pathways and guanylyl cyclase-regulated pathways in the fibroblast. Here, we demonstrate for the first time that elevations of cyclic GMP
block mitogen-induced activation of the MAP kinase pathway in
immortalized fibroblasts. We then demonstrate that platelet-derived growth factor (PDGF), fetal bovine serum (FBS), or fibroblast growth
factor (FGF) markedly blunt CNP-induced elevations of cyclic GMP in
either immortalized fibroblast cell lines or primary fibroblast cultures. The inhibitory effects of the growth factors or of serum on
cyclic GMP concentrations are rapid (within 5 min) and extensive and
are mediated by a direct and stable inhibition of GC-B. Intriguingly, an inhibition of both ligand-independent and CNP-dependent
GC-B activity is evident. The opposing effect of the mitogens is also highly specific, since NO-stimulated elevations of cyclic GMP are not
altered by growth factors or serum. The results strongly suggest that a
temporal, antagonistic relationship exists between a specific guanylyl
cyclase receptor (GC-B) and various mitogens during fibroblast
activation and that this occurs in the presence or absence of the
ligand, CNP. Since cyclic GMP inhibits the MAP kinase pathway
independent of its source of synthesis, the results also suggest that
growth factor-induced inhibition of the NO-regulated pathway is not
required for mitogen action.
Materials--
C-type natriuretic peptide and
des-[Cys105,Cys121]atrial natriuretic
peptide-(104-126) were from Peninsula Labs; BALB/3T3 (clone A31) and
A-10 cell lines were from ATCC; NIH/3T3 cells overexpressing rat
guanylyl cyclase-B (GC-B/3T3) were as recently described (14). A
BALB/3T3 fibroblast cell line overexpressing the NO-stimulated, rat
heterodimeric ( Cell Culture Conditions--
Cells were grown and maintained
using standard techniques. BALB/3T3 cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing
antibiotic/antimycotics and 10% calf serum or 10% fetal bovine serum
(FBS) as indicated. NIH/3T3 cells overexpressing rat GC-B (GCB/3T3) and
BALB/3T3 cells overexpressing the rat soluble guanylyl cyclase
(sgc/3T3) cell lines were maintained in DMEM/antibiotic/antimycotic, 10% FBS, and 0.1 mg/ml G418. Confluent cells were considered quiescent after maintenance in 0.5% FBS for 36-48 h or after 24 h in 0.5% FBS followed by 18-24 h in the absence of FBS.
Intact Cell Studies--
Quiescent cells were treated with serum
(standard heat-inactivated for tissue culture) and growth factors for
varying times 2 to 4 h after adding fresh medium containing the
appropriate amount of serum. Vehicle alone or growth factors were added
and the cells treated as described in the figure legends. With the exception of FBS, volume additions did not exceed 1% of cell media volume. The cyclic GMP content of intact cells plus medium was determined as follows: IBMX (0.25 mM final) was added and,
where indicated, followed 10 min later by CNP (20 nM final)
and the cells incubated an additional 10 min. HClO4 (0.5 N final) was added, and the acidified extracts were
analyzed for cyclic GMP. Cyclic GMP was estimated by radioimmunoassay
following purification of the perchloric acid extracts (16).
Thymidine Incorporation--
Quiescent GCB/3T3 fibroblasts
(24-well plates) in serum-deprived media were incubated for 1 h
with 20 nM CNP followed with 0-1% FBS for 14 h, and
then 2 µCi/ml [3H]thymidine (Amersham Pharmacia
Biotech) was added for an additional 2 h. The cells were washed
with cold phosphate-buffered saline, incubated with 10%
trichloroacetic acid for 30 min at 4 °C, washed with 10%
trichloroacetic acid, and insoluble material dissolved in 1 N NaOH. Radioactivity in a 50-µl aliquot was determined
in a scintillation counter.
Preparation of Cell Homogenates and Estimation of Guanylyl
Cyclase Activity--
Quiescent cells in 60- or 100-mm dishes were
treated with specific growth factors, serum, or
Na3VO4 as indicated in the figure legends. The
cells were washed twice with cold phosphate-buffered saline, and the
dish was immersed in liquid N2 and stored at
Guanylyl cyclase activity was estimated at 37 °C in a final volume
of 100 µl. The standard reaction mixture contained, in final
concentrations, 50 mM Hepes, pH 7.5, 7 mM
MgCl2, 1 mM GTP, 1 mM ATP, 120 mM NaCl, 2% glycerol, 0.2 mM EDTA, 10 mM NaF,1 mM NaN3, and 1 mM Na3VO4. CNP, when present, was
20 nM. Maximal guanylyl cyclase activity in homogenates was
estimated under the above conditions with the exceptions that ATP was
omitted, MgCl2 was replaced with 5 mM
MnCl2, and 1% Triton X-100 was added. The guanylyl cyclase
reaction was initiated by the addition of homogenate (5-10 µg of
protein) to the prewarmed (37 °C) reaction mixture and terminated by
adding 0.5 ml of ice-cold 110 mM
Zn(C2H3O2)2 followed by
addition of 0.5 ml of 110 mM
Na2CO3. The samples were frozen, thawed, and the supernatant fluid (3,000 × g, 15 min) fractionated
by alumina chromatography. Cyclic GMP in the eluant was estimated by
radioimmunoassay as above. In all cases cyclic GMP formation was linear
with time and protein concentration.
Western Blot Analysis of Cell Extracts--
Following
incubations in 6- or 12-well plates, the cells were frozen and thawed
in 150 or 300 µl of homogenization medium containing 1% Triton
X-100. Detergent-soluble protein was extracted for 1 h on ice, the
extracts sonicated, and insoluble material removed by centrifugation
for 30 min at 16,000 × g. Proteins were electrophoretically resolved on 8% acrylamide, 0.1% SDS gels and transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore Corp.) The membranes were probed overnight with antibodies to
phosphorylated ERK1/2 (Promega), MEK1/2 (New England Biolabs) or ERK1
(PharMingen) according to the supplier's instructions. Bound
antibodies were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech).
Metabolic Labeling, Immunoprecipitation, and Phosphoamino Acid
Analyses--
Confluent, quiescent GCB/3T3 cells in 100-mm dishes were
incubated 15 h in 5 ml of phosphate-free DMEM containing 1 mCi of [32P]orthophosphate and 0.5% FBS followed by a 1-h
incubation in the absence or presence of 10% FBS or 0.1 mM
Na3VO4. The cells were washed twice with 5 ml
of ice-cold phosphate-buffered saline and frozen on liquid
N2. The frozen cells were thawed at 0-2 °C in 0.8 ml of
cold homogenization buffer containing 1% Triton X-100, passed through
a 25-gauge needle 10-15 times, and rocked at 4 °C for 2 h. The
mixture was centrifuged at 170,000 × g for 20 min at
4 °C and the pelleted material discarded. Fifteen µl of normal
rabbit serum and 50 µl of a 50% protein A-agarose (Pierce) slurry
were added, and the samples were incubated for 60 min at 4 °C.
Protein A-agarose was removed (14,000 × g, 5 min), and
20 µl of a rabbit polyclonal antibody to the C-terminal 14 amino acids of rat guanylyl cyclase-B was added, and the samples were incubated overnight at 4 °C. The protein A-agarose antibody complex was pelleted as above and thoroughly washed in cold homogenization buffer containing 1% Triton X-100. Thirty five µl of Laemmli sample buffer and 3.5 µl of CNP Antagonizes Serum Activation of MAP Kinase--
Treatment of
quiescent GC-B/3T3 fibroblasts with 20 nM
CNP2 prior to
addition of serum sharply decreased the phosphorylation of ERK1/2
without decreasing the amount of ERK protein (Fig.
1). This is the first observation that
activation of a cyclic GMP signaling pathway leads to inhibition of the
MAP kinase cascade in fibroblasts. The marked decline in phosphorylated
ERK1/2 was accompanied by a decreased phosphorylation of MEK (not
shown), the upstream activator of ERK1/2, suggesting that CNP
interferes with an early step in activation of the MAP kinase
cascade.3 It is evident from
Fig. 1 that CNP was most effective at low serum concentrations
(0-0.3%), less so at intermediate serum concentrations (0.5-1%),
and at higher serum concentrations (3-5%) only somewhat effective in
blocking ERK phosphorylation. In the absence of serum, treatment of
cells with 20 nM CNP for 14 h decreased
[3H]thymidine incorporation by an average of 18% and
consistent with serum antagonism of the CNP effects on ERK1/2
phosphorylation, 20 nM CNP decreased thymidine
incorporation 10% (0.1% serum), about 7% (0.5% serum), and
ineffectively at higher amounts of serum (not
shown).4
The natriuretic peptide clearance receptor (which binds natriuretic
peptides but does not possess guanylyl cyclase activity (20)) has been
reported to mediate ANP inhibition of mitogen activation of the MAP
kinase cascade in some (21) but not all (22) cell lines. To determine
if the above effects of CNP were mediated by the clearance receptor in
the GC-B/3T3 cell line, des-[Cys105,Cys121]ANP, a ligand selective
for the clearance receptor (23), was tested on these cells at
concentrations of 20-1000 nM and did not inhibit basal or
serum-stimulated ERK1/2 phosphorylation or DNA synthesis. These
observations and the ability of low CNP concentrations to inhibit
ERK1/2 phosphorylation (Fig. 1) in cells overexpressing GC-B
(conditions which would favor CNP acting through GC-B rather than the
clearance receptor) are clear evidence that the effects of CNP are
mediated by GC-B.
The diminished effectiveness of CNP at the higher serum concentrations
could be explained by serum antagonism of CNP signaling. If so, then
net signaling by CNP- and serum- stimulated pathways may reflect a
balance between these two opposing signaling systems. Experiments on
intact and broken cells were thus designed to determine if serum and
defined mitogens interfere with CNP signaling (as measured by cyclic
GMP elevation in intact cells).
Serum Antagonizes CNP Elevations of Cyclic GMP--
The addition
of serum (10%) to quiescent BALB/3T3 fibroblasts (0.5% serum) for
1 h decreased CNP-stimulated elevations of cyclic GMP by nearly
70% (Fig. 2, 0.5+10), whereas
the CNP response was unaffected in normally cycling cells (10% serum)
(Fig. 2, 10+10). The 25% decline in CNP-elevated cyclic GMP
levels in serum-starved cells compared with control cells (Fig. 2,
0.5+0.5 versus 10+10) is not due to a
decreased sensitivity to CNP (data not shown) but reflects partial cell
loss during serum starvation and possibly decreased expression of GC-B
and/or other proteins necessary for signaling.
PDGF Is a Potent Suppresser of CNP Signaling--
PDGF, at
concentrations of 300-500 pM, is the primary fibroblast
mitogen in serum, accounting for at least 50% of the mitogenic activity (24-26). PDGF binding to specific heterodimeric or
homodimeric cell-surface receptor tyrosine kinases results in
activation of the MAP kinase cascade, protein kinase C, and several
other distinct signaling pathways (27-29). Low concentrations of PDGF
(in the presence of 0.5% serum) rapidly and effectively interfered
with CNP signaling (as monitored by elevation of cyclic GMP) in
quiescent BALB/3T3 fibroblasts (Fig. 3).
PDGF inhibition was concentration-dependent (Fig.
3A) and, in sub-nanomolar amounts, was as effective as serum in inhibiting CNP signaling. These concentrations of PDGF are well
within the range that is mitogenic for this and other cells of
mesenchymal origin (30-32). The inset of Fig. 3A
shows, by Western blot analysis of phosphorylated ERKs 1 and 2, that
concentrations of PDGF that decrease CNP signaling also cause
near-maximal activation of the MAP kinase pathway. The inhibitory
effect of PDGF developed rapidly (short lag-time) (Fig. 3B)
as is the case with PDGF activation of the MAP kinase cascade (33).
Treatment of the quiescent cells with 0.3 nM PDGF (in the
presence of 0.5% serum) resulted in a rapid (evident within 5 min) and
sharp decline in CNP-stimulated elevation of cyclic GMP levels reaching
near-maximal inhibition within 60 min. The rapidity of PDGF inhibition
is clear from the inset where PDGF was added shortly
after CNP. The similar time courses and concentration
dependence of PDGF inhibition of the CNP signaling pathway and
stimulation of the MAP kinase cascade suggest that suppression of CNP
signaling through cyclic GMP is an early downstream consequence of PDGF
receptor activation.
That this inhibition of CNP by serum and defined mitogens is not
confined to immortalized cell lines was confirmed in rat aortic smooth
muscle cells (A-10) and early passage human dermal fibroblasts (not
shown). Such results suggest that inactivation of GC-B by mitogens
represents a general consequence of growth factor signaling serving to
limit cyclic GMP antagonism of growth factor-regulated cell function.
Nitric Oxide Signaling through Cyclic GMP Is Unaffected by
Mitogens--
The above experiments established that serum and PDGF
decrease the cyclic GMP signal generated in response to CNP stimulation of the cell-surface receptor guanylyl cyclase, GC-B. NO and
NO-sensitive cytosolic guanylyl cyclase, a key signaling pathway in
many cell types (34), have been reported as antimitogenic under some
conditions (8, 9) and, as with the cell-surface receptor GC-B, could be
negatively regulated by serum and other mitogens. A BALB/3T3 cell line
overexpressing the NO-sensitive rat Na3VO4 Decreases CNP Signaling in Intact
Cells--
Reversible protein-tyrosine phosphorylation is a common
mechanism of mitogen signaling (38). Na3VO4, a
cell permeant, non-selective protein-tyrosine-phosphatase inhibitor
mimics the effects of many ligands that activate
protein-tyrosine-kinases and the MAP kinase pathway (39-41). This
phosphatase inhibitor also mimics the effects of serum and PDGF on CNP
signaling in fibroblasts (Fig. 5).
Treatment of quiescent GC-B/3T3 fibroblasts with 5-50 µM
Na3VO4 for 50 min reduced CNP elevations of
cyclic GMP 50-80%,5
respectively, while activating the MAP kinase cascade
(inset). Serum, PDGF, and FGF also decrease CNP signaling in
these cells (not shown), the effects being qualitatively and
quantitatively similar to those found with the endogenously expressed
GC-B of the BALB/3T3 fibroblasts. A simple and plausible explanation of these results and those of Figs. 2 and 3 is that tyrosine
phosphorylation of GC-B inhibits guanylyl cyclase activity.
Alternatively, regulatory proteins "activated" directly or
indirectly by tyrosine phosphorylation could inhibit the cyclase by
stable association or by phosphorylation or dephosphorylation.
Guanylyl Cyclase-B Is Stably Inactivated by Serum and
Na3VO4--
Quiescent cells were incubated
with 10% serum or 100 µM Na3VO4
for various times, and guanylyl cyclase activity was estimated in the
subsequent
homogenate6 in
the absence or presence of 20 nM CNP (Fig.
6). Serum treatment of intact cells
decreased CNP-stimulated guanylyl cyclase activity within 5 min, and by
15 min this activity was depressed to that seen in the absence of CNP
(Fig. 6A). Reduction of activity in the absence of CNP was
not as rapid but declined to 50% of control values by 15 min. These
results are consistent with a stable inactivation of guanylyl cyclase
and provide a mechanism for the effects of serum on cyclic GMP levels
in intact cells. Na3VO4 produced essentially the same effects on basal and CNP-stimulated activities as serum (Fig.
6B) implying that both serum and
Na3VO4 employ identical or similar mechanisms
to disrupt CNP signaling in intact cells.
Decreases in Phosphoserine and Phosphothreonine Correlate with
Inactivation of GC-B--
Phosphorylation/dephosphorylation could
explain the apparent stable inactivation of GC-B (15). Quiescent cells
in 0.5% serum were metabolically labeled with 32P and then
treated with 10% serum or 100 µM
Na3VO4 for 1 h. Autoradiography, immunoquantitation by Western blot analysis, and phosphoamino acid
analyses of immunoprecipitated GC-B (Fig.
7) showed large decreases in
32P content of GC-B (autoradiograph, upper
panel), with no loss of GC-B protein (Western blot
(WB), middle panel). Significantly, GC-B
contained no detectable
phosphotyrosine7
prior to or after mitogen treatment (phosphoamino acid analysis, lower panel), and thus the loss of 32P was
attributable to decreases in phosphoserine and phosphothreonine. Decreased phosphoserine and phosphothreonine correlate with decreased CNP stimulation in intact cells following serum or
Na3VO4 treatment yielding end points similar to
those seen for ligand-induced dephosphorylation and desensitization to
ligand of both GC-A and GC-B (15, 42). Specific phosphoamino acids in
GC-B have been identified as necessary for ligand-induced signaling and
undergo ligand-stimulated dephosphorylation, and thus dephosphorylation
of one or more of these residues may account for mitogen-induced
inactivation of GC-B (43).
The results of the preceding studies show that CNP markedly elevates
cyclic GMP in both quiescent (serum-limited) and normal cycling
(serum-replete) cells (Fig. 2). However, relatively high serum or
defined growth factors added to quiescent cells rapidly and sharply
decreases CNP stimulation of GC-B in intact cells (Figs. 2 and 3) by
directly lowering cyclase activity (Fig. 6) in the absence of changes
in GC-B expression (Fig. 7). It is clear then that mitogens
substantially disrupt signaling through inactivation of GC-B,
consequently suppressing CNP elevation of cyclic GMP in whole cells or
in broken cells. Conversely, CNP antagonism of serum activation of the
MAP kinase cascade (Fig. 1) demonstrates that CNP and mitogens are
antagonists at least in the "resting" or G0/early
G1 phase of the cell
cycle.8 The data in Fig. 2
also imply that serum or mitogen inhibition of CNP signaling is acute
in that it therefore appears reversible under chronic conditions.
Adaptation to Mitogens--
Reversible changes in signaling
pathways are important, and since fibroblasts in the proximity of a
wound are continuously exposed to high levels of mitogens (1), it is
important to determine the effects of such conditions on CNP
responsiveness. Basal, CNP-stimulated, and
Mn2+/Triton-stimulated guanylyl cyclase activities in
homogenates were determined at different times during an 8-h exposure
of quiescent GC-B/3T3 fibroblasts to 10% serum (Fig.
8). Basal and CNP-stimulated guanylyl
cyclase activities sharply decreased during the initial 1 h of
serum treatment but recovered to control levels by 6 h despite the
continued presence of serum. The recovery of guanylyl cyclase activity
corresponded well with the ability of CNP to elevate cyclic GMP in
intact cells (not shown) and the phosphorylation state of the cyclase
(not shown). This is consistent with covalent regulation of natriuretic
peptide receptor-guanylyl cyclases through phosphorylation previously
seen in broken cells (44). Although CNP responses changed with time,
both total guanylyl cyclase activity, as measured in the presence of
Mn2+/Triton, and the expression level of the cyclase
remained constant. The cyclase therefore appears to be reversibly
regulated by covalent modification catalyzed by one or more protein
kinases/phosphoprotein phosphatases, at least some of which are
mitogen-sensitive. Following rapid serum inactivation of the cyclase
CNP signaling is reestablished prior to the onset of DNA synthesis and
mitosis,9 coinciding with the
initiation and decline of immediate early gene transcription (30). As
the cell exits mitosis and is again sensitive to extracellular
mitogens,10 it again
responds to CNP. The simultaneous, similar, and consistent but opposite
effects of serum, PDGF, and Na3VO4 on CNP- and
mitogen-signaling pathways strongly suggest that suppression of CNP
signaling and activation of the MAP kinase cascade are functionally
linked.
Rapid changes in mitogen levels likely occur at the site of a wound as
platelets release large amounts of mitogenic/chemotactic factors such
as PDGF. In this context, where fibroblasts are attracted to the wound
or stimulated to proliferate, signaling pathways antagonistic to
proliferation or migration are likely suppressed. Clearly the acute
suppression of GC-B ligand-dependent as well as
CNP-independent activity by mitogens is an early event in their signaling pathways. The reversible nature of the inhibition is also
physiologically important, but the mechanism of reversibility (mitogen
receptor desensitization or signaling pathway component desensitization) remains unknown.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
1/
1) cytosolic guanylyl
cyclase was from Dr. Peter Yuen (University of Tennessee, Memphis) and
early passage human dermal fibroblasts were from G. Skuta and F. Grinnell (University of Texas Southwestern Medical Center, Dallas, TX).
Nucleotides were from Boehringer Mannheim and Sigma. Cell culture
materials were from Life Technologies, Inc. PDGFbb and basic FGF were
from R & D Systems. Antibodies to the phosphorylated forms of ERK1/2 and MEK1/2 were from Promega and anti-ERK1 antibodies were from PharMingen. All other reagents were obtained from Sigma unless noted otherwise.
80 °C.
The frozen cells were thawed at 0-2 °C in 0.3 (60-mm dish) or 0.5 ml (100 mm dish) of ice-cold homogenization buffer (50 mM
Hepes, pH 7.5, 10% glycerol, 100 mM NaCl, 10 µg/ml each
of leupeptin, pepstatin, and aprotinin, 50 mM NaF, 1 mM EDTA, and 1 mM
Na3VO4), scraped from the dish, and sonicated 3 times for 3 s. Protein concentration (bicinchoninic acid, Pierce)
was determined, and the homogenates were aliquoted, frozen in liquid
N2, and stored at
80 °C.
-mercaptoethanol were added, and the samples boiled and immunoprecipitated GC-B was isolated by SDS-PAGE (0.1% sodium dodecyl sulfate, 8% polyacrylamide). The resolved proteins were
transferred to polyvinylidene difluoride membranes and the membrane
probed overnight with the same polyclonal antibody as above. Goat
anti-rabbit IgG coupled to horseradish peroxidase was visualized by
chemiluminescence (Amersham Pharmacia Biotech). Following
autoradiography, the bands corresponding to GC-B were excised and
phosphoamino acid analyses performed on the acid hydrolysates (17).
RESULTS AND DISCUSSION
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Fig. 1.
CNP antagonizes serum-stimulated elevation of
phosphorylated ERK1/2. Confluent GCB/3T3 cells were maintained
24 h in DMEM + 0.5% FBS and then 18 h in DMEM alone. Fresh
DMEM was added followed in 2 h by addition of FBS to the final
concentrations indicated in the figure. Where indicated, the cells were
treated with 20 nM CNP for 1 h prior to FBS addition.
Cell extracts were prepared and immunoblotted for phosphorylated ERK1/2
(pERK1/2) and total ERK1 (ERK1) as described
under "Experimental Procedures." Each lane represents
approximately 6 µg of solubilized extract protein.
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Fig. 2.
CNP signaling in serum-starved fibroblasts is
inhibited by 10% serum. Confluent, serum-starved BALB/3T3
fibroblasts were switched from 10 to 0.5% FBS or to fresh 10% FBS for
36 h. Then fresh 0.5% FBS (0.5+0.5) or fresh 10% FBS
(0.5+10; 10+10) was added for 1 h. Following
a 10-min incubation with 20 nM CNP and 0.25 mM
IBMX, HClO4 was added, and cyclic GMP levels were
determined as described under "Experimental Procedures." Cyclic GMP
levels are given as the average of duplicate determinations (± range).
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Fig. 3.
PDGF rapidly inhibits CNP signaling in
serum-starved fibroblasts. Quiescent, serum-starved BALB/3T3
fibroblasts were used for these studies. A, cells were
treated for 30 min with increasing PDGF concentration and then
incubated with 20 nM CNP and 0.25 mM IBMX for
10 min, and HClO4 added, and cyclic GMP concentration
determined. Total incubation time with PDGF was 50 min;
inset, cells were treated with PDGF for 15 min,
rinsed, frozen, and immunoreactive phosphorylated ERK1/2
(pERK1/2) in 5 µg of protein analyzed by Western blot as
described under "Experimental Procedures." B, cells were
treated with 0.3 nM PDGF for varying times and then
incubated with 20 nM CNP and cyclic GMP measured as above.
The abscissa indicates total time in the presence
of PDGF; inset, 20 nM CNP was added at "0
min" (after 10 min with 0.25 mM IBMX), and the cells were
incubated for the indicated times and HClO4 added. At 2 min, vehicle (squares) or 0.3 nM PDGF
(triangles) was added and the incubation continued as above.
Cyclic GMP levels (A and B, means (±S.E.),
n = 3; B, inset, average of duplicates (± range) was measured as described under "Experimental
Procedures."
1/
1
heterodimeric soluble guanylyl cyclase (BALB/3T3 fibroblasts normally
lack an endogenous NO-sensitive soluble guanylyl cyclase (35, 36)) was
treated with serum, PDGF, or with fibroblast growth factor (FGF), a
receptor tyrosine kinase ligand synthesized and released by activated
macrophages and keratinocytes in the vicinity of a wound (2, 37), and
all decreased CNP elevations of cyclic GMP without diminishing NO
stimulation of cyclic GMP (Fig. 4). Thus,
the CNP-stimulated cell-surface cyclase, GC-B, is a specific target of
the mitogens, and increased degradation of cyclic GMP is not a
mechanism of serum or PDGF inhibition of CNP elevations of cyclic
GMP.
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Fig. 4.
Serum and mitogens do not inhibit NO
stimulation of soluble guanylyl cyclase in quiescent BALB/3T3
cells. Cells, overexpressing rat 1/
1
soluble guanylyl cyclase, were incubated in the absence or presence of
10% FBS, 0.3 nM PDGF, or 0.6 nM basic FGF for
30 min prior to a 10-min incubation with 0.25 mM IBMX then,
vehicle (
), 20 nM CNP, or 100 µM sodium
nitroprusside (SNP) were added for an additional 10 min to
control wells (CNP or SNP) or to those with FBS,
PDGF, or SNP (+ CNP or + SNP as indicated by the horizontal
arrows). Cyclic GMP contents of HClO4 extracts
of duplicate wells were determined as given under "Experimental
Procedures." Cyclic GMP levels are given as the average (± range).
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Fig. 5.
Na3VO4 is a potent
inhibitor of CNP signaling in serum-starved fibroblasts. Quiescent
GCB/3T3 fibroblasts were incubated 30 min with the indicated
concentrations of Na3VO4 followed by a 10-min
incubation with 20 nM CNP and 0.25 mM IBMX.
HClO4 was added and cyclic GMP accumulation determined as
given under "Experimental Procedures." Values for cyclic GMP are
means (± S.E.) of triplicate determinations. Inset,
quiescent cells were incubated for 15 min with the indicated amounts of
Na3VO4 and 6 µg protein analyzed for
phosphorylated ERK1/2 (pERK1/2) by immunoblotting as in Fig.
3.
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Fig. 6.
Serum and Na3VO4
treatment of serum-starved fibroblasts results in a rapid and stable
inhibition of ligand-dependent and CNP-independent guanylyl
cyclase activity. Quiescent, serum-starved GCB/3T3 fibroblasts in
60-mm dishes were incubated for the indicated times with 10% FBS
(A), or 100 µM Na3VO4
(B), then frozen in liquid N2 and
homogenates prepared as described under "Experimental Procedures."
Guanylyl cyclase activity in homogenates (5-10 µg protein) was
measured after a 4-min incubation in the absence, squares,
or presence, triangles, of 20 nM CNP. Cyclic GMP
values are the average of duplicate incubations (± range).
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Fig. 7.
Serum and Na3VO4
treatments of serum-starved fibroblasts result in dephosphorylation of
GC-B. Quiescent, serum-starved GC-B/3T3 fibroblasts metabolically
labeled with 32P were treated for 1 h with 10% FBS or
100 µM Na3VO4, and GC-B isolated
by immunoprecipitation and SDS-PAGE as described under "Experimental
Procedures." Upper panel, Western blot
(WB) and autoradiograph of immunoprecipitated GC-B.
Lower panel, 32P-labeled bands from the control,
serum-treated, and Na3VO4-treated
immunoprecipitates above were hydrolyzed for 60 min in 5.7 N HCl and phosphoamino acid resolved by two-dimensional
electrophoresis and visualized by autoradiography.
Pi, inorganic phosphate; P-S,
phosphoserine; P-T, phosphothreonine; O,
origin.
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Fig. 8.
CNP signaling fully recovers during prolonged
serum treatment of serum-starved fibroblasts. Quiescent,
serum-starved GCB/3T3 fibroblasts in 0.5% FBS were treated with 10%
FBS for the indicated times and guanylyl cyclase activity in
homogenates determined as described under "Experimental
Procedures." Guanylyl cyclase activity was determined in a 4-min
incubation with no additions (squares), 20 nM
CNP (triangles), or Mn2+/Triton (inverted
triangles). Cyclic GMP values are the averages (± range) of
duplicate determinations of single cell treatments.
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FOOTNOTES |
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* 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: Howard Hughes Medical
Institute, University of Texas Southwestern Medical Center, 5323 Harry
Hines Blvd., Dallas, TX 75325-9050. Tel.: 214-648-5086; Fax:
214-648-5087; E-mail: Ted.Chrisman{at}emailswmed.edu.
The abbreviations used are: GC-B, guanylyl cyclase-B; CNP, C-type natriuretic peptide; GC-A, guanylyl cyclase-A; ANP, atrial natriuretic peptide; FBS, fetal bovine serum; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; IBMX, 1-methyl-3-isobutylxanthine; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK kinase; DMEM, Dulbecco's modified Eagle's medium.
2 20 nM CNP stimulation of GC-B results in high levels of cyclic GMP in these cells (Fig. 5).
3 Under these conditions, we were unable to detect the presence of mitogen-activated protein kinase phosphatase-1, the MAP kinase phosphatase reportedly induced by ANP in mesangial cells (18).
4 Others (19) also have noted that NIH/3T3 cell lines are relatively resistant to antimitogenic effects of natriuretic peptides, possibly explained by rapid homologous desensitization of GC-A or GC-B (15).
5 Basal and CNP-stimulated activities of GC-B in cell homogenates are not inhibited by 1 mM Na3VO4 or by 10% FBS suggesting that their effects in intact cells are not due to direct inhibition of the cyclase.
6 The same results were seen with washed 100,000 × g pellets.
7 Phosphotyrosine immunoblots did not detect phosphotyrosine associated with immunoprecipitated GC-B under these conditions.
8 Similar effects were seen with BALB/3T3 fibroblasts and early passage human dermal fibroblasts.
9 In BALB/3T3 fibroblasts "early G1" ends at 6 h and S phase begins about 12 h after PDGF exposure (30, 45).
10 As cells pass the restriction point in G1, they become refractory to extracellular mitogens presumably regaining full mitogen sensitivity upon completion of the cell cycle (46).
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REFERENCES |
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