Reciprocal antagonism coordinates C-type natriuretic peptide and mitogen-signaling pathways in fibroblasts.

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 cyclaseregulated pathways in the fibroblast. Here, we demonstrate for the first time that elevations of cyclic GMP block mitogeninduced 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 ligandindependent 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.

EXPERIMENTAL PROCEDURES
Materials-C-type natriuretic peptide and des-[Cys 105 ,Cys 121 ]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 (␣ 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 * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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.
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. HClO 4 (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 Ci/ml [ 3 H]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-l 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 Na 3 VO 4 as indicated in the figure legends. The cells were washed twice with cold phosphate-buffered saline, and the dish was immersed in liquid N 2 and stored at Ϫ80°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 g/ml each of leupeptin, pepstatin, and aprotinin, 50 mM NaF, 1 mM EDTA, and 1 mM Na 3 VO 4 ), 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 N 2 , and stored at Ϫ80°C.
Guanylyl cyclase activity was estimated at 37°C in a final volume of 100 l. The standard reaction mixture contained, in final concentrations, 50 mM Hepes, pH 7.5, 7 mM MgCl 2 , 1 mM GTP, 1 mM ATP, 120 mM NaCl, 2% glycerol, 0.2 mM EDTA, 10 mM NaF,1 mM NaN 3 , and 1 mM Na 3 VO 4 . 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, MgCl 2 was replaced with 5 mM MnCl 2 , and 1% Triton X-100 was added. The guanylyl cyclase reaction was initiated by the addition of homogenate (5-10 g of protein) to the prewarmed (37°C) reaction mixture and terminated by adding 0.5 ml of ice-cold 110 mM Zn(C 2 H 3 O 2 ) 2 followed by addition of 0.5 ml of 110 mM Na 2 CO 3 . The samples were frozen, thawed, and the supernatant fluid (3,000 ϫ 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 6or 12-well plates, the cells were frozen and thawed in 150 or 300 l 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 ϫ 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 [ 32 P]orthophosphate and 0.5% FBS followed by a 1-h incubation in the absence or presence of 10% FBS or 0.1 mM Na 3 VO 4 . The cells were washed twice with 5 ml of ice-cold phosphate-buffered saline and frozen on liquid N 2 . 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 ϫ g for 20 min at 4°C and the pelleted material discarded. Fifteen l of normal rabbit serum and 50 l 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 ϫ g, 5 min), and 20 l 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 l of Laemmli sample buffer and 3.5 l of ␤-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). 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 [ 3 H]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-[Cys 105 ,Cys 121 ]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)   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 serumstimulated 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).

CNP Antagonizes Serum Activation of MAP
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, 0.5ϩ10), whereas the CNP response was unaffected in normally cycling cells (10% serum) (Fig. 2, 10ϩ10). The 25% decline in CNP-elevated cyclic GMP levels in serum-starved cells compared with control cells (Fig.  2, 0.5ϩ0.5 versus 10ϩ10) 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)(28)(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 lagtime) (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 ␣ 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. Na 3 VO 4 Decreases CNP Signaling in Intact Cells-Reversible protein-tyrosine phosphorylation is a common mechanism of mitogen signaling (38). Na 3 VO 4 , 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 M Na 3 VO 4 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 Na 3 VO 4 -Quiescent cells were incubated with 10% serum or 100 M Na 3 VO 4 for various times, and guanylyl cyclase activity was estimated in the subsequent homogenate 6 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. Na 3 VO 4 produced essentially the same effects on basal and CNP-stimulated activities as serum (Fig. 6B) implying that both serum and Na 3 VO 4 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 32 P and then treated with 10% serum or 100 M Na 3 VO 4 for 1 h. Autoradiography, immunoquantitation by Western blot analysis, and phosphoamino acid analyses of immunoprecipitated GC-B (Fig. 7) showed large decreases in 32 P 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 phosphotyrosine 7 prior to or after mitogen treatment (phosphoamino acid analysis, lower panel), and thus the loss of 32 P was attributable to decreases in phosphoserine and phosphothreonine. Decreased phosphoserine and phosphothreonine correlate with decreased CNP stimulation in intact cells following serum or Na 3 VO 4 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 5 Basal and CNP-stimulated activities of GC-B in cell homogenates are not inhibited by 1 mM Na 3 VO 4 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 ϫ g pellets. 7 Phosphotyrosine immunoblots did not detect phosphotyrosine associated with immunoprecipitated GC-B under these conditions.

FIG. 7. Serum and Na 3 VO 4 treatments of serum-starved fibroblasts result in dephosphorylation of GC-B.
Quiescent, serumstarved GC-B/3T3 fibroblasts metabolically labeled with 32 P were treated for 1 h with 10% FBS or 100 M Na 3 VO 4 , 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, 32 P-labeled bands from the control, serum-treated, and Na 3 VO 4 -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. P i , inorganic phosphate; P-S, phosphoserine; P-T, phosphothreonine; O, origin.

FIG. 6. Serum and Na 3 VO 4 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 M Na 3 VO 4 (B), then frozen in liquid N 2 and homogenates prepared as described under "Experimental Procedures." Guanylyl cyclase activity in homogenates (5-10 g 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 (Ϯ range).

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 G 0 /early G 1 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 Mn 2ϩ /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 Mn 2ϩ /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 Na 3 VO 4 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 liganddependent 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.
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 Mn 2ϩ /Triton (inverted triangles).
Cyclic GMP values are the averages (Ϯ range) of duplicate determinations of single cell treatments.