Reciprocal Antagonism Coordinates C-type Natriuretic Peptide and Mitogen-signaling Pathways in Fibroblasts*

Ted D. ChrismanDagger and David L. Garbers

From the Howard Hughes Medical Institute and Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050

    ABSTRACT
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Abstract
Introduction
References

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.

    INTRODUCTION
Top
Abstract
Introduction
References

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.

    EXPERIMENTAL PROCEDURES

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 (alpha 1/beta 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.

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 -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.

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 beta -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

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


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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.

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.


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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).

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.


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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."

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 alpha 1/beta 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 alpha 1/beta 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).

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.


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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.

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.


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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).

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).


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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.

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.


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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.

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.

    FOOTNOTES

* 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.

Dagger 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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Abstract
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