Atrial Natriuretic Peptide Induces the Expression of MKP-1, a Mitogen-activated Protein Kinase Phosphatase, in Glomerular Mesangial Cells

Atrial natriuretic peptide (ANP) has been shown to inhibit the proliferation of various types of cells including glomerular mesangial cells. The activation of mitogen-activated protein kinase (MAPK) is one of the main signal transduction systems leading to cell proliferation. MAPK is tightly regulated by the activating kinase, MEK, and specific phosphatase MKP-1. Constitutive expression of MKP-1 has been shown to inhibit cell proliferation by suppressing MAPK activity. In order to understand the mechanism of the anti-proliferative effect of ANP, we examined whether ANP could inhibit MAPK by inducing MKP-1 in cultured rat glomerular mesangial cells. ANP increased the expression of MKP-1 mRNA in a dose-dependent (10 nM maximum) and time-dependent, with a peak stimulation at 30 min, manner. Receptor for ANP is a transmembrane guanylyl cyclase. Activation of guanylyl cyclase of ANP receptor by ligand plays an essential role in ANP signal transduction. 8-Bromo-cGMP, a cell permeable analogue of cyclic GMP, and sodium nitroprusside, an activator of soluble guanylyl cyclase, could mimic the effects of ANP and were able to induce the expression of MKP-1 in a similar time course as ANP. The protein expression of MKP-1 was maximally stimulated by ANP at 120 min. Treatment of the cells with ANP for 120 min resulted in an inhibition of phorbol ester-induced activation of MAPK, while the activation of MEK was not affected by ANP. These results indicate that ANP might inhibit the proliferation of mesangial cells by inactivating MAPK through the induction of MKP-1.

Atrial natriuretic peptide (ANP) has been shown to inhibit the proliferation of various types of cells including glomerular mesangial cells. The activation of mitogen-activated protein kinase (MAPK) is one of the main signal transduction systems leading to cell proliferation. MAPK is tightly regulated by the activating kinase, MEK, and specific phosphatase MKP-1. Constitutive expression of MKP-1 has been shown to inhibit cell proliferation by suppressing MAPK activity. In order to understand the mechanism of the anti-proliferative effect of ANP, we examined whether ANP could inhibit MAPK by inducing MKP-1 in cultured rat glomerular mesangial cells. ANP increased the expression of MKP-1 mRNA in a dose-dependent (10 nM maximum) and time-dependent, with a peak stimulation at 30 min, manner. Receptor for ANP is a transmembrane guanylyl cyclase. Activation of guanylyl cyclase of ANP receptor by ligand plays an essential role in ANP signal transduction. 8-Bromo-cGMP, a cell permeable analogue of cyclic GMP, and sodium nitroprusside, an activator of soluble guanylyl cyclase, could mimic the effects of ANP and were able to induce the expression of MKP-1 in a similar time course as ANP. The protein expression of MKP-1 was maximally stimulated by ANP at 120 min. Treatment of the cells with ANP for 120 min resulted in an inhibition of phorbol ester-induced activation of MAPK, while the activation of MEK was not affected by ANP. These results indicate that ANP might inhibit the proliferation of mesangial cells by inactivating MAPK through the induction of MKP-1.
Atrial natriuretic peptide (ANP) 1 is a potent vasorelaxing peptide which regulates not only the vascular tone but glomerular filtration rates by inducing the relaxation or inhibiting the contraction of vascular smooth muscle cells (1) and glomerular mesangial cells (2). ANP was also found to inhibit the proliferation of vascular smooth muscle cells and mesangial cells (3). ANP is known to bind to the specific receptors which couple to particulate guanylyl cyclase and to increase intracellular cGMP in cultured mesangial cells (4,5). We have previously reported that ANP is able to inhibit the proliferation of mesangial cells in a cGMP-dependent manner (5). However, the mechanism of this anti-proliferative action of ANP is still poorly understood.
Mitogen-activated protein kinase (MAPK), also known as the extracellular signal regulated protein kinase (ERK), is a member of a family of serine/threonine kinases which are activated by various growth factors (6 -9). We and others have reported that MAPK is also activated by mitogens such as endothelin-1 in cultured mesangial cells (10,11). The activation of MAPK was found to induce the phosphorylation of nuclear transcription factors and protein kinases involved in the regulation of cell growth (6 -9), suggesting an essential role of MAPK in a signal transduction leading to cell proliferation. A single protein kinase, MAPK or ERK kinase (MEK) (12), was shown to activate MAPK by phosphorylating threonine 183 and tyrosine 185 (13) and the phosphorylation in both residues was found to be essential for MAPK to exert its full enzyme activity (14).
Recently, several laboratories have identified a family of inducible protein phosphatases, MKP-1/CL100/HVH1/erp (15)(16)(17)(18)(19) and PAC-1 (20), with dual protein-tyrosine/threonine specificity and selectivity for MAPK (21). The constitutive expression of MKP-1 was found to attenuate serum-or oncogenic ras-induced MAPK activation (16,22), to block MAPK-dependent gene expression (23,24), and to inhibit cell proliferation (19,22), suggesting that the dephosphorylation of MAPK in vivo by MKP-1 could have a negative effect on cell proliferation. The recent report suggests that the constitutive expression of MKP-1 also inhibits Jun kinase activity (25). We have hypothesized that the anti-proliferative action of ANP might be mediated by the induction of MKP-1. To prove this hypothesis, we examined the effect of ANP on the expression of MKP-1 and the activation of MAPK in cultured mesangial cells.
We report here that ANP induces the expression of MKP-1 mRNA and protein and inhibits the activation of MAPK cascade at the level of MAPK in cultured glomerular mesangial cells in concentrations enough to inhibit the proliferation of mesangial cells (5). These data could provide a new mechanism of anti-proliferative effect of ANP. * 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.
Mesangial Cells Culture and Experimental Protocol-Glomerular mesangial cells were obtained from a culture of glomeruli, isolated from male Sprague-Dawley rats weighing 100 -150 g by sieving method, in RPMI 1640 medium containing 20% fetal bovine serum (FBS) and antibiotics as described previously (30). Cells at passage 2 to 9 were grown until 80% confluency in 100-mm dishes and then made quiescent by reducing the concentrations of FBS to 0.4% for 72 h. For experiments, the quiescent cells were incubated in an incubation medium (RPMI 1640 medium with 0.4% fatty acid free bovine serum albumin and 20 mM HEPES, pH 7.4) at 37°C for 30 min. The cells were then exposed to various agents in the incubation medium at 37°C for indicated time interval. The reactions were stopped by rapid aspiration of the medium and by washing twice with ice-cold phosphate-buffered saline. The cells were used for Northern blot analysis, immunoblot analysis, and immunoprecipitation as follows.
Northern Blot Analysis-Ten g of total RNA extracted using TRIzol Reagent (Life Technologies, Inc.) was electrophoresed through a 1% agarose formaldehyde gel and transferred on nylon filter (Nytran, Schleicher and Schuell). The filter was prehybridized in Church buffer (500 mM NaPO 4 , pH 7.0, 7% SDS, 1% bovine serum albumin, 1 mM EDTA) at 65°C for 2 h and hybridized with 1 ϫ 10 6 cpm/ml radiolabeled HVH1 cDNA (18) at 65°C for 24 h in a rotating hybridization oven. The filter was washed twice with wash buffer A (40 mM NaPO 4 , pH 7.0, 5% SDS, 0.5% bovine serum albumin, 1 mM EDTA) for 10 min at 65°C, twice with wash buffer B (40 mM NaPO4, pH 7.0, 1% SDS, 1 mM EDTA) in the same conditions, and exposed to Kodak X-AR film with intensifying screen at Ϫ70°C. The intensity of the bands was quantified by a densitometric analysis and signals were normalized to glyceraldehyde-3-phosphate dehydrogenase signals.
Immunoprecipitation and Kinase Assay-The cell lysate (about 400 g of protein) was incubated with 5 l of anti-MEK antisera or 1 g of anti-ERK2 antibody for 2 h at 4°C. The immunoprecipitates were recovered by incubating with protein G-Sepharose (20 l, Pharmacia) for 1 h at 4°C, centrifuging, and washing three times with cell lysis buffer and once with a kinase buffer (18 mM HEPES, pH 7.5, 10 mM magnesium acetate, and 50 M ATP) without ATP. The activities of MAPK or MEK were determined by MBP kinase assay as described previously (26 -28). The immunoprecipitates with anti-ERK2 antibody were incubated with 20 g of MBP in 40 l of a kinase buffer containing 2 Ci of [␥-32 P]ATP for 15 min at 30°C. The immunoprecipitates with anti-MEK antisera were incubated first with 2 g of GST-MAPK in a kinase buffer at 30°C for 15 min and then with 2 Ci of [␥-[ 32 P]ATP and 20 g of MBP at 30°C for an additional 10 min.
Statistical Analysis-Results were expressed as mean Ϯ S.D. Analysis of variance (ANOVA) with subsequent Scheffe's test was used to determine significant differences in multiple comparisons.

Effects of ANP on MKP-1 Expression in Cultured Mesangial
Cells-We first examined whether MKP-1 mRNA could be induced in mesangial cells by the stimuli known to be effective in other types of cells. As shown in Fig. 1, fetal bovine serum (2.5%) and PDBu (100 nM) were able to rapidly induce MKP-1 mRNA when examined by Northern blot analysis using HVH-1 cDNA, a human homologue of 3CH134, as a probe. We next examined the effect of ANP on the expression of MKP-1 in cultured mesangial cells. As shown in Fig. 2, ANP (100 nM) increased the expression of MKP-1 mRNA rapidly and the maximal induction was obtained at 30 min. ANP-induced expression of MKP-1 mRNA was observed in a concentration-dependent manner of ANP with a maximal response at 10 -100 nM (Fig. 3), concentrations enough to inhibit the proliferation of mesangial cells (5).
ANP has been reported to activate receptor guanylyl cyclase and to increase intracellular cyclic GMP (cGMP) in cultured mesangial cells (4,5). In order to know the mechanism of ANP-induced expression of MKP-1 mRNA, we examined the effect of cGMP on MKP-1 mRNA expression in mesangial cells. When the mesangial cells were incubated with 8-Br-cGMP, a cell permeable analogue of cGMP, MKP-1 mRNA expression was increased in a similar time course as ANP (Fig. 4A). However, C-ANP, an analogue specific to clearance receptors for ANP (5), failed to induce MKP-1 mRNA expression (data not shown). SNP, an activator of soluble guanylyl cyclase, was also able to induce the expression of MKP-1 mRNA (Fig. 4B).
The expression of MKP-1 protein was next examined by an immunoblot analysis. The 39-kDa protein, the same molecular mass protein as MKP-1 reported in PC12 cells (23), was detected from rat mesangial cell lysate. This protein could not be detected when the antibody was preincubated with recombinant MKP-1, GST-HVH-1 (data not shown). As shown Fig. 5, ANP (100 nM) induced the expression of MKP-1 protein with a maximal stimulation at 120 min. ANP also induced the expression of the protein with 42-44 kDa molecular mass. This protein might be MKP-2 reported in PC12 cells (23).

Effects of ANP on MAPK Cascade in Rat Mesangial
Cells-We next examined the effect of ANP on the activities of MAPK and MEK. The mesangial cells were treated with 100 nM ANP for 120 min prior to stimulation with PDBu for 10 min and then the activities of MAPK and MEK were measured. PDBu rapidly increased MAPK and MEK (10 min maximum) in a concentration-dependent manner (10 -100 nM maximum) in rat mesangial cells (data not shown). PDBu-induced activation of MAPK was significantly inhibited in cells treated with ANP for 120 min, while basal activities of MAPK remained unchanged (Fig. 6A). In contrast, PDBu-induced activation of MEK was not affected by ANP (Fig. 6B), indicating that PDBu-induced activation of MAPK cascade was blocked by ANP at the level of MAPK. DISCUSSION ANP, a potent vasorelaxing peptide, is able to inhibit the proliferation of glomerular mesangial cells by cGMP-dependent mechanism (5). The present study was performed to clarify the mechanism of anti-proliferative action of ANP and the results indicate that ANP is able to induce the expression of MAPK phosphatase, MKP-1, by cGMP-dependent mechanism in concentrations enough to inhibit the proliferation of mesangial cells. Furthermore, PDBu-induced activation of MAPK cascade was inhibited by ANP at the level of MAPK.
MKP-1 (also called as 3CH134, CL100, erp, or HVH1) is a dual specificity phosphatase that selectively dephosphorylates MAPK in vitro (15,18) or in vivo (16,31). In mesangial cells, MKP-1 mRNA was induced by fetal bovine serum or phorbol ester, the agents known to activate MAPK (10,11). The induction of MKP-1 by these growth-promoting agents may be responsible for the down-regulation of MAPK after growth stimuli as suggested by Sun et al. (16). In the present study, we demonstrated that ANP, which could not activate MAPK (10), rapidly increased the expression of MKP-1 mRNA and MKP-1 protein in cultured mesangial cells. This is the first report that the anti-proliferative agent could induce MKP-1 gene expression.
ANP-induced expression of MKP-1 might be mediated by cGMP-dependent pathway, because 8-Br-cGMP and SNP were also able to induce the expression of MKP-1, while C-ANP, an analogue specific to clearance receptors of ANP, was without effect. It has been reported that MKP-1 mRNA expression is induced by the activation of protein kinase C or cAMP-dependent kinase (protein kinase A) cascade (19). The activation of protein kinase C or cAMP-dependent kinase (protein kinase A) has been shown to activate transcription factor(s) which bind to the 12-O-tetradecanoylphorbol-13-acetate responsive element or to cAMP responsive element. However, little information is available on how the cGMP-dependent signal transduction may influence gene expression. Recently, nitric oxide-releasing agents and the membrane permeable analogue of cGMP have been reported to activate transcription from AP-1 responsive promoters in rodent fibroblast and epithelial cell line (32). Since the human MKP-1 gene (CL100) contains one AP-1 site in the region upstream of the transcription start site (33), we hypothesize that ANP may induce the expression of the MKP-1 gene through the activation of this AP-1 site.
Constitutive expression of MKP-1 has been shown to attenuate serum-or oncogenic ras-induced MAPK activation (16,22), to block MAPK-dependent gene expression (23,24), and to inhibit cell proliferation (19,22), suggesting that the inactivation of MAPK in vivo by MKP-1 has a negative effect on cell proliferation. In the present study, when MKP-1 protein was maximally induced by exposing the cells to ANP for 120 min, phorbol ester-induced activation of MAPK was inhibited, while the activation of MEK was not affected, indicating that MAPK cascade was blocked by ANP at the level of MAPK. These date indicate that ANP may negatively regulate MAPK through the induction of MKP-1, leading to the inhibition of the proliferation of mesangial cells. We have previously reported that phorbol ester-induced activation of MAPK was inhibited by ANP in cultured mesangial cells when the cells were treated with ANP for only 10 min (10). Ten-min incubation periods might not be enough for ANP to induce the expression of MKP-1 protein and ANP has been shown to directly suppress phorbol ester-induced activation of protein kinase C in mesangial cells (34). Therefore, ANP is able to attenuate phorbol ester-induced activation of MAPK through two independent mechanisms; one might be the inhibition at a step proximal to MAPK as a short term effect and the other is due to the induction of MKP-1 expression as a relatively long term effect shown in the present study. Thus, MKP-1 induction by ANP shown in the present study might provide a new mechanism of anti-proliferative action of ANP in glomerular mesangial cells.