(Received for publication, September 14, 1995; and in revised form, October 27, 1995)
From the
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) ()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, 7, 8, 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, 7, 8, 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
activities of MAPK or MEK were determined by MBP kinase assay as
described previously(26, 27, 28) . The
immunoprecipitates with anti-ERK2 antibody were incubated with 20
µg of MBP in 40 µl of a kinase buffer containing 2 µCi of
[-
P]ATP for 15 min at 30 °C. The
immunoprecipitates with anti-MEK antisera were incubated first with 2
µg of GST-MAPK in a kinase buffer at 30 °C for 15 min and then
with 2 µCi of [
-[
P]ATP and 20
µg of MBP at 30 °C for an additional 10 min.
Figure 1:
Effects of
FBS or PDBu on MKP-1 mRNA expression in cultured glomerular mesangial
cells. Mesangial cells were made quiescent by reducing the
concentrations of FBS for 72 h and treated with 2.5% FBS (lane
2), 100 nM PDBu (lane 3), or vehicle (lane
1) for 60 min. Total RNA (10 µg) was subjected to Northern
blotting analysis. The filter was hybridized with a P-labeled HVH-1 probe and then rehybridized with a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe as
indicated.
Figure 2: Time course of ANP-induced expression of MKP-1 mRNA in cultured glomerular mesangial cells. The quiescent mesangial cells were treated with 100 nM ANP for the indicated time interval. Total RNA (10 µg) was subjected to Northern blot analysis as described in the legend to Fig. 1. A representative autoradiogram is shown in A and the results of a densitometric analysis of MKP-1 mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA are shown in B (mean ± S.D., n = 3).
Figure 3: Dose dependence of ANP-induced expression of MKP-1 mRNA in glomerular mesangial cells. The quiescent mesangial cells were treated with various concentrations of ANP for 30 min. Total RNA (10 µg) was subjected to Northern blot analysis as described in the legend to Fig. 1. A representative autoradiogram is shown in A and the results of a densitometric analysis of four independent experiments are shown in B (mean ± S.D.). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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).
Figure 4: Effects of 8-Br-cGMP, a cell permeable analogue of cGMP, or SNP on MKP-1 mRNA expression in glomerular mesangial cells. The quiescent mesangial cells were treated with 1 mM 8-Br-cGMP (A) or 0.1 mM SNP (B) for the indicated time interval. Total RNA (10 µg) was subjected to Northern blot analysis as described in the legend to Fig. 1. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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) .
Figure 5: Effect of ANP on MKP-1 protein expression in glomerular mesangial cells. The quiescent mesangial cells were treated with 100 nM ANP for the indicated time interval and then immunoblot analysis was performed using anti MKP-1 antibody. A representative result from three experiments is shown.
Figure 6: Effect of ANP on PDBu-induced activation of MAPK cascade in glomerular mesangial cells. The quiescent mesangial cells were exposed to 100 nM ANP or vehicle for 120 min and then stimulated 10 nM PDBu for 10 min. After immunoprecipitation, the activities of MAPK (A) were measured using MBP as a substrate and the activities of MEK (B) were evaluated by measuring its ability to activate GST-MAPK. Values shown are mean ± S.D., n = 3. *, p < 0.05 versus control; **, p < 0.01 versus control, #p < 0.05 versus PDBu.
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.