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Angiotensin II-Induced Mitogen-Activated Protein Kinase Phosphatase-1 Expression in Bovine Adrenal Glomerulosa Cells: Implications in Mineralocorticoid Biosynthesis

Division of Endocrinology, Diabetology and Nutrition (A.J.C., A.M.C., C.F.W.-B.) and Department of Gynecology and Obstetrics (S.R.), University Hospital, CH-1211 Geneva 14, Switzerland

Address all correspondence and requests for reprints to: Prof. Alessandro M. Capponi, Division of Endocrinology, Diabetology and Nutrition, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: alessandro.capponi{at}medecine.unige.ch.

	Abstract

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Angiotensin II (AngII) stimulates aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. AngII also triggers the MAPK pathways (ERK1/2 and p38). Because ERK1/2 phosphorylation is a transient process, phosphatases could play a crucial role in the acute steroidogenic response. Here we show that the dual specificity (threonine/tyrosine) MAPK phosphatase-1 (MKP-1) is present in bovine adrenal glomerulosa cells in primary culture and that AngII markedly increases its expression in a time- and concentration-dependent manner (IC₅₀ = 1 nM), a maximum of 548 ± 10% of controls being reached with 10 nM AngII after 3 h (n = 3, P < 0.01). This effect is completely abolished by losartan, a blocker of the AT₁ receptor subtype. Moreover, this AngII-induced MKP-1 expression is reduced to 250 ± 35% of controls (n = 3, P < 0.01) in the presence of U0126, an inhibitor of ERK1/2 phosphorylation, suggesting an involvement of the ERK1/2 MAPK pathway in MKP-1 induction. Indeed, shortly after AngII-induced phosphorylation of ERK1/2 (220% of controls at 30 min), MKP-1 protein expression starts to increase. This increase is associated with a reduction in ERK1/2 phosphorylation, which returns to control values after 3 h of AngII challenge. Enhanced MKP-1 expression is essentially due to a stabilization of MKP-1 mRNA. AngII treatment leads to a 53-fold increase in phosphorylated MKP-1 levels and a doubling of MKP-1 phosphatase activity. Overexpression of MKP-1 results in decreased phosphorylation of ERK1/2 and aldosterone production in response to AngII stimulation. These results strongly suggest that MKP-1 is the specific phosphatase induced by AngII and involved in the negative feedback mechanism ensuring adequate ERK1/2-mediated aldosterone production in response to the hormone.

	Introduction

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ALDOSTERONE, THE MAIN mineralocorticoid hormone, is synthesized primarily in the zona glomerulosa of the adrenal cortex, under the control of two major physiological stimuli, the octapeptide hormone angiotensin II (AngII) and extracellular potassium (K⁺) (1). Aldosterone is synthesized from cholesterol, the common precursor of all steroid hormones, which is stored as cholesterol esters within intracellular lipid droplets and is subsequently mobilized to the mitochondrion, upon stimulation, after hydrolysis by cholesterol ester hydrolase (2). The rate-limiting step in the activation of steroidogenesis is the delivery of cholesterol from the outer mitochondrial membrane to the inner membrane, where the cytochrome P450 side-chain cleavage enzyme is located (3). Within the mitochondria, cholesterol undergoes an enzymatic cascade leading eventually to the formation of aldosterone.

AngII-induced aldosterone biosynthesis is mediated through the AT₁ receptor subtype (4). The intracellular signaling pathways leading from receptor binding of the hormone to the mitochondrial production of aldosterone are still not fully understood. In most of its target cells, AngII rapidly activates various members of the MAPK family, such as extracellular signal-regulated kinases ERK1/2, p38 MAPK, Janus kinase, or ERK5 (5, 6). In bovine adrenal zona glomerulosa (BAG) cells, AngII stimulation leads to a rapid and transient phosphorylation of ERK1/2 and p38 MAPK, peaking within 5 min and returning to basal levels after 1 h (2, 7, 8). This activation occurs via activation of protein kinase C and the ras/raf-1 kinase (7). Similar kinetics are observed under stimulation of bovine glomerulosa cells with lysophosphatidic acid, which also causes phosphorylation of Src, Pyk, epidermal growth factor receptor, and Akt (9). In rat glomerulosa cells, binding of some integrins to a fibronectin matrix results in activation of ERK1/2 and cell proliferation (10) and AngII stimulates protein synthesis and inhibits proliferation via opposite mechanisms implying both ERK1/2 and p38 MAPK (8).

Recent work performed in several steroidogenic cell lines has shown that cAMP or heat shock induce a transient expression of MKP-1 and that there is, in particular, an important cross-talk between the MAPK and the ACTH/cAMP pathway. In Y1 mouse adrenocortical tumor cells, ACTH potently induces the expression of MKP-1 (11, 12). In H295R human adrenocortical carcinoma cells, activation of the cAMP/protein kinase A (PKA) signaling pathway leads to a robust expression of MKP-1, which is required for transcriptional activation of a specific target gene, i.e. CYP17 (13). In mouse adrenocortical Y1 cells, the steroidogenic effect of forskolin, an activator of adenylyl cyclase, occurs via activation of ERK1/2, which phosphorylates steroidogenic factor 1, leading to increased steroidogenic acute regulatory (StAR) mRNA and protein expression and, as a result, to increased steroid production (14).

In genuine glomerulosa cells, ERK1/2 function is essentially related to mitogenic and cell growth responses (15). Although a link between AngII-induced ERK1/2 phosphorylation and aldosterone production has not yet been directly demonstrated (15), ERK1/2 and p38 are clearly involved in AngII-stimulated expression of the 3β-hydroxysteroid dehydrogenase (3β-HSD) and StAR protein in Long Evans rat adrenal glomerulosa cells (16), a finding that clearly indicates a contribution of these kinases to the mineralocorticoid response to AngII.

The transient phosphorylation of ERK1/2 by AngII in glomerulosa cells suggests the intervention of phosphatase(s). In vascular smooth muscle cells, ERK1/2 inactivation results from AngII-induced expression of MAPK phosphatase (MKP)-1, a nuclear dual-specificity phosphatase (17, 18), and this effect requires activation of yet another tyrosine kinase, Jak2 (19). Interestingly, in rat neuroendocrine cells, MKP-1 gene transcription is modulated by a block to elongation which is sensitive to changes in intracellular calcium, a signal that is also triggered by AngII (20). In Y1 mouse adrenocortical tumor cells, ACTH stimulates the expression of MKP-1 mRNA and protein, a response that is only partially mediated by cAMP and PKA (11). Finally, in H295R human adrenocortical carcinoma cells, a transient, PKA-dependent induction of MKP-1 contributes to cAMP-induced transcription of the CYP17 hydroxylase enzyme (13).

MAPKs are a large class of proteins involved in signal transduction pathways that are activated by a range of stimuli and mediate a number of physiological and pathological changes in the cell (21). The activation of ERK1/2 results from phosphorylation by MAPK kinase (MEK1/2). However, activation of MAPKs is a reversible process, even in the continued presence of activating stimuli, indicating that protein phosphatases are also likely to provide an important mechanism for control. MKPs, also called dual specificity phosphatases, are a subclass of the protein tyrosine phosphatase gene superfamily, which selectively dephosphorylate critical phosphothreonine and phosphotyrosine residues within MAPKs (17). MKPs gene expression is induced by a host of growth factors and cellular stresses, thereby negatively regulating the activity of MAPK superfamily members including MAPK/ERK, stress-activated protein kinase/Janus kinase, and p38 (17).

In the present work we have focused on MKP-1, a member of the MKP family, and examined whether this protein is present in BAG cells in primary culture and whether its expression is modulated by AngII, thus contributing to the fine tuning of aldosterone biosynthesis.

	Materials and Methods

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Bovine adrenal zona glomerulosa (BAG) cell culture and treatments
Bovine adrenal glands were obtained from a local slaughterhouse. Zona glomerulosa cells were prepared by enzymatic dispersion with dispase and purified on Percoll density gradient as previously reported (22). Primary cultures of purified glomerulosa cells were maintained in DMEM as described in detail elsewhere (22). The cells were grown in 6-cm Petri dishes (2 x 10⁶ cells per dish) and kept in serum-free medium for 24 h before experiments, which were performed on the third day of culture. Cells were then washed and incubated at 37 C in serum-free medium containing various agents, for varying periods of time as appropriate. At the end of the incubation period, the media were collected and cells were processed for protein or total RNA extraction as described hereafter.

Transfection of adrenal glomerulosa cell
BAG cells in primary culture were transiently transfected with a lipid-based method (Effectene Transfection Reagent; Qiagen GmbH, Hilden, Germany). Half a million cells were seeded in each well of a 12-well plate, and a total of 0.3 µg of DNA (corresponding to human pSG5-MKP-1-Myc; kindly provided by Dr. S. Keyse, University of Dundee, Scotland, UK) was transfected according to manufacturer’s protocol the day after cell seeding. On the following day, cells were treated with AngII for various periods of time.

Determination of aldosterone production
Aldosterone content in incubation media was measured by direct RIA using a commercially available kit (Diagnostic Systems Laboratories, Webster, TX). Aldosterone production was normalized and expressed per microgram of cellular protein.

Western blot analysis
For the determination of protein expression levels, bovine glomerulosa cells were washed twice in ice-cold PBS, and total cellular extracts were prepared as described elsewhere (23). Proteins were quantified using a protein microassay (Bio-Rad, Munich, Germany). Equal amounts of protein (15 µg) were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Western blot analyses of MKP-1 and of phosphorylated and total ERK1/2 proteins were carried out with polyclonal rabbit antiserums directed against MKP-1 (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated MKP-1 and phosphorylated ERK1/2 (New England Biolabs Inc., Beverly, MA), total ERK1/2 (Santa Cruz Biotechnology), and myc (24). The membranes were then washed with the same buffer without milk and then incubated for 1 h with horseradish peroxidase-labeled goat antirabbit (CovalAb, Oullins, France) or rabbit antigoat (Sigma-Aldrich, Buchs, Switzerland) antibodies. Immunoreactive proteins were visualized by the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Dübendorf, Switzerland). Results were normalized either by using a nonspecific band in the Western blots for MKP-1 or by stripping the membranes and labeling them again with an antibody directed against the auxiliary transcription factor TFIIE.

Total RNA extraction and semiquantitative RT-PCR
Glomerulosa cell total RNA was extracted using the TRIzol Reagent (Invitrogen, Basel, Switzerland) according to the instructions of the manufacturer. This system consistently yields 50–80 µg total RNA/10⁷ cells. RT-PCR was used to evaluate MKP-1 mRNA abundance in response to various treatments. Total RNA (500 ng) was amplified with the one-step RT-PCR Access system (Promega Corp., Madison, WI) according to the instructions of the manufacturer. The primers were: human/bovine MKP-1 5'-CAA GCG CGA CGG CAC CCT-3' (forward) and 5'-GCC CAT GAA GCT GAA GTT-3' (reverse), corresponding to positions +521–538 and +1125–1142 (National Center for Biotechnology Information, GenBank, accession no. X68277), respectively; bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 5'-ATG GTG AAG GTC GGA GTG-3' (forward) and 5'-TGC AGA GAT GAT GAC CCT C-3' (reverse), corresponding to positions +82–99 and +426–444 (National Center for Biotechnology Information, GenBank, accession no. NM002046), respectively. Primers yielded products of the expected size corresponding to 362 bp for GAPDH and 621 bp for MKP-1. Ten microliters of the PCR products were analyzed on 1.2% agarose gel and quantified by densitometry. All mRNA levels were normalized to GAPDH mRNA.

Measurement of phosphatase activity
Phosphorylated MKP-1 activity was determined by measuring its ability to hydrolyze p-nitrophenyl phosphate (p-NPP) as described elsewhere (25). Phosphorylated MKP-1 was first immunoprecipitated from total bovine glomerulosa cell extracts with a specific phospho-MKP-1 antibody linked to agarose beads (MiniLeak slurry; Kem En tec, Copenhagen, Denmark), according to the instructions of the manufacturer. Total cellular protein extract was incubated with lysis buffer without phosphatase inhibitors for 10 min on ice, vortexed, placed again on ice for 10 min, and centrifuged for 5 min at 12,000 rpm in an Eppendorf centrifuge. Two hundred microgram proteins were adjusted to a volume of 400 µl of lysis buffer and incubated overnight with the antibody-slurry complex at 4 C on a tube rotator. The mixture was finally washed four times with lysis buffer. Immunoprecipitated phospho-MKP-1 phosphatase activity was then measured in 200 µl of solution containing 10 mM imidazole (pH 7.5), 0.1% β-mercaptoethanol, and 10 mM p-NPP. After 30 min of incubation at 30 C, the reaction was stopped by addition of 500 µl of 0.25 M NaOH. Absorbance at 410 nm was then measured. Nonspecific p-NPP hydrolysis in immunoprecipitates obtained with an unrelated antibody (anti-TFIIE) was subtracted from the data.

Analysis of data
Results are expressed as means ± SEM. Mean values were compared by ANOVA using Fisher’s test. A value of P < 0.05 was considered as statistically significant. Quantification of immunoblots and autoradiograms was performed using a Molecular Dynamics (Sunnyvale, CA) computing densitometer.

	Results

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MKP-1 is present in BAG cells and its expression is modulated by AngII in a time- and concentration-dependent manner
We first examined whether bovine glomerulosa cells in primary culture express MKP-1 protein. Indeed, as shown in Fig. 1A, resting BAG cells do express MKP1, although at rather low levels, as determined by Western blot analysis. AngII treatment led to a robust, concentration-dependent induction of MKP-1 protein expression, a maximal stimulation of approximately 5.5-fold being reached with 10 nM AngII (n = 3, P < 0.01). In addition, this stimulatory effect of AngII was prevented by a simultaneous treatment with 10 µM losartan, an antagonist of the AT₁ receptor subtype (150 ± 35% of control, n = 3, NS). A role for the AT₂ receptor subtype in AngII-induced MKP-1 expression was excluded by the fact that, when BAG cells were treated with AngII in the presence and in the absence of 10 µM PD123319, an antagonist of the AT₂ receptor subtype, AngII-induced MKP-1 expression was not affected (Fig. 1B).

View larger version (17K): [in this window] [in a new window]   FIG. 1. AngII induces MKP-1 protein expression in a concentration-dependent manner via the AT1 receptor subtype. A, BAG cells were treated for 3 h with either increasing concentrations of AngII or with 10 nM AngII and 1 µM losartan. Top, Representative Western blot, performed as described in Materials and Methods; bottom, densitometric analysis. B, Bovine glomerulosa cells were treated with either 10 nM AngII or 1 µM PD123319 or both AngII and PD123319 for 3 h. Top, Representative Western blot; bottom, densitometric analysis. All results were normalized with the top nonspecific band (NS). Results are expressed as means ± SEM, n = 3. * and **, Significantly different from control value with P < 0.05 and P < 0.01, respectively.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Kinetics of MKP-1 protein induction and ERK1/2 phosphorylation by AngII. Adrenal glomerulosa cells were treated with 10 nM AngII for the indicated periods of time. Total proteins were extracted and Western blot for MKP-1 was performed as described in Materials and Methods. The same membrane was then stripped and reprobed for phosphorylated ERK1/2 (P-ERK1/2) proteins. A, Representative Western blots for MKP-1 and P-ERK1/2. B, Densitometric analysis of data from three separate cell preparations. All results were normalized with the top nonspecific band (NS). Results are expressed as means ± SEM. * and **, Significantly different from control value with P < 0.05 and P < 0.01 for MKP-1 protein expression, respectively. , Significantly different from control value with P < 0.01 for P-ERK1/2.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Effect of AngII on MKP-1 mRNA stability. Bovine glomerulosa cells were first incubated without or with 10 nM AngII for 10 min; at this point (time 0'), cells were then treated with 2.5 µg/ml actinomycin D in the presence or in the absence of 10 nM AngII for various periods of time. Total RNA was extracted and semiquantitative RT-PCR using gene-specific primers for MKP-1 was performed as described in Materials and Methods. A, Representative Northern blot. B, Densitometric analysis. Results were normalized to the GAPDH mRNA levels. Results are expressed as means ± SEM from three separate experiments. * and **, Significantly different from control value with P < 0.05 and P < 0.01, respectively.

View larger version (15K): [in this window] [in a new window]   FIG. 4. AngII-induced MKP-1 protein expression requires ERK1/2 phosphorylation. BAG cells were treated for 3 h with 10 nM AngII after a 15-min preincubation without or with 10 µM U0126. Total proteins were extracted and Western blot for MKP-1 was performed as described in Materials and Methods. A, Representative Western blot. B, Densitometric analysis. Results are expressed as means ± SEM, n = 3. **, Significantly different with P < 0.01.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Effect of blockade of transcription and protein synthesis blockade on MKP-1 protein levels and ERK1/2 phosphorylation in AngII-treated BAG cells. Bovine glomerulosa cells were first incubated without or with 10 nM AngII for 10 min, and were then treated with 2.5 µg/ml actinomycin D (Act D) or 10 µg/ml cycloheximide (CHX), in the presence or in the absence of 10 nM AngII, for 3 h. Total proteins were extracted and Western blot for MKP-1 and phosphorylated ERK1/2 was performed as described in Materials and Methods. A, Representative Western blots. B, Densitometric analysis of MKP-1 Western blots from three independent experiments. C, Densitometric analysis of P-ERK1/2 (n = 3). Results are expressed as means ± SEM. **, Significantly different from control value with P < 0.01.

View larger version (13K): [in this window] [in a new window]   FIG. 6. Effect of AngII on MKP-1 phosphorylation. BAG cells were treated for 3 h with 10 nM AngII after a 15-min preincubation without or with 10 µM U0126. Total proteins were extracted and Western blot for phosphorylated MKP-1 was performed as described in Materials and Methods. A, Representative Western blots. B, Densitometric analysis of MKP-1 Western blots from three independent experiments. Western blotting of the auxiliary transcription factor TFIIE was used for normalization. Results are expressed as means ± SEM. **, Significantly different from control value with P < 0.01.

View larger version (11K): [in this window] [in a new window]   FIG. 7. Modulation of MKP-1 phosphatase activity under AngII treatment. BAG cells were treated for 3 h with 10 nM AngII after a 15-min preincubation without or with 10 µM U0126. Total proteins were extracted and phospho-MKP-1 was immunoprecipitated. Precipitates were then used for Western blotting or for a colorimetric assay of phosphatase activity as described in Materials and Methods. A, Western blot showing specificity of immunoprecipitation; a phospho-MKP-1 band is observed after treatment with a specific anti-phospho-MKP-1 antibody but not with an unrelated antibody directed against the auxiliary transcription factor TFIIE. B, Colorimetric measurement of phosphatase activity. Results are expressed as means ± SEM of two separate experiments performed in triplicates.

View larger version (9K): [in this window] [in a new window]   FIG. 8. Inhibition of ERK1/2 activation prevents AngII-induced aldosterone production. BAG cells were treated for 3 h with 10 nM AngII after a 15-min preincubation without or with 10 µM U0126. Aldosterone production in the incubation medium was then determined as described in Materials and Methods. Results are expressed as means ± SEM from three independent cell preparations. **, Significantly different with P < 0.01.

View larger version (16K): [in this window] [in a new window]   FIG. 9. Effect of MKP-1 overexpression on aldosterone production. Bovine adrenal glomeruosa cells were transiently transfected with MKP-1 cDNA as described under Materials and Methods. Two days later, cells were challenged with 10 nM AngII for 2 h. Total RNA was extracted, and semiquantitative RT-PCR using gene-specific primers for MKP-1 was performed as described in Materials and Methods. In parallel samples, total proteins were extracted and Western blot for MKP-myc was performed. Aldosterone production was determined in the incubation media. A, Representative agarose gel of PCR products. NT, Nontransfected cells; pSG5, cells transfected with empty vector; pSG5-MKP-1, cells transfected with vector containing MKP-1-myc cDNA. B, Representative Western blot. Coomassie blue staining was performed to ensure identical loading. C, Aldosterone production. Results are expressed as means ± SEM from three independent experiments. NS, Not significantly different; **, significantly different with P < 0.01; , significantly different from respective nonstimulated control with P < 0.01.

Effect of MKP-1 overexpression on ERK1/2 activation
We next examined ERK1/2 phosphorylation levels by Western blot analysis in response to AngII. As shown in Fig. 10, A and B, AngII treatment for 15 min—a time corresponding approximately to the maximal activation of ERK1/2 in control cells (Fig. 2)—led to a significant increase in ERK1/2 phosphorylation levels in nontransfected and mock-transfected bovine glomerulosa cells, reaching 317 ± 51 and 309 ± 32%, respectively (n = 3, P < 0.01 vs. nonstimulated cells for both values). In contrast, this AngII-induced phosphorylation of ERK1/2 was markedly reduced in cells overexpressing MKP-1, reaching only 205 ± 29% of nonstimulated controls (n = 3, P < 0.01 vs. AngII-induced ERK1/2 phosphorylation in mock-transfected cells). Thus, the levels of ERK1/2 phosphorylation were inversely correlated with MKP-1 expression levels.

View larger version (16K): [in this window] [in a new window]   FIG. 10. Effect of MKP-1 overexpression on ERK1/2 activation. MKP-1 was overexpressed in bovine glomerulosa cells as expressed in Fig. 9. Cells were then treated with 10 nM AngII for 15 min. Total proteins were then extracted for Western blot analysis of P-ERK1/2 and aldosterone content of the incubation media was determined. A, Representative Western blot of P-ERK1/2 and total ERK1/2. B, Densitometric analysis of MKP-1 Western blots from three independent experiments. C, Aldosterone production (n = 3). **, Significantly different with P < 0.01; , significantly different from respective nonstimulated control with P < 0.01.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two major original conclusions can be drawn from the present work: 1) ERK1/2 activation is an important mediator of AngII-induced aldosterone biosynthesis, and 2) AngII triggers a powerful negative feedback on its own response via a robust increase in MKP-1 expression, phosphorylation levels, and phosphatase activity.

The present work directly substantiates previous results showing an involvement of the ERK1/2 pathway in AngII-induced expression of two proteins that are crucial for aldosterone biosynthesis, 3β-HSD and StAR (16). It has been known for some time that AngII challenge leads to a rapid and transient phosphorylation of both ERK1/2 (p42/44) and p38 MAPK (MAPK) in bovine (7, 26) and murine (8, 10, 16) adrenal glomerulosa cells. This AngII-induced ERK1/2 and p38 MAPK activation has been clearly shown to play a crucial role in rat glomerulosa cell adhesion (10), protein synthesis, and inhibition of glomerulosa cell proliferation (8). Similarly, the steroidogenic response to factors activating the cAMP signaling pathway is also mediated by ERK1/2 activation, at least in mouse Y1 adrenocortical cells, where this results in increased expression of the StAR protein (14).

In BAG cells, AngII-mediated activation of ERK1/2 occurs via protein kinase C and Ras/Raf-1 kinase activation (7), in contrast to other AngII target cells, where epidermal growth factor receptor transactivation is the main process of ERK1/2 activation (27). Incidentally, epidermal growth factor receptor transactivation also mediates lysophosphatidic acid-induced ERK1/2 phosphorylation in bovine glomerulosa cells (9).

AngII-induced ERK1/2 activation in bovine glomerulosa cells is a reversible process involving most likely the intervention of some phosphatase(s). In the present work, we have also investigated the mechanism responsible for the fact that this AngII-induced ERK1/2 activation is only transient in BAG cells in primary culture. For this purpose, we focused our attention on the role of one likely candidate phosphatase, MKP-1, in this process. Several conclusions can be drawn in this respect: 1) MKP-1 protein expression and phosphorylation levels are robustly induced in BAG cells in primary culture in response to AngII; 2) AngII stabilizes MKP-1 mRNA; 3) AngII-induced MKP-1 expression depends upon ERK1/2 activation and is inversely correlated with ERK1/2 phosphorylation levels; 4) AngII enhances MKP-1 phosphorylation and phosphatase activity; and 5) MKP-1 overexpression prevents AngII-induced mineralocorticoid biosynthesis.

First, MKP-1 is induced by AngII in glomerulosa cells. Several studies have extensively described the ability of MKPs to inactivate MAPKs by protein dephosphorylation in various cell systems (17, 18). In the present work, AngII induced a rapid and transient activation ERK1/2 and a delayed, robust expression and phosphorylation of MKP-1 protein. Although AngII effects on MKP-1 expression have been reported in other AngII target cell types such as cardiomyocytes (28) and vascular smooth muscle cells (19, 29), to date no description of MKP-1 protein expression and phosphatase activity in response to AngII in a steroidogenic model has been presented.

Second, AngII increased MKP-1 protein levels essentially by stabilizing MKP-1 mRNA. MKP-1 protein expression has been shown to be under tight transcriptional control in various cell systems (20, 30, 31). In this study, we report rapid increases in MKP-1 mRNA levels within 30 min in response to AngII challenge, in agreement with previous kinetic observations in other cellular models (11, 20, 32). Interestingly, as observed, for example, with other proteins that are rapidly induced by AngII in vascular smooth muscle cells (33), the mechanism underlying the observed increase in MKP-1 mRNA and protein expression in bovine glomerulosa cells involved mainly a stabilization of MKP-1 mRNA, although we cannot exclude some transcriptional effect on the MKP-1 gene. A rapid MKP-1 mobilization thus appears to be required for an appropriate control of acutely induced aldosterone biosynthesis.

Third, the fact that maximum ERK1/2 activity in response to AngII correlates with increased amounts of MKP-1 protein, suggests that MKP-1 expression depends upon ERK1/2 phosphorylation and activation. Indeed, as shown here by using specific inhibitors, AngII effects on MKP-1 expression are mediated through ERK1/2 activation. On the other hand, ERK1/2 has been described as a target for MKP-1 (19, 34, 35). Therefore, we hypothesized that this could also be the case in bovine glomerulosa cells and that MKP-1 could be the specific phosphatase involved in the negative feedback mechanism of ERK1/2 inactivation after AngII treatment. Using pharmacological tools to block transcription and translation, we observed that when MKP-1 mRNA or protein synthesis were inhibited, ERK1/2 phosphorylation levels were sustained after AngII stimulation, in contrast with the transient response recorded under normal conditions. Interestingly, under basal conditions, protein or mRNA synthesis blockade induced a significant increase in ERK1/2 phosphorylation levels, probably by blocking any basal MKP-1 activity. Taken together, these results corroborate our hypothesis of a negative feedback for the control of ERK1/2 activity in response to AngII involving the MKP-1 phosphatase in glomerulosa cells.

Fourth, AngII treatment led to a marked increase in MKP-1 phosphorylation levels. Although phosphatases often undergo a phosphorylation-dependent modulation of their activity (36, 37), phosphorylation of MKP-1 on serine residues 359 and 364 has been shown to prolong the half-life of MKP-1 in hamster fibroblasts without directly altering its catalytic activity (25). In the end, however, such reduced MKP-1 degradation will lead to increased phosphatase activity. Indeed, we observed that the increase in MKP-1 expression under AngII challenge was associated with a marked enhancement of MKP-1 phosphorylation state, which was prevented by pharmacological blockade of ERK1/2 activation. As expected, this increased phosphorylation of the enzyme led to a doubling of the specific MKP-1 phosphatase activity in extracts from cells treated with AngII, as assessed by the ability to hydrolyze p-NPP after immunoprecipitation. Thus a positive correlation was observed between MKP-1 expression, phosphorylation and phosphatase activity.

Fifth, MKP-1 overexpression led to a partial suppression of AngII-induced aldosterone production. Concomitantly, we observed reduced phosphorylation levels of ERK1/2, a finding that strongly supports our view that MKP-1 is set to work to prevent exaggerated ERK1/2 and aldosterone responses to AngII. The fact that aldosterone production is only partially, although highly significantly reduced may reflect a less-than-optimal efficacy of the MKP-1 transfection system used in these experiments. Alternatively, other signaling pathways [Ca²⁺ (38), 12-lipoxygenase (39), etc.] may also contribute independently to activating the steroidogenic cascade.

In summary, we have identified MKP-1 as an important component of the signaling cascade mediating the steroidogenic response to AngII in BAG cells. MKP-1 induction by AngII appears to be critical in ensuring that AngII-elicited acute aldosterone production remains adequate and does not reach excessive levels.

	Acknowledgments

 
We thank Dr. S. Keyse (University of Dundee) for his generous gift of pSG5-MKP-1-Myc and Manuella Rey and Rachel Porcelli for their excellent technical assistance.

	Footnotes

 
This work was supported by Swiss National Science Foundation Grant 3100A0-112373 (to A.M.C.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 9, 2007

Abbreviations: AngII, Angiotensin II; BAG, bovine adrenal zona glomerulosa; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 3β-HSD, 3β-hydroxysteroid dehydrogenase; MEK, MAPK kinase; MKP, MAPK phosphatase; P-ERK1/2, phosphorylated ERK1/2; PKA, protein kinase A; p-NPP; p-nitrophenyl phosphate; StAR, steroidogenic acute regulatory.

Received February 20, 2007.

Accepted for publication July 25, 2007.

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