Angiotensin II Stimulates Protein Synthesis and Inhibits Proliferation in Primary Cultures of Rat Adrenal Glomerulosa Cells
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内分泌学杂志 2005年第2期
Service of Endocrinology (M.O., N.G.-P.), Department of Medicine, and Department of Physiology and Biophysics (S.C., M.D.P.), Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Faculty of Medicine, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail: nicole.gallo-payet@usherbrooke.ca.
Abstract
Angiotensin II (Ang II) is one of the most important stimuli of rat adrenal glomerulosa cells. The aim of the present study was to investigate whether Ang II can stimulate cell proliferation and/or hypertrophy and investigate pathways and intracellular targets. A 3-d treatment with Ang II (5–100 nM), through the Ang II type 1 receptor subtype, abolished cell proliferation observed in control cells but increased protein synthesis. Preincubation with PD98059 (a MAPK kinase inhibitor) abolished basal proliferation and had no effect on basal protein synthesis but did reverse the effect of Ang II on protein synthesis. The p38 MAPK inhibitor SB203580 reversed the inhibitory effect on cell proliferation and abolished the increase in protein synthesis, whereas the c-Jun N-terminal kinase inhibitor SP600125 had no effect. Time-course studies revealed that Ang II stimulated phosphorylation of both p42/p44mapk and p38 MAPK but did not activate c-Jun N-terminal kinase. Ang II had no effect on the level of cyclin E expression but increased the expression of the cyclin-dependent kinase, p27Kip1, an effect abolished in cells preincubated with SB203580 and PD98059. In conclusion, in cultured rat glomerulosa cells, a 3-d treatment with Ang II increases protein synthesis, with a concomitant decrease in proliferation. These effects are mediated by both the p42/p44mapk and p38 MAPK pathways, which increase expression of the steroidogenic enzymes, steroidogenic acute regulatory protein and 3?-hydroxysteroid dehydrogenase and p27Kip1, a protein known to block the cell cycle in G1 phase. Together these results support the key role of Ang II as a stimulus of steroid synthesis rather than a proliferating factor.
Introduction
THE ADULT ADRENAL cortex is composed of three concentric layers (zona glomerulosa, zona fasciculata, and zona reticularis), all of which have specific functional and morphological properties. The zona glomerulosa is specialized in aldosterone production and, in contrast to corticosterone secretion by the zona fasciculata, is under multifactorial regulation, with angiotensin II (Ang II) being the most important stimulus in vivo (for review see Refs.1, 2). In addition, glomerulosa cells have the ability to adapt their morphology to environmental conditions. For instance, it is well known that chronic administration of a low-sodium diet increases the number of Ang II type 1 (AT1) receptors (3, 4) and expands not only aldosterone secretion machinery (5) but also the width of the zona glomerulosa, an effect mainly associated with Ang II action (6, 7, 8, 9, 10, 11). However, the direct role of Ang II on either proliferation and/or hypertrophy is not yet clearly established. Indeed, McEwan et al. (11), using in vivo treatment with Ang II alone or in the presence of an AT1 receptor antagonist, demonstrated that Ang II stimulates proliferation but not hypertrophy of the glomerulosa cells. However, in rats receiving a sodium-deficient diet, the AT1 receptor antagonist DUP753 (Losartan, DuPont Merck Pharmaceuticals, Paris, France) failed to prevent hyperplasia of glomerulosa cells, in conditions in which proliferation in zona reticularis and smooth muscle cells were respectively blocked (11).
Both type 1 (AT1) and type 2 (AT2) Ang II receptors are present in rat adrenal zona glomerulosa, at a ratio varying from 60 to 80% AT1 and from 40 to 20% AT2 (12, 13, 14). Much of Ang II action on aldosterone secretion is largely due to activation of the Gq/phospholipase C-dependent hydrolysis of phosphatidylinositol 4,5 bisphosphate, leading to the production of inositol phosphates and diacylglycerol (2). On the other hand, as in several other G protein-coupled receptors, Ang II is able to activate the MAPK pathway, p42/p44mapk (also called ERK1/2) in bovine (15, 16) and rat (17, 18) glomerulosa cells. In vascular smooth muscle cells (VSMCs) and cardiomyocytes, Ang II likely activates both p38 MAPK (19) and c-Jun N-terminal kinase (JNK) (20, 21, 22).
Whereas it is well known that glomerulosa cells are often considered as the progenitor cells of the cortex, exhibiting the ability to both proliferate and migrate (23, 24, 25), the specific role of Ang II on either proliferation and/or hypertrophy is not yet clearly established. Some studies have suggested that Ang II may act as a direct mitogen. For instance, Ang II stimulates cell proliferation of cultured bovine fasciculata cells (16, 26) or in a bovine adrenocortical cell clone (27). To date, all studies addressing the effects of Ang II on adrenocortical cells have been performed using bovine glomerulosa or fasciculata cells. By contrast, there are no studies assessing these effects with rat adrenal glomerulosa cells. Previous studies from our group have shown that, in contrast to fasciculata cells, glomerulosa cells, either from rat or human origin, have the propensity to easily proliferate in culture, doubling their number after 3 d, even in a medium containing only 2% fetal serum (28, 29). Due to the important role of the zona glomerulosa in maintaining electrolyte homeostasis, defining how cell physiology (growth and steroidogenesis) is regulated by Ang II is a key concern because it is the main stimulus of aldosterone secretion. Thus, the objective of this study was to determine, using primary cultures of rat glomerulosa cells, whether Ang II induces cell proliferation and/or hypertrophy and clarify the role of the three MAPK pathways (ERK1/2, JNK, and p38 MAPK) in these events.
Materials and Methods
Chemicals
The chemicals used in the present study were obtained from the following sources: collagenase, MEM (MEM Eagle’s medium) and OPTI-MEM medium from Life Technologies (Burlington, Canada); Ang II from Bachem (Marina Delphen, CA); horseradish peroxidase-conjugated antirabbit and antimouse antibodies, enhanced chemiluminescence detection system and L-[2,3,4,5,6-3H] phenylalanine from Amersham Pharmacia Biotech (Oakville, Canada); Apoptag kit from Chemicon International Inc. (Temecula, CA); PD98059, the antibodies antiphosphorylated p42/p44mapk, antiphosphorylated p38, antiphosphorylated JNK, anti-p42/p44mapk, anti-p38, and anti-JNK from New England Biolabs, Inc. (Mississauga, Canada); the antibodies anticyclin E and anti-p27Kip1 from Santa Cruz Biotechnologies (Santa Cruz, CA); antitubulin, SB203580, and SP600125 from Chemicon (Mississauga, Canada); 5-bromo-2-deoxyuridine (BrdU) anti-BrdU Alexa Fluor-594, Alexa Fluor-594 phalloidin, and 4',6'-diamino-2-phenylindole (DAPI) from Molecular Probes (Eugene, OR); crystal violet, deoxyribonuclease, PD98059, and PD123319 from Sigma Chemical Co. (St. Louis, MO); polyvinylidene difluoride membranes from Millipore (Bedford, MA); and DC protein assay from Bio-Rad Laboratories (Hercules, CA). All other chemicals were of grade A purity.
Preparation of glomerulosa cell cultures
Zona glomerulosa cells were obtained from adrenal glands of female Long Evans rats weighing 200–250 g and isolated according to the method previously described in detail (30). All protocols were approved by the Animal Care and Ethics Committee of our faculty. Isolation and cell dissociation of the zona glomerulosa was performed in MEM medium (supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin). After a 20-min incubation at 37 C with collagenase (2 mg/ml) and deoxyribonuclease (25 μg/ml), cells were disrupted by gentle aspiration with a sterile 10-ml pipette, filtered, and centrifuged for 10 min at 100 x g. The cell pellet was then resuspended in OPTI-MEM medium supplemented with 2% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Depending on experiments, cells were plated at various densities in plastic 35-mm petri dishes (5 x 105 cells/dish) for Western blots analyses, 24-well plates (70 x 103 cells/well) for protein synthesis measurements, and 96-multiwell plates (70 x 103 cells/plate) for proliferation assays. Cells were cultured at 37 C in a humidified atmosphere composed of 95% air-5% CO2. The culture medium was changed daily, and cells were used after 3 d of culture. Except in specific experiments, cells were stimulated with 5 nM Ang II for 3-d treatments and 100 nM for acute stimulations. Cells were examined daily, and phase-contrast images were taken using a microscope (Leica Corp., Deerfield, IL) equipped with a x32 objective.
Proliferation assays
The method used to evaluate cell proliferation was adapted from that described by Gillies et al. (31) and Campbell et al. (32). Cells were plated on plastic 96-well plates at a concentration of 30 x 103 cells/well. Cells were treated daily for 3 d without or with Ang II alone or in the presence of various inhibitors. Concentrations used for the inhibitors corresponded to their dissociation constant, i.e. to inhibit specific function, without altering cell viability: PD98059 (5, 10, and 20 μM); SB203580 and SP600125 (1, 5, and 10 μM). On the third day, cells were fixed with 3.7% (vol/vol) formaldehyde in Hanks’ buffered saline (HBS) (in millimoles): NaCl 130; KCl 3.5; CaCl2/2H2O 2.3; MgCl2/6H2O 0.98; HEPES 5; EGTA 0.5) for 10 min at room temperature. After HBS washing, cells were incubated for 10 min at room temperature with 0.1% crystal violet/H2O. After extensive washes, cells were lysed for 10 min in 100 μl of 1% sodium dodecyl sulfate solution and ODs read at 595 nm with a μQuant spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT). For each experiment, a standard curve was performed using serial dilutions of a cell solution in which cell number was evaluated with the aid of a hemacytometer.
Cell proliferation was also measured using fluorescence BrdU incorporation, as described elsewhere (33). Cells were plated on plastic 96-well plates at a concentration of 30 x 103 cells/well. Cells were treated daily for 3 d without or with Ang II alone or in the presence of various inhibitors. After 24 h of culture, 10 μM BrdU was added to the culture medium 4 h before stimulation with Ang II. On the third day, cells were fixed with 3.7% (vol/vol) formaldehyde in HBS for 10 min at room temperature and permeabilized for 10 min with 0.2% Triton X-100 in HBS. Cells were then incubated with anti-BrdU Alexa Fluor-594 (1:500). Fluorescence intensity was determined using a microplate fluorescence reader FL600 (Bio-Tek) (excitation 56,040 nm; emission 64,540 nm). Results are expressed as the percent changes from basal conditions using six culture wells for each experimental condition.
Protein synthesis measurements
Cells were plated on plastic 24-well plates at a concentration of 70 x 103 cells/well, in quadruplicate. Cells were treated daily with hormones without or with various inhibitors for 3 d, as described for proliferation assays. A first duplicate cell series was washed twice with ice-cold HBS buffer and lysed in 50 μl of 50 mM HEPES (pH 7.8) and 1% Triton X-100. Protein content for each experimental condition was determined using the Bio-Rad DC method. The second duplicate cell series was used to evaluate exact cell number with the aid of a hemacytometer.
The relative amount of protein synthesis was also determined by assessing tritiated phenylalanine incorporation. Cells were plated on plastic 24-well plates at a concentration of 70 x 103 cells/well. After 24 h of culture, 1 μCi/ml [-3H]phenylalanine was added to the media 2 h before stimulation with Ang II. After 3 d, the medium was aspirated and cells washed three times with cold HBS solution, followed by addition of trichloroacetic acid solution (TCA) (20%) for 20 min on ice. After centrifugation (3000 x g, 15 min, 4 C), the TCA-insoluble fraction was washed twice with TCA solution and solubilized in 0.1 N NaOH solution for 1 h on ice. Incorporation of radioactivity was measured by liquid scintillation counting (Beckman counter, Beckman Coulter, Inc., Fullerton, CA). Incorporation of radioactivity was measured by liquid scintillation counting (Beckman counter). Data were normalized as maximum of phenylalanine incorporation in control conditions for the same number of cells (1 x 105 cells), as described elsewhere (34).
In situ apoptosis detection
Cells were processed according to the protocol provided by the manufacturer of the Apoptag kit (Chemicon) using terminal deoxynucleotidyl transferase biotin-deoxyuridine 5-triphosphate nick end labeling fluorescence, as used previously (35). Cells were stimulated and fixed as described in proliferation assays. After fixation and equilibration, cells were incubated for 1 h and 30 min at 37 C in a solution containing the terminal deoxynucleotidyl transferase enzyme and the reaction buffer containing digoxigenin-labeled nucleotides. Cells were then washed twice in HBS and incubated with an antidigoxigenin fluorescein isothiocyanate-coupled antibody. Triple immunofluorescence was performed to identify microfilaments using Alexa Fluor-594 phalloidin (1:60) (60 min incubation at room temperature) and with DAPI (1:300) (5 min incubation) to visualize nuclei. After washing, cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and examined on a Nikon Eclipse 300 microscope (Mississauga, Canada) equipped with a CoolSnap fx digital camera (Roper Scientific, Tucson, AZ). Images were taken using a x20 objective. To better quantify apoptotic cells present in dishes, apoptotic nuclei were counted in 10 fields for each condition (about 1500 cells) and plotted against the total number of cells present in each field.
Planar cell surface areas
The larger and lower diameters of the cells were measured semiautomatically using the MetaMorph Imaging System 4.5 software package (Universal Imaging Corp., Westchester, PA). Cell area was calculated using a cell parameter analysis module. Graphs and distribution curve fitting were drawn by fitting a Gaussian function to the histograms using the Origin software (Rockware Inc., Golden, CO). Results are expressed as means ± SE of 192 cells for each experimental condition.
Western blotting
Cells were cultured in 35-mm petri dishes at a concentration of 5 x 105 cells/petri and used after 3 d for short-term stimulation without or with Ang II, in the absence or presence of various inhibitors introduced 30 min before Ang II treatment, namely, PD98059, SB203580, and SP600125, all at 10 μM (for p42/p44mapk, p38, and JNK phosphorylation studies). After 3 d of culture, cell number reached approximately 1 x 106 cells/petri. After hormonal stimulation in culture medium, cells were washed twice with HBS buffer and incubated for 10 min on ice with 1 ml of HBS-glucose solution containing 100 nM staurosporine and 1 mM sodium orthovanadate to block kinase and phosphatase activities. The solution was removed and cells were lysed with 30 μl of 50 mM HEPES (pH 7.8), 100 nM staurosporine, 1 mM sodium orthovanadate, 1% Triton X-100, 0.04 U/ml aprotinin, and 1 mM benzamidin. The cells were then scraped with a rubber policeman and transferred into Eppendorf tubes for 30 min on ice. The insoluble material was pelleted at 15,000 x g for 15 min at 4 C. Samples from an equivalent number of cells (from 30 μg protein) were compared in each experiment and processed as described previously (36). Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and proteins transferred electrophoretically onto polyvinylidene difluoride membranes. Membranes were blocked with 1% gelatin and 0.05% Tween 20 in Tris-buffered saline (pH 7.5). After three washes with Tris-buffered saline-Tween 20 (0.05%), membranes were incubated with antiphospho p42/p44mapk (dilution 1:1000), antiphospho p38 (dilution 1:500), antiphospho JNK (dilution 1:500), anti-p42/p44mapk (dilution 1:1000), anti-p38 (dilution 1:500), anti-JNK (dilution 1:500), anticyclin E (dilution 1:1000), anti-p27Kip1 (dilution 1:1000), or antitubulin (dilution 1:500). Detection was performed by reaction with horseradish peroxidase-conjugated secondary antibody and visualized by enhanced chemiluminescence according to the manufacturer’s instructions. The immunoreactive bands were scanned by laser densitometry and expressed in arbitrary units.
Data analysis
The data are presented as means ± SE of the number of experiments indicated in parentheses. Statistical analyses of the data were performed using Sigma Stat program (Systat Software Inc., Point Richmond, CA) to perform one-way ANOVA. Homogeneity of variance was assessed by Bartlett’s test, and P values were determined using Tukey’s posttest for significant difference.
Results
Ang II inhibits cell proliferation and stimulates protein synthesis
Glomerulosa cells have the propensity to easily proliferate in culture, doubling their number after 3 d (from 30 x 103 cells/well at the outset of the experiments to 57 x 103 cells/well after 3 d in culture) in a medium containing only 2% fetal serum (Fig. 1A). Measurement of cell proliferation using BrdU labeling or cell counting revealed that addition of Ang II for 3 d decreased the number of cells in a dose-dependent manner with a maximal effect observed at 10 nM (Fig. 1B), lowering cell number to that observed at time of plating (Fig. 1A). In the same experimental conditions, Ang II increased phenylalanine incorporation in a dose-dependent manner (maximum effect at 10 nM) with 10 nM Ang II inducing a significant increase of 158.5 ± 2.2% (P < 0.001, n = 3) over basal protein synthesis (Fig. 1C). As shown in Fig. 2, when stimulated with 5 nM Ang II, the number of apoptotic cells was low and without variation among the various experimental conditions. The increase in cell protein content was also concomitant with a significant increase in cell size as observed by phase-contrast microscopy of cells stimulated for 3 d with 5 nM Ang II (Fig. 3, A and B). Measurement of cell area indicated a normal Gaussian-type distribution, with mean values increasing from 300 ± 5 μm2 in control cells to 384 ± 5 μm2 (P < 0.001, n = 192) in Ang II-treated cells (Fig. 3, C and D).
FIG. 1. Effect of Ang II on proliferation (A and B) and protein synthesis (C) in rat glomerulosa cells. Cells were plated in 96-well plates for proliferation studies (30 x 103 cells/well) and 24-well plates for protein content studies (70 x 103 cells/well). A, Dose-dependent effect of Ang II (0.1–100 nM) on cell proliferation, measured by BrdU incorporation. B, Cells were stimulated without or with Ang II (5 nM) (hatched bars) daily for 3 d. Cell number was determined using a colorimetric method. C, Dose-dependent effect of Ang II (0.1–100 nM) on protein synthesis was determined by [3H]-phenylalanine incorporation, as explained in Materials and Methods. Results are expressed as the mean ± SE of three experiments, with each experimental condition containing six individual samples. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control cells. *, P < 0.01; **, P < 0.001.
FIG. 2. In situ apoptosis detection in rat glomerulosa cells. Cells (50 x 103 cells) were plated onto petri and stimulated without (A) or with Ang II (5 nM) (B) (hatched bars in C) every day for 3 d. A and B, In situ terminal deoxynucleotidyl transferase biotin-deoxyuridine 5-triphosphate nick end labeling is represented by green labeling of nuclei, compared with DAPI (blue) for visualization of all nuclei, and F-actin labeling with Alexa-Fluor 594-phalloidin. C, Results are reported as the mean ± SE of the total number of cells vs. apoptotic cells.
FIG. 3. Effect of Ang II on rat glomerulosa cell surface. Cells were plated at an initial concentration of 70 x 103 cells and were stimulated daily for 3 d without (A and C) or in the presence of 5 nM Ang II (B and D). A and B, Phase-contrast morphology of cells cultured for 3 d. Scale bars, 20 μm. C and D, Distribution of cell surface area from glomerulosa cells cultured in the absence (C) or presence of Ang II (D). Calculations are from experimental conditions illustrated in A and B. Continuous lines were drawn by fitting a Gaussian function to the histograms as described in Materials and Methods. Results are expressed as the mean ± SE of 192 cells from three independent experiments, for each experimental condition.
Because several studies have shown that glomerulosa cells contain both AT1 and AT2 receptors (12, 13, 14), cells were stimulated for 3 d with or without Ang II in the absence or presence of 1 μM DUP753 (a specific antagonist of the AT1 receptor) or 1 μM PD123319 (a specific antagonist of the AT2 receptor). Results reveal that the opposite effect of Ang II on proliferation (Fig. 4A) and phenylalanine incorporation (Fig. 4B) was blocked by the AT1 receptor antagonist DUP753 but not by the AT2 receptor antagonist PD123319.
FIG. 4. Involvement of AT1 and AT2 receptors in the effect of Ang II on proliferation (A) and protein synthesis (B) in rat glomerulosa cells cultured for 3 d. Cells were plated in 96-well plates for proliferation studies (30 x 103 cells/well) and 24-well plates for protein synthesis studies (70 x 103 cells/plate). Cells were stimulated for 3 d without or with Ang II (5 nM) (hatched bars), in the absence or presence of 1 μM DUP753 (a specific antagonist of the AT1 receptor) or with 1 μM PD123319 (a specific antagonist of the AT2 receptor), introduced 30 min before Ang II. A, Cell proliferation was measured using fluorescence BrdU incorporation. B, Protein synthesis was determined by assessing the incorporation of [3H]-phenylalanine. Results are expressed as the mean ± SE of three experiments, each experimental condition containing six individual samples. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control cells (*, P < 0.001) and between Ang II- and inhibitor plus Ang II-treated cells, compared with control cells (##, P < 0.001).
p42/p44mapk and p38 MAPK, but not JNK, are stimulated by Ang II
Activation of p42/p44mapk, p38 MAPK, and JNK MAPK pathways has been reported to be signaling cascades triggered by Ang II, with an action described both on cell proliferation and cell hypertrophy, depending on cell type and/or experimental conditions (for review see Refs.37, 38). Thus, activation of the above MAPK pathways was assessed and compared in glomerulosa cells maintained in primary culture for 3 d. Ang II induced a time-dependent increase in p42/p44mapk phosphorylation, as determined by Western blotting using antibodies against the phosphorylated active forms of ERK1/2. Maximal stimulation was observed after 5 min, with a stimulation ratio of 17.3 and remained 4.5-fold higher than basal values after 30 min exposure to Ang II (Fig. 5, A and C). The effect was dose dependent, with a significant increase observed at 1 nM (Fig. 5B). This Ang II effect was mediated by the AT1 receptor because preincubation with DUP753 abolished the response, whereas preincubation with the AT2 receptor antagonist PD123319 did not significantly change Ang II-induced p42/p44mapk phosphorylation. Preincubation with PD98059, a specific inhibitor of MAPK kinase (MEK), abrogated Ang II stimulation (Fig. 5, D and E). Ang II also stimulated, in a dose-dependent manner, the phosphorylated form of p38 MAPK in rat glomerulosa cells. Activation occurred rapidly (2 min), exhibiting an increased ratio of 9.2-fold over basal values, followed by a return to basal levels thereafter (Fig. 6, A and C). The effect was dose dependent (Fig. 6B) and, similar to p42/p44mapk, was mediated by AT1 receptor activation (Fig. 6D). In contrast to p42/p44mapk and p38 MAPK activation, Ang II had no effect on JNK activation throughout the entire time course (data not shown).
FIG. 5. Time-course and dose-dependent effect of Ang II on the phosphorylation of p42/p44mapk in rat glomerulosa cells. A, Three-day cultured cells (1 x 106 cells) were stimulated in the absence (none) or presence of 100 nM Ang II for intervals ranging from 1 to 30 min. Cell lysates containing equal amounts of protein (30 μg) were subjected to Western blot analyses with antibodies against phosphorylated p42/p44mapk (upper panels of blots). Lower panels represent the same blots reprobed for total p42/p44mapk. B, Cells were stimulated with various concentrations of Ang II ranging from 0.1 to 100 nM for 5 min. C, Time course of p42/p44mapk phosphorylation as analyzed by densitometry. D, Cells were stimulated without or with 100 nM Ang II for 5 min in absence or presence of 1 μM DUP753 (a specific antagonist of the AT1 receptor), 1 μM PD123319 (a specific antagonist of the AT2 receptor) or with 10 μM PD98059 (a specific inhibitor of MEK), introduced 30 min before Ang II. E, Densitometric analysis of the effect of inhibitors, DUP753, PD123319, and PD98059, on p42/p44mapk phosphorylation. All data represent the mean ± SE of three different experiments. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control (**, P < 0.001).
FIG. 6. Time-course and dose-dependent effect of Ang II on the phosphorylation of p38 MAPK in rat glomerulosa cells. A, Three-day cultured cells (1 x 106 cells) were stimulated in the absence (none) or presence of 100 nM Ang II for periods ranging from 1 to 30 min. Cell lysates containing equal amounts of protein (30 μg) were subjected to Western blot analyses with antibodies against phosphorylated p38 MAPK (upper panels of blots). Lower panels represent the same blots reprobed for total p38 MAPK. B, Cells were stimulated with various concentrations of Ang II ranging from 0.1 to 100 nM for 5 min. C, Time course of p38 MAPK phosphorylation as analyzed by densitometry. D, Cells were stimulated without or with 100 nM Ang II for 5 min in absence or presence of 1 μM DUP753 (a specific antagonist of the AT1 receptor), 1 μM PD123319 (a specific antagonist of the AT2 receptor), or with 10 μM SB203580 (a specific inhibitor of p38 MAPK), introduced 30 min before Ang II. All data represent the mean ± SE of three different experiments. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control (**, P < 0.001).
Subsequent experiments were also conducted to ascertain which type of cascade could be involved in the opposite action of Ang II on proliferation (decreased in cell number) and hypertrophy (increase in phenylalanine incorporation). Cells were stimulated with or without Ang II in the absence or presence of specific inhibitors of these three MAPK pathways. PD98059 decreased, in a dose-dependent manner, basal levels of proliferation but did not modify the level of inhibition induced by Ang II (Fig. 7A), although it did reverse the inhibitory effect of Ang II on phenylalanine incorporation (Fig. 7B). On the other hand, SB203580, a specific antagonist of the p38 MAPK, reversed the effects of Ang II, in a dose-dependent fashion, on both inhibition of cell proliferation (Fig. 7C) and the observed increase in protein synthesis (Fig. 7D). Finally, using similar experimental conditions, the JNK inhibitor SP600125 did not modify the effects of Ang II, on either proliferation or protein synthesis (Fig. 7, E and F).
FIG. 7. Involvement of p42/p44mapk, p38 MAPK, and JNK on the effect of Ang II on proliferation and protein synthesis of rat glomerulosa cells. Cells were plated in 96-well plates (30 x 103 cells/well) for proliferation studies (A, C, and E) and 24-well plates (70 x 103 cells/well) for protein content studies (B, D, and F). Cells were stimulated for 3 d without or with Ang II (5 nM) (hatched bars), in the absence or presence of three concentrations of inhibitors: 5, 10, and 20 μM PD98059 (a specific inhibitor of MEK); 1, 5, and 10 μM SB203580 (a specific inhibitor of p38 MAPK); and 1, 5, and 10 μM SP600125 (a specific inhibitor of JNK), introduced 30 min before Ang II. Cell proliferation was measured using fluorescence BrdU incorporation. Protein synthesis was determined by assessing the incorporation of [3H]-phenylalanine. Results are expressed as the mean ± SE of six individual samples. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control cells (*, P < 0.001) and between Ang II- and inhibitor plus Ang II-treated cells, compared with control cells (##, P < 0.001).
Ang II increases the expression of p27Kip1
The following experiments were aimed at understanding how Ang II produces these opposite effects on proliferation and protein synthesis. The fact that Ang II decreased cell proliferation but enhanced protein synthesis led us to consider that, during the 3-d incubation period, Ang II may act on one of the important steps of the cell cycle, possibly through one of the stimulated p42/p44mapk or p38 MAPK pathways. The effect of Ang II was therefore examined on the expression of cyclin E as a representative of the late events of the G1 phase of the cell cycle (39). As shown in Fig. 8A, expression of cyclin E did not vary significantly during the 3 d of culture, in both control and Ang II-treated cells.
FIG. 8. Effect of Ang II on cyclin E and p27Kip1 protein expression in rat glomerulosa cells cultured for 3 d. Cell lysates from equivalent numbers of cells (1 x 106 cells/petri) were separated on 10% polyacrylamide gels and subjected to Western blot analysis using specific antibodies against cyclin E and p27Kip1. Cells were cultured for 3 d in the absence or presence of 5 nM Ang II (A). B, Cells were cultured for 3 d in the absence or presence of 5 nM Ang II and either 10 μM SB203580 (a specific inhibitor of p38 MAPK) or 10 μM PD98059 (a specific inhibitor of MEK) introduced 30 min before Ang II.
Because cell cycle progression is controlled by cyclin/cyclin-dependent kinase (Cdk) complexes counterbalanced by Cdk inhibitors, expression and activity of G1-specific cyclin-dependent kinases can be inhibited by the presence of cyclin-dependent kinase inhibitors, including p27Kip1, which form complexes with cyclin/Cdk and could thereby block their interaction with substrates (34). As seen in Fig. 8A, Ang II increased the level of p27Kip1, measured by immunoblotting using specific antibody. In cells cultured in the presence of SB203580 or PD98059, the effect of Ang II on p27Kip1 level was abrogated, without affecting cyclin E levels (Fig. 8B).
Discussion
The present study provides compelling evidence that Ang II promotes cellular hypertrophy, but not proliferation, in rat adrenal glomerulosa cells maintained in primary culture for 3 d. The growth-promoting effect of Ang II occurs via AT1 receptor activation, without any participation of the AT2 receptor. Both p42/p44mapk and p38 MAPK are involved in the increase in protein synthesis and in the expression of p27Kip1, which, in turn, is responsible for inhibition of the cell cycle.
Proliferation vs. hypertrophy
Having previously shown that glomerulosa cells easily proliferate in culture, we used this model to directly assess the effects of Ang II on proliferation and protein synthesis. Measurement of cell proliferation using BrdU labeling or cell counting revealed that addition of Ang II during 3 d decreases cell number to the level observed at the outset of cell culture. These data on cell number could reflect the balance between the rate of cell proliferation and the rate of cell death via apoptosis. However, quantification of the number of apoptotic cells during the 3-d treatment with or without Ang II revealed that this number was low and did not differ between control and Ang II-treated cells. This decrease in cell proliferation is accompanied by a significant increase in protein synthesis and cell size. Pharmacological experiments indicate that the opposite effect of Ang II on proliferation and phenylalanine incorporation is mediated only by the AT1 receptor and not the AT2 receptor.
These results could appear provocative at first glance. Indeed, McEwan et al. (11) reported that Ang II infusion stimulates both cell proliferation and hypertrophy of glomerulosa cells and indicated that only proliferation is sensitive to inhibition by the AT1 receptor antagonist DUP753. However, such observations did not discriminate between direct effects of Ang II on glomerulosa cells or indirect effects through paracrine interaction with endothelial cells or fibroblasts, which also express Ang II AT1 receptors (1). Indeed, as recently reviewed by Bouzegrhane and Thibault (40), the proliferative effect of Ang II described in many studies may be caused by stimulation of growth factor synthesis or extracellular matrix components, such as fibronectin (41, 42). These hypotheses could be in agreement with the observations that in some cell types, such as cardiomyocytes, fibroblasts, or VSMC, Ang II up-regulates synthesis of extracellular matrix components, in particular collagen (43, 44), fibronectin (45, 46), and thrombospondin (22). Such matrix production and matrix assembly is necessary to support the capacity of Ang II to induce cell proliferation (47). This may explain why Tian et al. (16) observed proliferation only after 9 d in culture, suggesting that cells must express proteins necessary to generate AT1-receptor mediated proliferation.
The specific role of Ang II in proliferation and/or hypertrophy continues to be a subject of debate. Discrepancies in most of its targets tissues, including cardiomyocytes and VSMCs (38), could be explained in part by either cell type-specific action or a developmental regulatory process. For example, in immature ovine cardiomyocytes, Ang II stimulates hyperplasia but not hypertrophy (48). On the other hand, in adult human kidney mesangial cells, the hormone induces both hypertrophy and hyperplasia (49), whereas in adult cardiac tissue and VSMCs, it is now generally accepted that Ang II is implicated in hypertrophy but not in cell proliferation (34, 50, 51, 52, 53). In the present instance, we provide evidence that in rat adrenal glomerulosa cells in primary culture, Ang II, through the AT1 receptor, stimulates protein synthesis and inhibits proliferation normally observed during the 3 d in culture. Steroidogenic acute regulatory protein (StAR) and 3?-hydroxysteroid dehydrogenase (3?-HSD) are among the specific proteins stimulated (data not shown), corroborating previous observations made in bovine adrenal glomerulosa cells (54, 55) and in H295R cells (56, 57, 58). Such Ang II stimulation of StAR and 3?-HSD expression is associated with an increase in aldosterone secretion.
Molecular events involved in the hypertrophic effects of Ang II
The Ang II-promoting effect on protein synthesis occurs with a concomitant arrest in cell proliferation, suggesting a shift from pathways involved in proliferation to those involved in cell protein synthesis. We thus investigated which signaling pathway may be involved in such action and which intracellular proteins may be the targets of this shift from proliferation to hypertrophy. Our results indicate that Ang II induces a time-dependent increase in p42/p44mapk phosphorylation and a rapid and transient increase in p38 MAPK phosphorylation but has no effect on JNK.
Activation of the three MAPK pathways has been reported to be an important component in the signaling cascade triggered by Ang II, with an action described both on cell proliferation and cell hypertrophy, depending on cell type and/or experimental conditions (for review see Refs.37, 38). Whereas the role of p42/p44mapk is well documented in both Ang II-influenced proliferation and hypertrophy, the involvement of p38 MAPK in hypertrophy is a more recent finding. Our results indicate that specific inhibition of p42/p44mapk and p38 MAPK abrogated the effect of Ang II on tritiated phenylalanine incorporation. Thus, both pathways are essential in Ang II action on protein synthesis. In addition, MEK and p38 MAPK inhibitors both reverse the effect of Ang II on cell number. We did not observe any variation in JNK activity by Ang II, corroborating recent work in the field. Indeed, in H295R adrenocortical cells, Ang II leads to a dose-dependent increase in p38 MAPK activity but has no effect on JNK (59). Similar results have also been reported in bovine glomerulosa cells (60).
Although the p38 MAPK pathway has classically been associated with apoptosis and inflammatory reactions in response to cellular stress, there is increasing evidence that p38 MAPK is also activated by many G protein-coupled receptors, such as the AT1 receptor. For example, studies on neonatal cardiomyocytes demonstrated that activation of p38 MAPK-dependent pathways is associated with cell hypertrophy (61, 62), collagen synthesis, and fibrogenesis (44). Our results are thus in agreement with these studies demonstrating that p42/p44mapk but not p38 MAPK is involved in basal stimulation of DNA synthesis, whereas both p42/p44mapk and p38 MAPK are necessary in mediating Ang II-induced protein synthesis.
To confirm these results, we also measured the level of key proteins involved in regulating progression in cell cycle entry. Our data demonstrate that expression of cyclin E does not change throughout the 3 d in culture or during Ang II treatment. However, Ang II did increase the level of p27Kip1, an effect completely reversible when cells are coincubated with SB203580 or PD98059. This indicates that p38 MAPK and p42/p44mapk are responsible for the increase in p27Kip1 by Ang II, which in turn is responsible for inhibition of cell proliferation. A similar observation has also been recently described in vascular smooth muscle (62, 63, 64).
Altogether the present data clearly indicate that in cultured rat glomerulosa cells, a 3-d treatment with Ang II increases protein synthesis, with a concomitant decrease in cellular proliferation. These effects are mediated by both the p42/p44mapk and p38 MAPK pathways, which increase expression of the steroidogenic enzymes, StAR and 3?-HSD, and p27Kip1, a protein known to block the cell cycle in G1 phase. These results further support the key role of Ang II in maintaining the proper machinery necessary to support aldosterone biosynthesis, not only in acute situations but also during a more prolonged stimulation.
Acknowledgments
The authors thank Lyne Bilodeau and Lucie Chouinard for experimental assistance and Dr. Mylène C?té for stimulating discussions. We express our gratitude for the generous gift of antisera from our colleagues: cyclin E and p27Kip1, Dr. Nathalie Rivard (Département d’Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke; 3?-HSD, Dr. Van Luu-The (CHUL Research Center, Ste-Foy, Québec, Canada); and StAR, Dr. Douglas Stocco (Texas Tech University, Lubbock, Texas).
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Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Faculty of Medicine, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail: nicole.gallo-payet@usherbrooke.ca.
Abstract
Angiotensin II (Ang II) is one of the most important stimuli of rat adrenal glomerulosa cells. The aim of the present study was to investigate whether Ang II can stimulate cell proliferation and/or hypertrophy and investigate pathways and intracellular targets. A 3-d treatment with Ang II (5–100 nM), through the Ang II type 1 receptor subtype, abolished cell proliferation observed in control cells but increased protein synthesis. Preincubation with PD98059 (a MAPK kinase inhibitor) abolished basal proliferation and had no effect on basal protein synthesis but did reverse the effect of Ang II on protein synthesis. The p38 MAPK inhibitor SB203580 reversed the inhibitory effect on cell proliferation and abolished the increase in protein synthesis, whereas the c-Jun N-terminal kinase inhibitor SP600125 had no effect. Time-course studies revealed that Ang II stimulated phosphorylation of both p42/p44mapk and p38 MAPK but did not activate c-Jun N-terminal kinase. Ang II had no effect on the level of cyclin E expression but increased the expression of the cyclin-dependent kinase, p27Kip1, an effect abolished in cells preincubated with SB203580 and PD98059. In conclusion, in cultured rat glomerulosa cells, a 3-d treatment with Ang II increases protein synthesis, with a concomitant decrease in proliferation. These effects are mediated by both the p42/p44mapk and p38 MAPK pathways, which increase expression of the steroidogenic enzymes, steroidogenic acute regulatory protein and 3?-hydroxysteroid dehydrogenase and p27Kip1, a protein known to block the cell cycle in G1 phase. Together these results support the key role of Ang II as a stimulus of steroid synthesis rather than a proliferating factor.
Introduction
THE ADULT ADRENAL cortex is composed of three concentric layers (zona glomerulosa, zona fasciculata, and zona reticularis), all of which have specific functional and morphological properties. The zona glomerulosa is specialized in aldosterone production and, in contrast to corticosterone secretion by the zona fasciculata, is under multifactorial regulation, with angiotensin II (Ang II) being the most important stimulus in vivo (for review see Refs.1, 2). In addition, glomerulosa cells have the ability to adapt their morphology to environmental conditions. For instance, it is well known that chronic administration of a low-sodium diet increases the number of Ang II type 1 (AT1) receptors (3, 4) and expands not only aldosterone secretion machinery (5) but also the width of the zona glomerulosa, an effect mainly associated with Ang II action (6, 7, 8, 9, 10, 11). However, the direct role of Ang II on either proliferation and/or hypertrophy is not yet clearly established. Indeed, McEwan et al. (11), using in vivo treatment with Ang II alone or in the presence of an AT1 receptor antagonist, demonstrated that Ang II stimulates proliferation but not hypertrophy of the glomerulosa cells. However, in rats receiving a sodium-deficient diet, the AT1 receptor antagonist DUP753 (Losartan, DuPont Merck Pharmaceuticals, Paris, France) failed to prevent hyperplasia of glomerulosa cells, in conditions in which proliferation in zona reticularis and smooth muscle cells were respectively blocked (11).
Both type 1 (AT1) and type 2 (AT2) Ang II receptors are present in rat adrenal zona glomerulosa, at a ratio varying from 60 to 80% AT1 and from 40 to 20% AT2 (12, 13, 14). Much of Ang II action on aldosterone secretion is largely due to activation of the Gq/phospholipase C-dependent hydrolysis of phosphatidylinositol 4,5 bisphosphate, leading to the production of inositol phosphates and diacylglycerol (2). On the other hand, as in several other G protein-coupled receptors, Ang II is able to activate the MAPK pathway, p42/p44mapk (also called ERK1/2) in bovine (15, 16) and rat (17, 18) glomerulosa cells. In vascular smooth muscle cells (VSMCs) and cardiomyocytes, Ang II likely activates both p38 MAPK (19) and c-Jun N-terminal kinase (JNK) (20, 21, 22).
Whereas it is well known that glomerulosa cells are often considered as the progenitor cells of the cortex, exhibiting the ability to both proliferate and migrate (23, 24, 25), the specific role of Ang II on either proliferation and/or hypertrophy is not yet clearly established. Some studies have suggested that Ang II may act as a direct mitogen. For instance, Ang II stimulates cell proliferation of cultured bovine fasciculata cells (16, 26) or in a bovine adrenocortical cell clone (27). To date, all studies addressing the effects of Ang II on adrenocortical cells have been performed using bovine glomerulosa or fasciculata cells. By contrast, there are no studies assessing these effects with rat adrenal glomerulosa cells. Previous studies from our group have shown that, in contrast to fasciculata cells, glomerulosa cells, either from rat or human origin, have the propensity to easily proliferate in culture, doubling their number after 3 d, even in a medium containing only 2% fetal serum (28, 29). Due to the important role of the zona glomerulosa in maintaining electrolyte homeostasis, defining how cell physiology (growth and steroidogenesis) is regulated by Ang II is a key concern because it is the main stimulus of aldosterone secretion. Thus, the objective of this study was to determine, using primary cultures of rat glomerulosa cells, whether Ang II induces cell proliferation and/or hypertrophy and clarify the role of the three MAPK pathways (ERK1/2, JNK, and p38 MAPK) in these events.
Materials and Methods
Chemicals
The chemicals used in the present study were obtained from the following sources: collagenase, MEM (MEM Eagle’s medium) and OPTI-MEM medium from Life Technologies (Burlington, Canada); Ang II from Bachem (Marina Delphen, CA); horseradish peroxidase-conjugated antirabbit and antimouse antibodies, enhanced chemiluminescence detection system and L-[2,3,4,5,6-3H] phenylalanine from Amersham Pharmacia Biotech (Oakville, Canada); Apoptag kit from Chemicon International Inc. (Temecula, CA); PD98059, the antibodies antiphosphorylated p42/p44mapk, antiphosphorylated p38, antiphosphorylated JNK, anti-p42/p44mapk, anti-p38, and anti-JNK from New England Biolabs, Inc. (Mississauga, Canada); the antibodies anticyclin E and anti-p27Kip1 from Santa Cruz Biotechnologies (Santa Cruz, CA); antitubulin, SB203580, and SP600125 from Chemicon (Mississauga, Canada); 5-bromo-2-deoxyuridine (BrdU) anti-BrdU Alexa Fluor-594, Alexa Fluor-594 phalloidin, and 4',6'-diamino-2-phenylindole (DAPI) from Molecular Probes (Eugene, OR); crystal violet, deoxyribonuclease, PD98059, and PD123319 from Sigma Chemical Co. (St. Louis, MO); polyvinylidene difluoride membranes from Millipore (Bedford, MA); and DC protein assay from Bio-Rad Laboratories (Hercules, CA). All other chemicals were of grade A purity.
Preparation of glomerulosa cell cultures
Zona glomerulosa cells were obtained from adrenal glands of female Long Evans rats weighing 200–250 g and isolated according to the method previously described in detail (30). All protocols were approved by the Animal Care and Ethics Committee of our faculty. Isolation and cell dissociation of the zona glomerulosa was performed in MEM medium (supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin). After a 20-min incubation at 37 C with collagenase (2 mg/ml) and deoxyribonuclease (25 μg/ml), cells were disrupted by gentle aspiration with a sterile 10-ml pipette, filtered, and centrifuged for 10 min at 100 x g. The cell pellet was then resuspended in OPTI-MEM medium supplemented with 2% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Depending on experiments, cells were plated at various densities in plastic 35-mm petri dishes (5 x 105 cells/dish) for Western blots analyses, 24-well plates (70 x 103 cells/well) for protein synthesis measurements, and 96-multiwell plates (70 x 103 cells/plate) for proliferation assays. Cells were cultured at 37 C in a humidified atmosphere composed of 95% air-5% CO2. The culture medium was changed daily, and cells were used after 3 d of culture. Except in specific experiments, cells were stimulated with 5 nM Ang II for 3-d treatments and 100 nM for acute stimulations. Cells were examined daily, and phase-contrast images were taken using a microscope (Leica Corp., Deerfield, IL) equipped with a x32 objective.
Proliferation assays
The method used to evaluate cell proliferation was adapted from that described by Gillies et al. (31) and Campbell et al. (32). Cells were plated on plastic 96-well plates at a concentration of 30 x 103 cells/well. Cells were treated daily for 3 d without or with Ang II alone or in the presence of various inhibitors. Concentrations used for the inhibitors corresponded to their dissociation constant, i.e. to inhibit specific function, without altering cell viability: PD98059 (5, 10, and 20 μM); SB203580 and SP600125 (1, 5, and 10 μM). On the third day, cells were fixed with 3.7% (vol/vol) formaldehyde in Hanks’ buffered saline (HBS) (in millimoles): NaCl 130; KCl 3.5; CaCl2/2H2O 2.3; MgCl2/6H2O 0.98; HEPES 5; EGTA 0.5) for 10 min at room temperature. After HBS washing, cells were incubated for 10 min at room temperature with 0.1% crystal violet/H2O. After extensive washes, cells were lysed for 10 min in 100 μl of 1% sodium dodecyl sulfate solution and ODs read at 595 nm with a μQuant spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT). For each experiment, a standard curve was performed using serial dilutions of a cell solution in which cell number was evaluated with the aid of a hemacytometer.
Cell proliferation was also measured using fluorescence BrdU incorporation, as described elsewhere (33). Cells were plated on plastic 96-well plates at a concentration of 30 x 103 cells/well. Cells were treated daily for 3 d without or with Ang II alone or in the presence of various inhibitors. After 24 h of culture, 10 μM BrdU was added to the culture medium 4 h before stimulation with Ang II. On the third day, cells were fixed with 3.7% (vol/vol) formaldehyde in HBS for 10 min at room temperature and permeabilized for 10 min with 0.2% Triton X-100 in HBS. Cells were then incubated with anti-BrdU Alexa Fluor-594 (1:500). Fluorescence intensity was determined using a microplate fluorescence reader FL600 (Bio-Tek) (excitation 56,040 nm; emission 64,540 nm). Results are expressed as the percent changes from basal conditions using six culture wells for each experimental condition.
Protein synthesis measurements
Cells were plated on plastic 24-well plates at a concentration of 70 x 103 cells/well, in quadruplicate. Cells were treated daily with hormones without or with various inhibitors for 3 d, as described for proliferation assays. A first duplicate cell series was washed twice with ice-cold HBS buffer and lysed in 50 μl of 50 mM HEPES (pH 7.8) and 1% Triton X-100. Protein content for each experimental condition was determined using the Bio-Rad DC method. The second duplicate cell series was used to evaluate exact cell number with the aid of a hemacytometer.
The relative amount of protein synthesis was also determined by assessing tritiated phenylalanine incorporation. Cells were plated on plastic 24-well plates at a concentration of 70 x 103 cells/well. After 24 h of culture, 1 μCi/ml [-3H]phenylalanine was added to the media 2 h before stimulation with Ang II. After 3 d, the medium was aspirated and cells washed three times with cold HBS solution, followed by addition of trichloroacetic acid solution (TCA) (20%) for 20 min on ice. After centrifugation (3000 x g, 15 min, 4 C), the TCA-insoluble fraction was washed twice with TCA solution and solubilized in 0.1 N NaOH solution for 1 h on ice. Incorporation of radioactivity was measured by liquid scintillation counting (Beckman counter, Beckman Coulter, Inc., Fullerton, CA). Incorporation of radioactivity was measured by liquid scintillation counting (Beckman counter). Data were normalized as maximum of phenylalanine incorporation in control conditions for the same number of cells (1 x 105 cells), as described elsewhere (34).
In situ apoptosis detection
Cells were processed according to the protocol provided by the manufacturer of the Apoptag kit (Chemicon) using terminal deoxynucleotidyl transferase biotin-deoxyuridine 5-triphosphate nick end labeling fluorescence, as used previously (35). Cells were stimulated and fixed as described in proliferation assays. After fixation and equilibration, cells were incubated for 1 h and 30 min at 37 C in a solution containing the terminal deoxynucleotidyl transferase enzyme and the reaction buffer containing digoxigenin-labeled nucleotides. Cells were then washed twice in HBS and incubated with an antidigoxigenin fluorescein isothiocyanate-coupled antibody. Triple immunofluorescence was performed to identify microfilaments using Alexa Fluor-594 phalloidin (1:60) (60 min incubation at room temperature) and with DAPI (1:300) (5 min incubation) to visualize nuclei. After washing, cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and examined on a Nikon Eclipse 300 microscope (Mississauga, Canada) equipped with a CoolSnap fx digital camera (Roper Scientific, Tucson, AZ). Images were taken using a x20 objective. To better quantify apoptotic cells present in dishes, apoptotic nuclei were counted in 10 fields for each condition (about 1500 cells) and plotted against the total number of cells present in each field.
Planar cell surface areas
The larger and lower diameters of the cells were measured semiautomatically using the MetaMorph Imaging System 4.5 software package (Universal Imaging Corp., Westchester, PA). Cell area was calculated using a cell parameter analysis module. Graphs and distribution curve fitting were drawn by fitting a Gaussian function to the histograms using the Origin software (Rockware Inc., Golden, CO). Results are expressed as means ± SE of 192 cells for each experimental condition.
Western blotting
Cells were cultured in 35-mm petri dishes at a concentration of 5 x 105 cells/petri and used after 3 d for short-term stimulation without or with Ang II, in the absence or presence of various inhibitors introduced 30 min before Ang II treatment, namely, PD98059, SB203580, and SP600125, all at 10 μM (for p42/p44mapk, p38, and JNK phosphorylation studies). After 3 d of culture, cell number reached approximately 1 x 106 cells/petri. After hormonal stimulation in culture medium, cells were washed twice with HBS buffer and incubated for 10 min on ice with 1 ml of HBS-glucose solution containing 100 nM staurosporine and 1 mM sodium orthovanadate to block kinase and phosphatase activities. The solution was removed and cells were lysed with 30 μl of 50 mM HEPES (pH 7.8), 100 nM staurosporine, 1 mM sodium orthovanadate, 1% Triton X-100, 0.04 U/ml aprotinin, and 1 mM benzamidin. The cells were then scraped with a rubber policeman and transferred into Eppendorf tubes for 30 min on ice. The insoluble material was pelleted at 15,000 x g for 15 min at 4 C. Samples from an equivalent number of cells (from 30 μg protein) were compared in each experiment and processed as described previously (36). Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and proteins transferred electrophoretically onto polyvinylidene difluoride membranes. Membranes were blocked with 1% gelatin and 0.05% Tween 20 in Tris-buffered saline (pH 7.5). After three washes with Tris-buffered saline-Tween 20 (0.05%), membranes were incubated with antiphospho p42/p44mapk (dilution 1:1000), antiphospho p38 (dilution 1:500), antiphospho JNK (dilution 1:500), anti-p42/p44mapk (dilution 1:1000), anti-p38 (dilution 1:500), anti-JNK (dilution 1:500), anticyclin E (dilution 1:1000), anti-p27Kip1 (dilution 1:1000), or antitubulin (dilution 1:500). Detection was performed by reaction with horseradish peroxidase-conjugated secondary antibody and visualized by enhanced chemiluminescence according to the manufacturer’s instructions. The immunoreactive bands were scanned by laser densitometry and expressed in arbitrary units.
Data analysis
The data are presented as means ± SE of the number of experiments indicated in parentheses. Statistical analyses of the data were performed using Sigma Stat program (Systat Software Inc., Point Richmond, CA) to perform one-way ANOVA. Homogeneity of variance was assessed by Bartlett’s test, and P values were determined using Tukey’s posttest for significant difference.
Results
Ang II inhibits cell proliferation and stimulates protein synthesis
Glomerulosa cells have the propensity to easily proliferate in culture, doubling their number after 3 d (from 30 x 103 cells/well at the outset of the experiments to 57 x 103 cells/well after 3 d in culture) in a medium containing only 2% fetal serum (Fig. 1A). Measurement of cell proliferation using BrdU labeling or cell counting revealed that addition of Ang II for 3 d decreased the number of cells in a dose-dependent manner with a maximal effect observed at 10 nM (Fig. 1B), lowering cell number to that observed at time of plating (Fig. 1A). In the same experimental conditions, Ang II increased phenylalanine incorporation in a dose-dependent manner (maximum effect at 10 nM) with 10 nM Ang II inducing a significant increase of 158.5 ± 2.2% (P < 0.001, n = 3) over basal protein synthesis (Fig. 1C). As shown in Fig. 2, when stimulated with 5 nM Ang II, the number of apoptotic cells was low and without variation among the various experimental conditions. The increase in cell protein content was also concomitant with a significant increase in cell size as observed by phase-contrast microscopy of cells stimulated for 3 d with 5 nM Ang II (Fig. 3, A and B). Measurement of cell area indicated a normal Gaussian-type distribution, with mean values increasing from 300 ± 5 μm2 in control cells to 384 ± 5 μm2 (P < 0.001, n = 192) in Ang II-treated cells (Fig. 3, C and D).
FIG. 1. Effect of Ang II on proliferation (A and B) and protein synthesis (C) in rat glomerulosa cells. Cells were plated in 96-well plates for proliferation studies (30 x 103 cells/well) and 24-well plates for protein content studies (70 x 103 cells/well). A, Dose-dependent effect of Ang II (0.1–100 nM) on cell proliferation, measured by BrdU incorporation. B, Cells were stimulated without or with Ang II (5 nM) (hatched bars) daily for 3 d. Cell number was determined using a colorimetric method. C, Dose-dependent effect of Ang II (0.1–100 nM) on protein synthesis was determined by [3H]-phenylalanine incorporation, as explained in Materials and Methods. Results are expressed as the mean ± SE of three experiments, with each experimental condition containing six individual samples. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control cells. *, P < 0.01; **, P < 0.001.
FIG. 2. In situ apoptosis detection in rat glomerulosa cells. Cells (50 x 103 cells) were plated onto petri and stimulated without (A) or with Ang II (5 nM) (B) (hatched bars in C) every day for 3 d. A and B, In situ terminal deoxynucleotidyl transferase biotin-deoxyuridine 5-triphosphate nick end labeling is represented by green labeling of nuclei, compared with DAPI (blue) for visualization of all nuclei, and F-actin labeling with Alexa-Fluor 594-phalloidin. C, Results are reported as the mean ± SE of the total number of cells vs. apoptotic cells.
FIG. 3. Effect of Ang II on rat glomerulosa cell surface. Cells were plated at an initial concentration of 70 x 103 cells and were stimulated daily for 3 d without (A and C) or in the presence of 5 nM Ang II (B and D). A and B, Phase-contrast morphology of cells cultured for 3 d. Scale bars, 20 μm. C and D, Distribution of cell surface area from glomerulosa cells cultured in the absence (C) or presence of Ang II (D). Calculations are from experimental conditions illustrated in A and B. Continuous lines were drawn by fitting a Gaussian function to the histograms as described in Materials and Methods. Results are expressed as the mean ± SE of 192 cells from three independent experiments, for each experimental condition.
Because several studies have shown that glomerulosa cells contain both AT1 and AT2 receptors (12, 13, 14), cells were stimulated for 3 d with or without Ang II in the absence or presence of 1 μM DUP753 (a specific antagonist of the AT1 receptor) or 1 μM PD123319 (a specific antagonist of the AT2 receptor). Results reveal that the opposite effect of Ang II on proliferation (Fig. 4A) and phenylalanine incorporation (Fig. 4B) was blocked by the AT1 receptor antagonist DUP753 but not by the AT2 receptor antagonist PD123319.
FIG. 4. Involvement of AT1 and AT2 receptors in the effect of Ang II on proliferation (A) and protein synthesis (B) in rat glomerulosa cells cultured for 3 d. Cells were plated in 96-well plates for proliferation studies (30 x 103 cells/well) and 24-well plates for protein synthesis studies (70 x 103 cells/plate). Cells were stimulated for 3 d without or with Ang II (5 nM) (hatched bars), in the absence or presence of 1 μM DUP753 (a specific antagonist of the AT1 receptor) or with 1 μM PD123319 (a specific antagonist of the AT2 receptor), introduced 30 min before Ang II. A, Cell proliferation was measured using fluorescence BrdU incorporation. B, Protein synthesis was determined by assessing the incorporation of [3H]-phenylalanine. Results are expressed as the mean ± SE of three experiments, each experimental condition containing six individual samples. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control cells (*, P < 0.001) and between Ang II- and inhibitor plus Ang II-treated cells, compared with control cells (##, P < 0.001).
p42/p44mapk and p38 MAPK, but not JNK, are stimulated by Ang II
Activation of p42/p44mapk, p38 MAPK, and JNK MAPK pathways has been reported to be signaling cascades triggered by Ang II, with an action described both on cell proliferation and cell hypertrophy, depending on cell type and/or experimental conditions (for review see Refs.37, 38). Thus, activation of the above MAPK pathways was assessed and compared in glomerulosa cells maintained in primary culture for 3 d. Ang II induced a time-dependent increase in p42/p44mapk phosphorylation, as determined by Western blotting using antibodies against the phosphorylated active forms of ERK1/2. Maximal stimulation was observed after 5 min, with a stimulation ratio of 17.3 and remained 4.5-fold higher than basal values after 30 min exposure to Ang II (Fig. 5, A and C). The effect was dose dependent, with a significant increase observed at 1 nM (Fig. 5B). This Ang II effect was mediated by the AT1 receptor because preincubation with DUP753 abolished the response, whereas preincubation with the AT2 receptor antagonist PD123319 did not significantly change Ang II-induced p42/p44mapk phosphorylation. Preincubation with PD98059, a specific inhibitor of MAPK kinase (MEK), abrogated Ang II stimulation (Fig. 5, D and E). Ang II also stimulated, in a dose-dependent manner, the phosphorylated form of p38 MAPK in rat glomerulosa cells. Activation occurred rapidly (2 min), exhibiting an increased ratio of 9.2-fold over basal values, followed by a return to basal levels thereafter (Fig. 6, A and C). The effect was dose dependent (Fig. 6B) and, similar to p42/p44mapk, was mediated by AT1 receptor activation (Fig. 6D). In contrast to p42/p44mapk and p38 MAPK activation, Ang II had no effect on JNK activation throughout the entire time course (data not shown).
FIG. 5. Time-course and dose-dependent effect of Ang II on the phosphorylation of p42/p44mapk in rat glomerulosa cells. A, Three-day cultured cells (1 x 106 cells) were stimulated in the absence (none) or presence of 100 nM Ang II for intervals ranging from 1 to 30 min. Cell lysates containing equal amounts of protein (30 μg) were subjected to Western blot analyses with antibodies against phosphorylated p42/p44mapk (upper panels of blots). Lower panels represent the same blots reprobed for total p42/p44mapk. B, Cells were stimulated with various concentrations of Ang II ranging from 0.1 to 100 nM for 5 min. C, Time course of p42/p44mapk phosphorylation as analyzed by densitometry. D, Cells were stimulated without or with 100 nM Ang II for 5 min in absence or presence of 1 μM DUP753 (a specific antagonist of the AT1 receptor), 1 μM PD123319 (a specific antagonist of the AT2 receptor) or with 10 μM PD98059 (a specific inhibitor of MEK), introduced 30 min before Ang II. E, Densitometric analysis of the effect of inhibitors, DUP753, PD123319, and PD98059, on p42/p44mapk phosphorylation. All data represent the mean ± SE of three different experiments. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control (**, P < 0.001).
FIG. 6. Time-course and dose-dependent effect of Ang II on the phosphorylation of p38 MAPK in rat glomerulosa cells. A, Three-day cultured cells (1 x 106 cells) were stimulated in the absence (none) or presence of 100 nM Ang II for periods ranging from 1 to 30 min. Cell lysates containing equal amounts of protein (30 μg) were subjected to Western blot analyses with antibodies against phosphorylated p38 MAPK (upper panels of blots). Lower panels represent the same blots reprobed for total p38 MAPK. B, Cells were stimulated with various concentrations of Ang II ranging from 0.1 to 100 nM for 5 min. C, Time course of p38 MAPK phosphorylation as analyzed by densitometry. D, Cells were stimulated without or with 100 nM Ang II for 5 min in absence or presence of 1 μM DUP753 (a specific antagonist of the AT1 receptor), 1 μM PD123319 (a specific antagonist of the AT2 receptor), or with 10 μM SB203580 (a specific inhibitor of p38 MAPK), introduced 30 min before Ang II. All data represent the mean ± SE of three different experiments. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control (**, P < 0.001).
Subsequent experiments were also conducted to ascertain which type of cascade could be involved in the opposite action of Ang II on proliferation (decreased in cell number) and hypertrophy (increase in phenylalanine incorporation). Cells were stimulated with or without Ang II in the absence or presence of specific inhibitors of these three MAPK pathways. PD98059 decreased, in a dose-dependent manner, basal levels of proliferation but did not modify the level of inhibition induced by Ang II (Fig. 7A), although it did reverse the inhibitory effect of Ang II on phenylalanine incorporation (Fig. 7B). On the other hand, SB203580, a specific antagonist of the p38 MAPK, reversed the effects of Ang II, in a dose-dependent fashion, on both inhibition of cell proliferation (Fig. 7C) and the observed increase in protein synthesis (Fig. 7D). Finally, using similar experimental conditions, the JNK inhibitor SP600125 did not modify the effects of Ang II, on either proliferation or protein synthesis (Fig. 7, E and F).
FIG. 7. Involvement of p42/p44mapk, p38 MAPK, and JNK on the effect of Ang II on proliferation and protein synthesis of rat glomerulosa cells. Cells were plated in 96-well plates (30 x 103 cells/well) for proliferation studies (A, C, and E) and 24-well plates (70 x 103 cells/well) for protein content studies (B, D, and F). Cells were stimulated for 3 d without or with Ang II (5 nM) (hatched bars), in the absence or presence of three concentrations of inhibitors: 5, 10, and 20 μM PD98059 (a specific inhibitor of MEK); 1, 5, and 10 μM SB203580 (a specific inhibitor of p38 MAPK); and 1, 5, and 10 μM SP600125 (a specific inhibitor of JNK), introduced 30 min before Ang II. Cell proliferation was measured using fluorescence BrdU incorporation. Protein synthesis was determined by assessing the incorporation of [3H]-phenylalanine. Results are expressed as the mean ± SE of six individual samples. Statistical significance: one-way ANOVA showed a significant effect of Ang II-treated cells, compared with control cells (*, P < 0.001) and between Ang II- and inhibitor plus Ang II-treated cells, compared with control cells (##, P < 0.001).
Ang II increases the expression of p27Kip1
The following experiments were aimed at understanding how Ang II produces these opposite effects on proliferation and protein synthesis. The fact that Ang II decreased cell proliferation but enhanced protein synthesis led us to consider that, during the 3-d incubation period, Ang II may act on one of the important steps of the cell cycle, possibly through one of the stimulated p42/p44mapk or p38 MAPK pathways. The effect of Ang II was therefore examined on the expression of cyclin E as a representative of the late events of the G1 phase of the cell cycle (39). As shown in Fig. 8A, expression of cyclin E did not vary significantly during the 3 d of culture, in both control and Ang II-treated cells.
FIG. 8. Effect of Ang II on cyclin E and p27Kip1 protein expression in rat glomerulosa cells cultured for 3 d. Cell lysates from equivalent numbers of cells (1 x 106 cells/petri) were separated on 10% polyacrylamide gels and subjected to Western blot analysis using specific antibodies against cyclin E and p27Kip1. Cells were cultured for 3 d in the absence or presence of 5 nM Ang II (A). B, Cells were cultured for 3 d in the absence or presence of 5 nM Ang II and either 10 μM SB203580 (a specific inhibitor of p38 MAPK) or 10 μM PD98059 (a specific inhibitor of MEK) introduced 30 min before Ang II.
Because cell cycle progression is controlled by cyclin/cyclin-dependent kinase (Cdk) complexes counterbalanced by Cdk inhibitors, expression and activity of G1-specific cyclin-dependent kinases can be inhibited by the presence of cyclin-dependent kinase inhibitors, including p27Kip1, which form complexes with cyclin/Cdk and could thereby block their interaction with substrates (34). As seen in Fig. 8A, Ang II increased the level of p27Kip1, measured by immunoblotting using specific antibody. In cells cultured in the presence of SB203580 or PD98059, the effect of Ang II on p27Kip1 level was abrogated, without affecting cyclin E levels (Fig. 8B).
Discussion
The present study provides compelling evidence that Ang II promotes cellular hypertrophy, but not proliferation, in rat adrenal glomerulosa cells maintained in primary culture for 3 d. The growth-promoting effect of Ang II occurs via AT1 receptor activation, without any participation of the AT2 receptor. Both p42/p44mapk and p38 MAPK are involved in the increase in protein synthesis and in the expression of p27Kip1, which, in turn, is responsible for inhibition of the cell cycle.
Proliferation vs. hypertrophy
Having previously shown that glomerulosa cells easily proliferate in culture, we used this model to directly assess the effects of Ang II on proliferation and protein synthesis. Measurement of cell proliferation using BrdU labeling or cell counting revealed that addition of Ang II during 3 d decreases cell number to the level observed at the outset of cell culture. These data on cell number could reflect the balance between the rate of cell proliferation and the rate of cell death via apoptosis. However, quantification of the number of apoptotic cells during the 3-d treatment with or without Ang II revealed that this number was low and did not differ between control and Ang II-treated cells. This decrease in cell proliferation is accompanied by a significant increase in protein synthesis and cell size. Pharmacological experiments indicate that the opposite effect of Ang II on proliferation and phenylalanine incorporation is mediated only by the AT1 receptor and not the AT2 receptor.
These results could appear provocative at first glance. Indeed, McEwan et al. (11) reported that Ang II infusion stimulates both cell proliferation and hypertrophy of glomerulosa cells and indicated that only proliferation is sensitive to inhibition by the AT1 receptor antagonist DUP753. However, such observations did not discriminate between direct effects of Ang II on glomerulosa cells or indirect effects through paracrine interaction with endothelial cells or fibroblasts, which also express Ang II AT1 receptors (1). Indeed, as recently reviewed by Bouzegrhane and Thibault (40), the proliferative effect of Ang II described in many studies may be caused by stimulation of growth factor synthesis or extracellular matrix components, such as fibronectin (41, 42). These hypotheses could be in agreement with the observations that in some cell types, such as cardiomyocytes, fibroblasts, or VSMC, Ang II up-regulates synthesis of extracellular matrix components, in particular collagen (43, 44), fibronectin (45, 46), and thrombospondin (22). Such matrix production and matrix assembly is necessary to support the capacity of Ang II to induce cell proliferation (47). This may explain why Tian et al. (16) observed proliferation only after 9 d in culture, suggesting that cells must express proteins necessary to generate AT1-receptor mediated proliferation.
The specific role of Ang II in proliferation and/or hypertrophy continues to be a subject of debate. Discrepancies in most of its targets tissues, including cardiomyocytes and VSMCs (38), could be explained in part by either cell type-specific action or a developmental regulatory process. For example, in immature ovine cardiomyocytes, Ang II stimulates hyperplasia but not hypertrophy (48). On the other hand, in adult human kidney mesangial cells, the hormone induces both hypertrophy and hyperplasia (49), whereas in adult cardiac tissue and VSMCs, it is now generally accepted that Ang II is implicated in hypertrophy but not in cell proliferation (34, 50, 51, 52, 53). In the present instance, we provide evidence that in rat adrenal glomerulosa cells in primary culture, Ang II, through the AT1 receptor, stimulates protein synthesis and inhibits proliferation normally observed during the 3 d in culture. Steroidogenic acute regulatory protein (StAR) and 3?-hydroxysteroid dehydrogenase (3?-HSD) are among the specific proteins stimulated (data not shown), corroborating previous observations made in bovine adrenal glomerulosa cells (54, 55) and in H295R cells (56, 57, 58). Such Ang II stimulation of StAR and 3?-HSD expression is associated with an increase in aldosterone secretion.
Molecular events involved in the hypertrophic effects of Ang II
The Ang II-promoting effect on protein synthesis occurs with a concomitant arrest in cell proliferation, suggesting a shift from pathways involved in proliferation to those involved in cell protein synthesis. We thus investigated which signaling pathway may be involved in such action and which intracellular proteins may be the targets of this shift from proliferation to hypertrophy. Our results indicate that Ang II induces a time-dependent increase in p42/p44mapk phosphorylation and a rapid and transient increase in p38 MAPK phosphorylation but has no effect on JNK.
Activation of the three MAPK pathways has been reported to be an important component in the signaling cascade triggered by Ang II, with an action described both on cell proliferation and cell hypertrophy, depending on cell type and/or experimental conditions (for review see Refs.37, 38). Whereas the role of p42/p44mapk is well documented in both Ang II-influenced proliferation and hypertrophy, the involvement of p38 MAPK in hypertrophy is a more recent finding. Our results indicate that specific inhibition of p42/p44mapk and p38 MAPK abrogated the effect of Ang II on tritiated phenylalanine incorporation. Thus, both pathways are essential in Ang II action on protein synthesis. In addition, MEK and p38 MAPK inhibitors both reverse the effect of Ang II on cell number. We did not observe any variation in JNK activity by Ang II, corroborating recent work in the field. Indeed, in H295R adrenocortical cells, Ang II leads to a dose-dependent increase in p38 MAPK activity but has no effect on JNK (59). Similar results have also been reported in bovine glomerulosa cells (60).
Although the p38 MAPK pathway has classically been associated with apoptosis and inflammatory reactions in response to cellular stress, there is increasing evidence that p38 MAPK is also activated by many G protein-coupled receptors, such as the AT1 receptor. For example, studies on neonatal cardiomyocytes demonstrated that activation of p38 MAPK-dependent pathways is associated with cell hypertrophy (61, 62), collagen synthesis, and fibrogenesis (44). Our results are thus in agreement with these studies demonstrating that p42/p44mapk but not p38 MAPK is involved in basal stimulation of DNA synthesis, whereas both p42/p44mapk and p38 MAPK are necessary in mediating Ang II-induced protein synthesis.
To confirm these results, we also measured the level of key proteins involved in regulating progression in cell cycle entry. Our data demonstrate that expression of cyclin E does not change throughout the 3 d in culture or during Ang II treatment. However, Ang II did increase the level of p27Kip1, an effect completely reversible when cells are coincubated with SB203580 or PD98059. This indicates that p38 MAPK and p42/p44mapk are responsible for the increase in p27Kip1 by Ang II, which in turn is responsible for inhibition of cell proliferation. A similar observation has also been recently described in vascular smooth muscle (62, 63, 64).
Altogether the present data clearly indicate that in cultured rat glomerulosa cells, a 3-d treatment with Ang II increases protein synthesis, with a concomitant decrease in cellular proliferation. These effects are mediated by both the p42/p44mapk and p38 MAPK pathways, which increase expression of the steroidogenic enzymes, StAR and 3?-HSD, and p27Kip1, a protein known to block the cell cycle in G1 phase. These results further support the key role of Ang II in maintaining the proper machinery necessary to support aldosterone biosynthesis, not only in acute situations but also during a more prolonged stimulation.
Acknowledgments
The authors thank Lyne Bilodeau and Lucie Chouinard for experimental assistance and Dr. Mylène C?té for stimulating discussions. We express our gratitude for the generous gift of antisera from our colleagues: cyclin E and p27Kip1, Dr. Nathalie Rivard (Département d’Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke; 3?-HSD, Dr. Van Luu-The (CHUL Research Center, Ste-Foy, Québec, Canada); and StAR, Dr. Douglas Stocco (Texas Tech University, Lubbock, Texas).
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