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Endocrinology Vol. 147, No. 11 5460-5469
Copyright © 2006 by The Endocrine Society

Differential Involvement of Cytoskeleton and Rho-Guanosine 5'-Triphosphatases in Growth-Promoting Effects of Angiotensin II in Rat Adrenal Glomerulosa Cells

Mélissa Otis and Nicole Gallo-Payet

Service of Endocrinology, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4

Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service d’Endocrinologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail: nicole.gallo-payet{at}usherbrooke.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glomerulosa cells readily proliferate in primary culture. However, 3-d treatment with angiotensin II (Ang II) promotes cellular hypertrophy with a concomitant decrease in proliferation. The aim of the present study was to investigate the manner by which cytoskeleton and Rho-GTPase proteins may be involved in Ang II-induced growth and MAPK activation. Preincubation with Y27632 (an inhibitor of Rho-associated kinase) decreased basal proliferation, as did Ang II, whereas toxin B, which inhibits Rho-GTPases, enhanced the inhibitory effect of Ang II. Conversely, toxin B inhibited protein synthesis induced by Ang II, whereas Y27632 had no effect. Ang II induced a rapid but transient activation of RhoA/B, an effect abolished in Y27632-preincubated cells. Activation of Rac appeared biphasic, with an early activation at 1 min, followed by a more sustained effect at 10 min. Toxin B abolished Rac activation. Immunofluorescence studies revealed that Y27632 and toxin B disrupted the F-actin network similarly to Ang II. Y27632 also abolished the cortical F-actin ring induced by Ang II. Preincubation of cells with toxin B abolished p38 MAPK phosphorylation and early activation of p42mapk (ERK2) and decreased p44mapk (ERK1) induced by Ang II. In contrast, Y27632, abolished p44mapk (ERK1) but had no effect on p42mapk (ERK2) or p38. Together these results indicate that, in rat adrenal glomerulosa cells, specific Rho/Rho-associated kinase-dependent activation of p44mapk (ERK1) and an intact cytoskeletal organization are necessary in mediating basal cell proliferation, whereas activation of p42/p44mapk, p38 MAPK, and Rac are essential in mediating Ang II-induced protein synthesis (steroidogenic acute regulatory protein and 3ß-hydroxysteroid dehydrogenase).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOTENSIN II (ANG II) is considered to be the main hormonal stimulus of the zona glomerulosa of the adrenal cortex (1, 2), acting on both aldosterone secretion and zona glomerulosa morphology (3, 4, 5). These actions are mediated by the Ang II type 1 receptor subtype of Ang II (6, 7). The effect on aldosterone secretion clearly involves calcium and protein kinase C as second messengers (8, 9), whereas the effects on growth alternatively involve the MAPK pathways (5, 7, 10, 11). Activation of Ras/Raf-1-dependent p42/p44mapk by Ang II in bovine glomerulosa cells is protein kinase C dependent and partly mediated by a pertussis toxin-sensitive Gi protein (7, 10, 12). Some studies have suggested that Ang II may act as a direct mitogen for glomerulosa cells (5, 7). However, in a recent study, our group revealed evidences that Ang II promotes cellular hypertrophy but not proliferation in rat adrenal glomerulosa cells maintained in culture for 3 d (13). This effect of Ang II on protein synthesis involved both p42/p44mapk and p38 MAPK pathways as well as important changes in cell morphology (13). On the other hand, we and others have also shown that the cytoskeleton modulates the action of Ang II in the adrenal gland. Indeed, cytochalasin and colchicine (which respectively depolymerize microfilaments and microtubules) decrease Ang II-induced inositol phosphates production and aldosterone secretion (14, 15). In recent years, an increasing number of studies have shown cytoskeletal involvement in signal transduction and the activation of MAPK pathways [p42/p44mapk, c-Jun N-terminal kinase (JNK), and p38 MAPK] (16, 17, 18) (for review see Ref. 19). Among the key proteins engaged in these processes are Rho-GTPases, Rho, Rac, and Cdc42 (20).

Rho-GTPases constitute a distinct subclass within the Ras-related small GTPase superfamily and are ubiquitous to all eukaryotic cells. Twenty-two mammalian genes encoding Rho-GTPases have been described including three Rho isoforms A, B, and C; three Rac isoforms 1, 2, and 3; and Cdc42 (21, 22). Active GTP-bound forms of Rho-GTPases achieve their regulatory function through a conformation-specific interaction with target proteins. Rho-GTPases are likely to play an integral regulatory role in cytoskeleton organization. Rho and its downstream effector Rho-associated kinase (ROCK) act as regulators of stress fibers and focal adhesion assembly. Rac, on the other hand, regulates the formation of lamellipodia protrusions and membrane ruffles, whereas Cdc42 triggers filopodia at the cell periphery (for review see Ref. 22). In addition, to date, more than 50 effectors have been identified for Rho, Rac, and Cdc42 including serine/threonine kinases, tyrosine kinases, lipid kinases, lipases oxidases, and scaffold proteins (22). In particular, strong evidence indicates that Rho plays a pivotal role in cytoskeletal-mediated activation of several intracellular proteins involved in the regulation of MAPK pathways (23, 24, 25, 26, 27). For example, in vascular smooth muscle cells (VSMCs), ROCK was reported to be involved in p42/p44mapk activation (28), whereas Rac was alternatively involved in p38 MAPK activation resulting in transduction of growth and hypertrophy (29, 30, 31). Rac is also essential for Ang II-induced vascular remodeling and Ang II-induced activation of p42/p44mapk, whereas JNK pathways are also Rac dependent in VSMCs (32).

In light of these regulatory interactions, the aim of the present study was therefore to explore whether and how Rho-GTPases and the cytoskeleton may be involved in the growth-promoting effects of Ang II in rat adrenal glomerulosa cells (13). More specifically, the study aimed at verifying their involvement in the activation of both p42/p44mapk and p38 MAPK pathways as well as the effect of Ang II on the inhibition of basal proliferation and accrued protein synthesis, including steroidogenic enzymes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
The chemicals used in the present study were obtained from the following sources: collagenase, MEM Eagle’s medium, and OPTI-MEM medium from Life Technologies (Burlington, Canada); deoxyribonuclease from Sigma Chemical Co. (St. Louis, MO); Ang II from Bachem (Marina Delphen, CA); antiphosphorylated p42/p44mapk, antiphosphorylated p38 MAPK, anti-p42/p44mapk, anti-p38 MAPK, and antipaxillin antibodies from New England Biolabs, Inc. (Mississauga, Ontario, Canada); 5-bromo-2-deoxyuridine (BrdU), anti-BrdU Alexa Fluor-594, antimouse Alexa Fluor 488, phalloidin Alexa Fluor-594, and 4',6'-diamino-2-phenylindole from Molecular Probes (Eugene, OR). Clostridium difficile toxin B and Y27632 were from Calbiochem (La Jolla, CA). Rho and Rac assay kits were from Upstate Biotechnology (Lake Placid, NY); 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); polyvinylidene difluoride membranes from Millipore (Bedford, MA); and Bio-Rad DC protein assay from Bio-Rad Laboratories (Hercules, CA). The steroidogenic acute regulatory protein (StAR) antibody was a kind gift from Dr. Douglas Stocco (Texas Tech University, Lubbock, TX) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) from Dr. Van Luu-The (CHUL Research Center, Ste-Foy, Québec, Canada). 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 (33). All protocols were approved by the Animal Care and Ethics Committee of our institution. Isolation and cell dissociation of the zona glomerulosa were performed in MEM (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. According to experiments, cells were plated at various densities either in plastic 35-mm petri dishes for Western blots (5 x 105 cells/dish) and indirect fluorescence analyses (5 x 104 cells/dish), 24-well plates (70 x 103 cells/well) for protein synthesis measurements, or 96-multiwell plates (30 x 103 cells/well) 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 in culture. Except in specific experiments, cells were stimulated with 5 nM Ang II for 3-d treatments (concentration for which stimulation of aldosterone secretion, protein synthesis, and inhibition of proliferation all reach a plateau) (13, 33, 34, 35) and 100 nM for acute stimulations (in which maximal effects were obtained during a 2-h stimulation period) (13). Cells were incubated in the absence or presence of C. difficile toxin B (3 ng/ml, 60 min), which inhibits all Rho-GTPases (36), or Y27632 (20 µM, 30 min), a specific inhibitor of the Rho-kinase family (37). Doses and exposure times were chosen according to criteria of reversibility after washing and without affecting cell viability. Cells were examined daily, and phase-contrast images were acquired using a Leica Corp. microscope (Deerfield, IL) equipped with a x32 objective.

Proliferation assays
Cell proliferation was measured using fluorescence BrdU incorporation (13). 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 were added to the culture medium 4 h before stimulation with Ang II (5 nM). On the third day, cells were fixed with 3.7% (vol/vol) formaldehyde in Hanks’-buffered saline (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 percent changes from basal conditions using six culture wells for each experimental condition.

Protein synthesis measurements
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 (5 nM). 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 with a counter (Beckmann, Fullerton, CA). Data were normalized as maximum of phenylalanine incorporation in control conditions for the same number of cells (1 x 105 cells), as described by Otis et al. (13).

Determination of RhoA and Rac activation
Rho-GTPases exhibit both GDP-bound inactive and GTP-bound active forms. Measurement of Rac and RhoA/B activities was performed as described by Yamaguchi et al. (38). Activity of Rho-GTPases was determined using the glutathione S-transferase (GST)-fused Rho-binding domain (RBD) of rhotekin (39) for GTP-bound RhoA and GST-fused Cdc42/Rac-interactive binding domain of p21-activated kinase (PAK1) (GST-PAK1) for GTP-bound Rac1 (38). After 3 d of culture, cell density reached approximately 3 x 106 cells/petri. Cells were harvested for short-term stimulation without or with Ang II (100 nM) and subsequently lysed with lysis buffer [50 mM Tris-HCl (pH 7.2), 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 500 mM NaCl, 10 mM MgCl2 with protease inhibitors]. Total lysate proteins (800 µg) were clarified by centrifugation at 10,000 x g at 4 C for 10 min, after which equal volumes of supernatants were incubated with GST-RBD beads (20 µg) for Rho-GTP or with GST-PAK1 beads (20 µg) for Rac-GTP at 4 C for 60 min. The beads were washed three times with wash buffer [50 mM Tris-HCl (pH 7.2), 1% Triton X-100, 150 mM NaCl, 10 mM MgCl2 with protease inhibitors]. The eluted proteins were resolved by SDS-gel electrophoresis. The concentration of bound active GTP-loaded small GTPase was analyzed by immunoblotting using the following primary antibodies: anti-RhoA/B and anti-Rac antibodies (1:1000). Control assays were performed by incubating lysates of unstimulated cells with either guanosine 5'-3-O-(thio)triphosphate (positive control) or GDP (negative control) for 15 min at 25 C before incubation with GST-RBD or GST-PAK1 beads.

Immunofluorescence studies
For actin cytoskeleton studies, cells were plated on plastic petri dishes (density of 5 x 104 cells after 3 d in culture) and treated with the appropriate stimuli. Cells were fixed with 3.7% (vol/vol) formaldehyde in HBS buffer for 15 min at 4 C, permeabilized for 10 min in HBS-0.2% Triton X-100, and blocked for 45 min in HBS/0.5% BSA. Cells were then incubated with antipaxillin (1:1000) and Alexa Fluor-594 phalloidin (1:60) (for visualization of microfilaments) for 60 min at room temperature. After washings, cells were further incubated for 60 min at room temperature with a secondary conjugated anti-IgG antibody coupled with Alexa Fluor-488 nm. Cells were then incubated with 4',6'-diamino-2-phenylindole (1:300) for 5 min. After washings, cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlington, Ontario, Canada) and images were acquired with an ORCA-ER digital camera (Hamamatsu, Bridgewater, NJ) mounted on a Nikon Eclipse TE-2000 inverted microscope (Nikon Canada, Mississauga, Canada) equipped for epiillumination. Images were acquired using a x100 objective.

Western blotting
Cells were cultured in 35-mm petri dishes at a concentration of 5 x 105 cells/petri, with cell density reaching approximately 1 x 106 cells/petri after 3 d of culture. Cells were used after 3 d for short-term stimulation without or with Ang II (100 nM) in the absence or presence of various inhibitors introduced before Ang II treatment, namely cytochalasin B (10 µM, 30 min), C. difficile toxin B (3 ng/ml, 60 min), or Y27632 (20 µM, 30 min). For expression of StAR and 3ß-HSD, glomerulosa cells were cultured for 3 d without or with PD98059 (10 µM) [inhibitor of MAPK kinase (MEK)] or with SB203580 (10 µM) (inhibitor of p38 MAPK) introduced 30 min before Ang II (5 nM). After hormonal stimulation in culture medium, 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. 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 amount of protein (30 µg protein) were separated on 10% SDS-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 MAPK (dilution 1:500), anti-p42/p44mapk (dilution 1:1000), anti-p38 MAPK (dilution 1:500), anti-StAR (dilution 1:1000), anti-3-ßHSD (dilution 1:500), or anti-tubulin (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 one-way ANOVA, followed by a test of simple effects when appropriate. Homogeneity of variance was assessed by Bartlett’s test and P values were obtained by Tukey honestly significant differences. For simple comparisons between two groups, a Student’s t test was performed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of the cytoskeleton and Rho-GTPases on Ang II-induced proliferation and protein synthesis
In a previous report, we demonstrated that Ang II decreases basal proliferation but enhances protein synthesis (13). In the present study, cells were treated without or with Ang II in the absence or presence of 10 µM cytochalasin B, a disruptor of actin cytoskeleton. As previously shown, Ang II decreased basal proliferation (Fig. 1AGo) but enhanced protein synthesis (Fig. 2AGo) (13). Preincubation with cytochalasin B had no effect on cell viability but decreased basal cell number, which was further reduced by the addition of Ang II (Fig. 1AGo). Preincubation with C. difficile toxin B (3 ng/ml, 60 min), which inhibits all Rho-GTPases, had no effect on proliferation but further decreased the effect of Ang II, as did cytochalasin II (Fig. 1BGo). In contrast, preincubation with Y27632 (20 µM, 30 min), a specific inhibitor of ROCK, inhibited basal cell proliferation (Fig. 1BGo).


Figure 1
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  		FIG. 1. Involvement of actin (A) and Rho-GTPases (B) on proliferation of 3-d cultured rat glomerulosa cells. Cells were plated in 96-well plates for proliferation studies (30 x 103 cells/well). Cells were then stimulated for 3 d without or with Ang II (5 nM) (hatched bars), in the absence or presence of cytochalasin B (Cyto B; 10 µM) (A) or C. difficile toxin B (3 ng/ml) (B) or with Y27632 (20 µM) (B) introduced 30 min before Ang II. Cell proliferation was measured using fluorescence BrdU incorporation as described in Materials and Methods. Results are expressed as the mean ± SE of four experiments, each experimental condition containing six individual samples. Statistical significance: one-way ANOVA revealed a significant effect of Ang II- or inhibitor-treated cells, compared with basal values (**, P < 0.001), and between Ang II- and inhibitor plus Ang II-treated cells, compared with Ang II-treated cells (*, P < 0.01).

 

Figure 2
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  		FIG. 2. Involvement of actin (A) and Rho-GTPases (B) on protein synthesis of 3-d cultured rat glomerulosa cells. Cells were plated in 24-well plates for protein synthesis studies (70 x 103 cells/well). Cells were then stimulated for 3 d without or with Ang II (5 nM) (hatched bars), in the absence or in the presence of cytochalasin B (Cyto B; 10 µM) (A) or C. difficile toxin B (3 ng/ml) (B) or with Y27632 (20 µM) (B) introduced 30 min before Ang II. Protein synthesis was determined by assessing the incorporation of [3H]phenylalanine as described in Materials and Methods. Results are expressed as the mean ± SE of four experiments, each experimental condition containing six individual samples. Statistical significance: one-way ANOVA revealed a significant effect of Ang II- or inhibitor-treated cells, compared with basal values, and between Ang II- and inhibitor plus Ang II-treated cells, compared with Ang II-treated cells (**, P < 0.001).

 
Conversely, preincubation with cytochalasin B decreased the effect of Ang II on protein content (Fig. 2AGo), whereas toxin B and Y27632 had no effect on basal protein content. However, toxin B inhibited the stimulatory effect of Ang II, whereas Y27632 had no effect (Fig. 2BGo). Together these results indicate that an intact cytoskeletal architecture and involvement of Rho-GTPases are necessary for mediating cell proliferation. The effect of Ang II on the stimulation of protein synthesis, on the other hand, requires Rac activation.

Activation of Rho-GTPases by Ang II
Ang II induced a rapid activation of RhoA/B (1–5 min) with a maximum observed at 2 min (Fig. 3Go, A and B). Preincubation with Y27632 abolished the activation of RhoA/B, whereas toxin B had no effect (Fig. 3Go, A and B). In contrast, Ang II induced a biphasic activation of Rac, with an initial early activation at 1 min and a second activation at 10 min. Toxin B completely abolished both phases, whereas Y27632 decreased the sustained phase only (Fig. 3Go, C and D). These results indicate specificity of the inhibitors, with Y27632 specifically inhibiting Rho and toxin B specifically inhibiting Rac.


Figure 3
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  		FIG. 3. Activation of Rho and Rac by angiotensin II in rat glomerulosa cells. After 3 d in culture, cells (2 x 106 cells) were preincubated without (control conditions) or with C. difficile toxin B (3 ng/ml, 60 min) or Y27632 (20 µM, 30 min) and then stimulated without (none) or with 100 nM Ang II for the indicated time periods. The activities of Rho (A) and Rac (C) were determined by measuring the amount of GTP-loaded protein bound to GST-Rhotekin or GST-PAK1 as described in Materials and Methods. Effect of Ang II on the time course of RhoA/B (B) or Rac (D) activation as analyzed by densitometry. Ang II stimulation was in the absence of inhibitor (bullet) or presence of toxin B ({circ}) or Y37632 ({blacktriangledown}). All data represent the means ± SE of six different experiments. Statistical significance: one-way ANOVA revealed a significant effect of Ang II- or inhibitor-treated cells, compared with basal values (none) (*, P < 0.001), and between inhibitor plus Ang II-treated cells, compared with Ang II-treated cells (#, P < 0.001).

 
Effect of Ang II and Rho-GTPases on cytoskeletal organization
To further investigate the mechanism by which Rho-GTPases affect cytoskeletal organization in addition to their involvement in MAPK activation, the effect of Ang II was assessed on the spatial localization of actin and paxillin, a protein present at focal adhesions initiating the connection between hormone-receptor binding and cytoskeletal organization at focal adhesion contacts (40). Cells were monitored using dual-labeling immunofluorescence microscopy with Alexa Fluor-594-phalloidin for visualization of actin and Alexa Fluor-488 antibody for visualization of paxillin. As described in a previous report (41), control cells had a flattened polygonal morphology, characterized by a discrete network of thin stress fibers crossing the entire cell and the presence of focal adhesion points evidenced by paxillin labeling (Fig. 4Go, A, D, and G). Ang II induced a rapid (visible after 1 min) (15) but transient (no longer apparent after 30 min) reorganization of the actin cytoskeleton. A 15-min stimulation with Ang II induced the formation of an intense ring of F-actin at the cell membrane, a disruption of the well-organized stress fiber network, and the formation of several thin filopodia, in lieu of focal adhesions (Fig. 4BGo, arrows). These changes were accompanied by a disappearance of paxillin labeling, which became diffuse throughout the cytoplasm (Fig. 4Go, E and H). Cells recovered their original control morphology after 30 min, even in the continuous presence of Ang II (Fig. 4Go, C, F, and I).


Figure 4
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  		FIG. 4. Effect of Ang II on immunofluorescence labeling of actin filaments and paxillin. Cells were plated on plastic petri dishes (1 x 105 cells) as described in Materials and Methods and then stimulated without (A, D, and G) (none) or with 100 nM Ang II for 15 (B, E, and H) and 30 min (C, F, and I). After formaldehyde fixation and permeabilization with 0.1% Triton X-100, cells were processed for immunofluorescence labeling using phalloidin coupled to Alexa Fluor-594 nm for visualization of F-actin (A–C) (red) and with antipaxillin antibody coupled to Alexa Fluor-488 nm for visualization of paxillin (D–F) (green). Merged images are shown in G–I. Images are representative illustrations of more than 50 cells originating from three different experiments. Bars, 13 µm.

 
The effect of the Rho-GTPase inhibitors was assessed on the time-course action of Ang II on actin organization. Preincubation of cells with toxin B abolished the anchoring of F-actin to focal adhesion points and shortened the length of actin filaments, which were no longer structured as parallel-oriented fibers (Fig. 5Go, A and E). Addition of Ang II in toxin B-treated cells accelerated the effect of Ang II, which became perceptible after 5 min (Fig. 5FGo) comparatively with 15 min (Fig. 5CGo). This effect remained transient, however, disappearing after 15 min (Fig. 5GGo), rather than the 30 min observed in control cells (Fig. 5DGo). After 30 min, cells exhibited the same morphology as control toxin B-treated cells (Fig. 5Go, H vs. D). Preincubation of cells with the ROCK inhibitor Y27632 induced: 1) a reduction in thickness and a shortening of actin filaments in control conditions (Fig. 5IGo); 2) abolished the formation of the actin cortical ring by Ang II (Fig. 5Go, J, K, and L); and 3) favored the formation of well-developed lamellipodia (Fig. 5Go, K and L). However, as shown in Fig. 5Go, K and L, and confirmed by paxillin labeling, cells remained attached to the substratum, indicating that focal adhesions were not specifically affected by inhibitors of the Rho family GTPases. These results suggest that Rho and its target kinase ROCK are essential for the formation of microfilaments but not the formation of focal adhesions.


Figure 5
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  		FIG. 5. Effect of various Rho GTPase inhibitors on Ang II-induced changes in actin filaments and paxillin localization. Cells were plated on plastic petri dishes (1 x 105 cells) as described in Materials and Methods. Cells were preincubated without (A–D) (control conditions) or with C. difficile toxin B (3 ng/ml, 60 min), a selective inhibitor of Rho, Rac, and Cdc42 (E–H) or Y27632 (20 µM, 30 min), a specific inhibitor for the Rho-kinase family (I–L) and then stimulated without (A, E, and I) (none) or with 100 nM Ang II for 5 (B, F, and J), 15 (C, G, and K), and 30 min (D, H, and L). After formaldehyde fixation and permeabilization with 0.1% Triton X-100, cells were processed for immunofluorescence labeling with antipaxillin antibody coupled to Alexa Fluor-488 nm for visualization of paxillin (green) and with phalloidin coupled to Alexa Fluor-594 nm for visualization of F-actin (red). Images are representative illustrations of more than 50 cells originating from three different experiments. Bars, 13 µm.

 
Role of the cytoskeleton and Rho-GTPases in Ang II-induced activation of MAPKs
As shown in Fig. 6AGo, Ang II, at a concentration of 100 nM, induced a rapid increase in p42/p44mapk phosphorylation (maximum observed at 5 min, 17.3-fold increase), followed by a sustained activation exceeding 30 min (4.5-fold increase) (13). Preincubation of cells with cytochalasin B (30 min with 10 µM) significantly decreased the sustained response to Ang II on p42/p44mapk activation (Fig. 6Go, B, E, and F), with no effect on the early phase of stimulation (Fig. 6Go, E and F). Preincubation with toxin B abolished the early phase of Ang II-stimulated phosphorylation of both p42 and p44mapk (Fig. 6Go, C, E, and F), without affecting the sustained response (Fig. 6Go, E and F). On the other hand, preincubation with the specific ROCK inhibitor Y27632 (20 µM, 30 min) abolished only phosphorylation of p44mapk (Fig. 6Go, D, E, and F) without affecting p42mapk phosphorylation (Fig. 6Go, E and F). These results indicate that the early and sustained phases of p42/p44mapk phosphorylation induced by Ang II are differentially controlled by the actin cytoskeleton and Rho-GTPases. Ang II also stimulated phosphorylation of p38 MAPK (Fig. 7AGo). However, this effect was transient (maximum observed at 2 min, 9.2-fold increase) followed by a return to basal levels after 10 min of incubation (Fig. 7CGo) (13). Preincubation of cells with cytochalasin B (Fig. 7BGo) and toxin B (Fig. 7DGo) abolished the effect of Ang II, whereas Y27632 had no effect on Ang II responses (Fig. 7Go, E and F).


Figure 6
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  		FIG. 6. Effect of cytochalasin B and Rho-GTPase inhibitors on the time-course effect of Ang II-induced p42/p44mapk activation. After 3 d in culture, cells (1 x 106 cells) were preincubated without (A) (control conditions) or with cytochalasin B (10 µM, 30 min) (B), C. difficile toxin B (3 ng/ml, 60 min), an inhibitor of Rho-GTPases (C), or Y27632 (20 µM, 30 min), a specific inhibitor of ROCK (D), followed by stimulation without (none) or with 100 nM Ang II for the indicated time periods. Cell lysates containing equal concentrations of protein were subjected to Western blot analyses with antibodies against phosphorylated p42/p44mapk (upper panels). Lower panels represent the same blots reprobed for total p42/p44mapk. Effect of cytochalasin B or Rho-GTPases inhibitors on the time-course phosphorylation of p44mapk (E) and p42mapk (F) as analyzed by densitometry. Ang II stimulation in the absence of inhibitor (bullet) or presence of cytochalasin B ({square}), toxin B ({circ}), or Y27632 ({blacktriangledown}). All data represent the means ± SE of six different experiments. Statistical significance: one-way ANOVA revealed a significant effect of Ang II- or inhibitor-treated cells, compared with basal values (none) (*, P < 0.001), and between inhibitor plus Ang II-treated cells, compared with Ang II-treated cells (#, P < 0.001).

 

Figure 7
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  		FIG. 7. Effect of cytochalasin B and Rho-GTPase inhibitors on the time-course effect of Ang II-induced p38 MAPK activation. After 3 d in culture, cells (1 x 106 cells) were preincubated without (A) (control conditions) or with cytochalasin B (10 µM, 30 min) (B), C. difficile toxin B (3 ng/ml, 60 min) (D), or Y27632 (20 µM, 30 min) (E), followed by stimulation without (none) or with 100 nM Ang II for the indicated time periods. Cell lysates containing equal concentrations of protein were subjected to Western blot analyses with antibodies to phosphorylated p38 MAPK (upper panels). Lower panels represent the same blots reprobed for total p38 MAPK. Effect of cytochalasin B or Rho-GTPases inhibitors on the time course of p38 MAPK phosphorylation (C and F) as analyzed by densitometry. Ang II stimulation in the absence of inhibitor (bullet) or presence of cytochalasin B ({square}), toxin B ({circ}), or Y27632 ({blacktriangledown}). All data represent the means ± SE of six different experiments. Statistical significance: one-way ANOVA revealed a significant effect of Ang II- or inhibitor-treated cells, compared with basal values (none) (*, P < 0.001), and between inhibitor plus Ang II-treated cells, compared with Ang II-treated cells (#, P < 0.001).

 
Role of MAPKs in steroidogenesis
To investigate whether the increase in protein synthesis involves steroidogenesis enzymes, cells were preincubated in the presence or absence of either 10 µM PD98059 (an inhibitor of MEK) or 10 µM SB203580 (an inhibitor of p38 MAPK). As shown in Fig. 8AGo, Ang II increased the expression levels of StAR and 3ß-HSD but were suppressed in cells preincubated with PD98059 (10 µM) or SB203580 (10 µM) (Fig. 8BGo). These results indicate that p42/p44mapk and p38 MAPK play a role in Ang II-stimulated aldosterone production by enhancing expression of StAR and 3ß-HSD proteins.


Figure 8
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  		FIG. 8. Effect of MAPK inhibitors on Ang II-induced expression of StAR and 3ß-HSD. Glomerulosa cells were cultured for 3 d without (A) or with (B) PD98059 (10 µM) (inhibitor of MEK) or SB203580 (10 µM) (inhibitor of p38 MAPK) introduced 30 min before Ang II (5 nM) stimulation. Cell lysates from equivalent numbers of cells (1 x 106 cells/petri dish) were separated on 10% polyacrylamide gels and subjected to Western blot analysis using specific antibodies against 3ß-HSD and StAR, as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In a previous report, we had shown 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 occurred via Ang II type 1 receptor activation and involved both p42/p44mapk and p38 MAPK activation. Indeed, specific inhibition of p42/p44mapk and p38 MAPK abrogated the effect of Ang II on tritiated phenylalanine incorporation. In addition, the effect of Ang II on protein synthesis occurred with a concomitant decrease in cell proliferation, these latter effects being mediated by an increase in the expression of p27Kip1, which, in turn, is responsible for inhibition of the cell cycle (13). The present study provides further evidences that basal proliferation requires a well-structured actin filament organization into stress fibers and a specific Rho/ROCK-dependent activation of p44mapk (ERK1). Conversely, the increase in cell protein content by Ang II entails the disruption of F-actin organization, formation of a cortical actin ring, and Rac-dependent activation of p42/p44mapk and p38 MAPK. This is the first demonstration of the differential involvement of cytoskeletal proteins Rho/ROCK and Rac on pp44mapk-induced proliferation vs. p42/p44mapk-induced protein synthesis in rat glomerulosa cells.

Involvement of Rho-GTPases in proliferation and protein synthesis
Glomerulosa cells, when cultured on plastic, typically exhibit a polygonal morphology, are well attached to the substratum, and readily proliferate, doubling their number after 3 d, even in medium containing only 2% fetal serum (42, 43). In the present study, preincubation with cytochalasin B, which disrupts F-actin organization (11, 15), decreased the sustained activation of p42/p44mapk, inhibited p38 MAPK activation, and decreased both basal proliferation and protein synthesis induced by Ang II. Thus, the effect of Ang II on actin filaments may account for its ability to inhibit proliferation and alternatively favor cell protein synthesis.

These observations are in keeping with the well-documented essential role of the cytoskeleton in overall cell physiology (44, 45). Indeed, it is well known that ACTH induced rounding up and actin disruption of at least adrenocortical Y1 cells (14, 23). This rounding up facilitates transport of cholesterol to the mitochondria and increase in steroid secretion. Known inhibitors of Rho-GTPases were also used to dissect the respective roles of Rho/ROCK and Rac in glomerulosa cell function. At the concentration used, Y27362 inhibited Rho activation, but not Rac, whereas toxin B inhibited Rac, but not RhoA/B, indicating that, in rat adrenal glomerulosa cells, these inhibitors are excellent tools for differentiating the distinctive involvement of these two Rho-GTPases. More importantly, the present results indicate that these two Rho-GTPases are differentially involved in proliferation and protein synthesis. Y27632 decreased basal proliferation, as did cytochalasin B and specifically abolishing phosphorylation of p44mapk (ERK1), hence suggesting that microfilaments and Rho, but not Rac, are central players in mediating proliferation.

These results clearly indicate that both phosphorylated kinases p42 and p44mapk, also known as ERK2 and ERK1, play a pivotal role in either proliferation or growth arrest, depending on the duration and intensity of p42/p44mapk activation (for review see Refs. 46, 47, 48). The two isoforms of p42 and p44mapk share an 84% identity at the amino acid level. The actual individual roles of p42 (ERK2) and p44mapk (ERK1) isoforms have emerged only recently, with the generation of p44mapk knockout (49) and p42mapk knockout mice (50). Disruption of the p42mapk locus leads to embryonic lethality early in mouse development (50), whereas p44mapk knockout mice are viable and fertile and of normal size (49). It thus appears that p44mapk is unable to compensate for p42mapk, whereas p42mapk or possibly other growth factors are able to compensate for p44mapk deletion.

Recent studies have shown that the MEK1/p42/p44mapk complex is mainly involved in proliferation, whereas the MEK2/p42/p44mapk module induces growth arrest, with massive induction of cyclin D expression (49, 51, 52). In p44mapk knockout mice, skin hyperproliferation induced by phorbol ester is reduced (52) and, in p44mapk knockout thymocytes, maturation and proliferation are also severely reduced in response to phorbol esters (49). The balance of MEK1 and MEK2 signaling (i.e. the upstream of p42/p44mapk) ultimately determines the kinetics and intensity of p42/p44mapk activation and the ensuing final outcome: proliferation or growth arrest. Ussar and Voss (51) suggested that the kinetic activation of p42/p44mapk by MEK1 promotes proliferation via cyclin D-CDK4/6 activation and that the sustained activation of p42/p44mapk by MEK2, leads to p21cip1- and p27kip-mediated growth arrest (51). These findings corroborate our previous observations in which the effect of Ang II on protein synthesis involves increased expression of p27kip and steroidogenic enzymes, including StAR and 3ß-HSD, hence associated with cell differentiation. Stimulation of protein synthesis by Ang II requires both p38 MAPK and p42/p44mapk activation (13). Moreover, among the proteins are those of steroidogenesis, namely StAR and 3ß-HSD. These results complement the observations that, in bovine adrenal glomerulosa cells, p42/p44mapk is involved in Ang II-induced phosphorylation of cholesterol ester hydrolase (53) and p38 MAPK in the Ang II-induced release of calcium from intracellular pools (54).

As in other studies (17, 55, 56, 57), the present results confirm that Rho/ROCK are upstream mediators of p42/p44mapk phosphorylation by Ang II. However, the role of Rho/ROCK in p42/p44mapk activation is not a general consensus. For instance, Ang II-induced activation of p42/p44mapk is not mediated through Rho/ROCK in VSMCs, (16). On the other hand, toxin B, which specifically inhibits Rac, was shown herein to abolish p38 MAPK phosphorylation as well as the early increase in p42/p44mapk phosphorylation and the effect of Ang II on protein synthesis. Conversely, Y29632 did not affect p38 MAPK nor did it modify the effect of Ang II on protein synthesis. These results are in agreement with those recently obtained by Touyz et al. (58). In VSMCs, Ang II was shown to mediate the activation of p38 MAPK and JNK, but not p42/p44mapk, all of these effects being reduced by cytochalasin B. Thus, whereas p38 MAPK and JNK require an intact actin cytoskeleton for full activation by Ang II, this does not appear to be the case for p42/p44mapk.

Activation of Rho/ROCK and Rac by Ang II
From the present results, proliferation clearly requires a well-structured network of microfilaments, attached to focal adhesions. Y29632 was observed to disrupt stress fibers and decrease proliferation, thus in agreement with the assertion that Rho/ROCK signaling pathways regulate the assembly of stress fibers (59), often initiated at focal adhesions. Both FAK and paxillin are present at these sites, together with integrins (17, 60). In bovine aortic endothelial cells, inhibition of ROCK (with Y27632) also suppressed stress fiber formation (61).

Toxin B induced a specific disruption of the anchoring of F-actin from focal adhesion points, without affecting the stress network per se. Such effects accelerated Ang II-induced modification of cell morphology and the formation of a peripheral actin ring. On the other hand, RhoA/B was rapidly and transiently activated by Ang II, whereas Y27632 abolished Ang II-induced actin accumulation at the membrane level. Finally, whereas Y27632 disrupted F-actin organization, it did not affect cell adherence through focal adhesions. This inhibition of ROCK also induced the formation of lamellipodia, suggesting that inhibition of the Rho pathway enhances the Rac/Cdc42 pathway. In light of the known functions of Rac and Rho (59), the above observations suggest that ROCK is critical for the formation of the cortical actin ring, shifting actin sensitivity from Rho to Rac. This hypothesis that Rac may in fact down-regulate Rho activity, as reported by Sander et al. (39), appears consistent with the present pharmacological results obtained with Y27632 and toxin B, considering that Y27362 inhibited Rho activation, but not Rac, whereas toxin B inhibited Rac, but not Rho.

The observation that inhibition of RhoA/ROCK impairs basal proliferation and that inhibition of Rac impairs Ang II-induced protein synthesis clearly indicates that Rho-GTPases play pivotal roles in dictating cell behavior. Reciprocal balance between Rho and Rac determines cell morphology and cell function, at least in regard to proliferation or hypertrophy. The model proposed in Fig. 9Go illustrates that basal proliferation requires a well-structured actin filament organization into stress fibers and the activation of both Rho/ROCK and p44mapk phosphorylation (Fig. 9AGo). Stimulation with Ang II induces growth arrest (increased expression of p27kip) and increases protein synthesis (including expression of StAR and 3ß-HSD) (13). This action of Ang II involves the disruption of F-actin organization, formation of a cortical actin ring, and Rac activation. Phosphorylation of p42/p44mapk is necessary during Ang II stimulation but requires interaction with p38 MAPK to trigger an increase in cell protein content (Fig. 9BGo). Further detailed studies will, however, be necessary to fully uncover the exact relationship between focal adhesions, cytoskeleton, and MAPK activation in basal conditions and under Ang II stimulation.


Figure 9
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  		FIG. 9. Actin cytoskeleton and the signaling machinery implicated in proliferation (A) and protein synthesis (B) of rat adrenal glomerulosa cells. Stress fiber organization and activation of Rho/ROCK and p44mapk are both necessary for proliferation. Stimulation with Ang II induces growth arrest and increases protein synthesis. The increase in cell protein content produced by Ang II involves the disruption of F-actin organization, formation of a cortical actin ring, and Rac activation. During Ang II stimulation, p42/p44mapk is essential but requires interaction with p38 MAPK to fully increase cell protein content. See text for additional details. FA, Focal adhesion; Cai, intracellular calcium; R-AT1, Ang II type 1 receptor; PLC, phospholipase C; PKC, protein kinase C.

 
In summary, by assessing cell function as a whole including morphology and biochemical data, this study provides evidences that basal proliferation, in rat adrenal glomerulosa cells, requires specific Rho/ROCK-dependent activation of p44mapk (ERK1) and an intact cytoskeletal organization. The increase in protein content by Ang II involves the disruption of F-actin organization, formation of a cortical actin ring, and Rac-dependent activation of p42/p44mapk and p38 MAPK. This is the first demonstration of the differential involvement of cytoskeletal proteins Rho/ROCK and Rac on p44mapk-induced proliferation vs. p42- and p44mapk-induced protein synthesis in rat glomerulosa cells. Providing answers to these mechanisms of cell signaling and function may help to understand why the zona glomerulosa is so unique, compared with the adjacent zona fasciculata. For example, as reviewed recently by Vinson (4, 62), the zona glomerulosa is the site of active proliferation and is often considered as the progenitor site for the entire cortex, although the exact location of proliferation is not clearly established (subcapsular, zona glomerulosa and/or zona intermedia). In this regard, glomerulosa cells have the inherent capability of producing a wealth of growth factors, which may contribute or explain their great plasticity and important morphological changes under specific conditions, such as a sodium-deficient diet.


   Acknowledgments
 
The authors thank Lyne Bilodeau and Lucie Chouinard for experimental assistance, Dr. Shirley Campbell for stimulating discussions, and Dr. Marcel D. Payet for the critical review of the manuscript. We express our gratitude for the generous gift of antisera from our colleagues Dr. Van Luu-The (CHUL Research Center, Ste-Foy, Québec, Canada; 3 ß-HSD) and Dr. Douglas Stocco (Texas Tech University, Lubbock, TX; StAR).


   Footnotes
 
This work was supported by grants from the Fondation des Maladies du Coeur du Québec and the Canadian Institute for Heath Research (to N.G.-P.) (MOP-10998). N.G.-P. is a recipient of a Canada Research Chair in Endocrinology of the Adrenal Gland.

A part of this study was presented as a poster presentation at the 86th Annual Meeting of The Endocrine Society, New Orleans, LA, June 16–19, 2004, and at the Société Française d’Endocrinologie, Strasbourg, France, October 9–12, 2005.

Disclosure statement: M.O. and N.G.-P. have nothing to declare.

First Published Online August 17, 2006

Abbreviations: Ang II, Angiotensin II; BrdU, 5-bromo-2-deoxyuridine; GST, glutathione S-transferase; HBS, Hanks’-buffered saline; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; PAK1, p21-activated kinase; RBD, Rho-binding domain; ROCK, Rho-associated kinase; SDS, sodium dodecyl sulfate; StAR, steroidogenic acute regulatory protein; TCA, trichloroacetic acid solution; VSMC, vascular smooth muscle cell.

Received June 8, 2006.

Accepted for publication August 8, 2006.


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 Results
 Discussion
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