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Endocrinology, doi:10.1210/en.2008-0282
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Endocrinology Vol. 149, No. 7 3435-3445
Copyright © 2008 by The Endocrine Society

In Adrenal Glomerulosa Cells, Angiotensin II Inhibits Proliferation by Interfering with Fibronectin-Integrin Signaling

Mélissa Otis, Shirley Campbell, Marcel D. Payet and Nicole Gallo-Payet

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, Quebec, 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, Quebec, Canada J1H 5N4. E-mail: Nicole.Gallo-Payet{at}USherbrooke.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (Ang II), through the Ang II type 1 receptor subtype, inhibits basal proliferation of adrenal glomerulosa cells by inducing the disruption of actin stress fiber organization. This effect is observed in cells cultured on plastic or on fibronectin. The aim of the present study was to investigate how Ang II may interfere with extracellular matrix/integrin signaling. In cells treated for 3 d with echistatin (EC) (a snake-venom RGD-containing protein that abolishes fibronectin binding to {alpha}5β1 or {alpha}vβ3 integrins), basal proliferation decreased by 38%, whereas Ang II was unable to abolish basal proliferation. In cells grown on fibronectin, Ang II decreased binding of paxillin to focal adhesions and, similarly to EC, induced a rapid dephosphorylation of paxillin (1 min), followed by an increase after 15 min. Fibronectin enhanced RhoA/B and Rac activation induced by Ang II, an effect abolished by EC. Under basal conditions, paxillin was more readily associated with RhoA/B than with Rac. Stimulation with Ang II induced a transient decrease in RhoA/B-associated paxillin (after 5 min), with a return to basal levels after 10 min, while increasing Rac-associated paxillin. Finally, results reveal that glomerulosa cells are able to synthesize and secrete fibronectin, a process by which cells can stimulate their own proliferative activity when cultured on plastic. Together, these results suggest that Ang II acts at the level of integrin-paxillin complexes to disrupt the well- developed microfilament network, a condition necessary for the inhibition of cell proliferation and initiation of steroidogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ZONA GLOMERULOSA of the adult adrenal cortex is not only specialized in aldosterone production but is also an important site of cell proliferation. Indeed, the zona glomerulosa is considered, together with the adjacent capsule and/or zona intermedia, as the progenitor region of the cortex (for review see Refs. 1 and 2). In addition, glomerulosa cells have the property to adapt their morphology to surrounding environmental conditions.

Angiotensin II (Ang II) is the most potent stimulus of aldosterone secretion (3, 4). Ang II also stimulates cell proliferation, at least in in vivo conditions (5, 6, 7). However, studies from our laboratory have shown that Ang II promotes cellular hypertrophy, but not proliferation, in rat adrenal glomerulosa cells maintained in primary culture for 3 d (8).

Several studies have shown that the extracellular microenvironment can orchestrate a number of cell functions such as proliferation, differentiation, migration, survival, and steroidogenesis (9, 10, 11). For instance, cells cultured on collagens, laminin (LN), and fibronectin (FN) proliferate at a higher rate than when cultured on plastic (11). These extracellular matrix proteins interact with cells through binding of integrin receptors localized on the plasma membrane. Integrins are transmembrane proteins comprised of two subunits, {alpha} and β, and constitute the predominant family of proteins mediating cell-matrix interactions (12), which are responsible for the transduction and activation of several intracellular cascades (13, 14). For example, FN is a specific ligand for integrin {alpha}5β1, although integrins {alpha}3β1, {alpha}4β1, {alpha}xβ2, {alpha}vβ3,and {alpha}vβ6 are also able to bind FN (15, 16). Several members of the integrin family, including integrins {alpha}5β1 and {alpha}vβ3, bind FN at specific recognition sites consisting of small amino acid sequences such as the RGD sequence (Arg-Gly-Asp) (17, 18). Integrins {alpha}V, {alpha}5, β1, and β3 subunits have all been shown to be expressed in rat adrenal glomerulosa cells (11, 19). The adhesion of glomerulosa cells to FN promotes proliferation and aldosterone secretion through an increase in intracellular calcium and activation of p42/p44mapk (19).

Interaction between extracellular matrix proteins and actin cytoskeleton is also mediated by integrins and is initiated at focal adhesion sites. Focal adhesions are large complexes of structural, enzymatic, and adaptor proteins and serve as the nucleation site for actin filaments. Stimulation of actin stress fiber formation is frequently associated with rapid tyrosine phosphorylation of cytoskeletal-associated proteins, such as paxillin, resulting in the formation of binding sites for other proteins that mediate structural and signaling events (20, 21). In many cell types, stress fiber formation, focal adhesion complex assembly, and rapid tyrosine phosphorylation of paxillin are dependent on RhoA activation, which is mediated by Rho-kinase (20, 22, 23). Recently, we demonstrated that the cytoskeleton, through Rho-GTPases, Rho/ROCK, and Rac, are involved in MAPK activation and in Ang II-induced cell growth. Indeed, in rat adrenal glomerulosa cells, the activation of RhoA, an intact cytoskeletal organization, and the activation of p42/p44mapk are necessary for mediating basal cell proliferation, whereas activation of Rac, p42/p44mapk, and p38 MAPK are essential for mediating Ang II-induced protein synthesis (24, 25).

Altogether, our previous studies indicate that 1) basal proliferation of glomerulosa cells in culture requires an intact cytoskeletal organization, 2) a FN matrix is important for triggering several glomerulosa cell functions (proliferation, increased intracellular calcium concentration, and aldosterone production) (19), and 3) Ang II induces a disruption of stress fiber organization necessary in mediating protein synthesis and steroid secretion. These observations thus raise the question as to how Ang II may act on FN matrix signaling. We therefore investigated whether Ang II could interfere with binding on FN and its integrins. In addition, to better understand the basal proliferative activities of glomerulosa cells, we also assessed whether glomerulosa cells are able to produce extracellular matrix proteins and, especially, FN.


   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, Eagle’s MEM, and OPTI-MEM from Life Technologies (Burlington, Canada); PC-1 medium from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD); Ang II from Bachem (Marina Delphen, CA); and RNA extraction kit from Ambion (Austin, TX). Matrix-coated dishes and coated 24- and 96-well plates (FN, LN, and collagen IV) were obtained from BD-VWR Canlab (Ville Mont-Royal, Quebec, Canada); antirabbit and antimouse IgG-fluorescein isothiocyanate (FITC) from ICN Biochemicals (Costa Mesa, CA); echistatin and anti-FN antibody from Chemicon International Inc. (Temecula, CA); anti-paxillin antibody from BD Transduction Laboratories (Mississauga, Ontario, Canada); and anti-phosphotyrosine monoclonal antibody (PY99) from Santa Cruz Biotechnology (Santa Cruz, CA). Antirabbit Alexa Fluor-488 nm, Alexa Fluor-594 nm phalloidin, 4',6'-diamino-2-phenylindole (DAPI), 5-bromo-2-deoxyuridine (BrdU), and anti-BrdU Alexa Fluor-594 nm were purchased from Molecular Probes (Eugene, OR); deoxyribonuclease, orthovanadate (Na3VO4), and PD123319 from Sigma Chemical Co. (St. Louis, MO); and DUP753 from Losartan, DuPont Merck Pharmaceuticals (Paris, France). Rho and Rac assay kits were from Upstate Biotechnology (Lake Placid, NY); horseradish peroxidase-conjugated antirabbit and antimouse antibodies, enhanced chemiluminescence detection system (ECL), recombinant protein G-Sepharose, and L-[2,3,4,5,6-3H] phenylalanine from GE Healthcare (Oakville, Canada); polyvinylidene difluoride membranes from Millipore (Bedford, MA); Bio-Rad DC protein assay from Bio-Rad Laboratories (Hercules, CA); and Vectashield from Vector Laboratories (Burlingame, 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 (26). All protocols were approved by the Animal Care Committee of our faculty. Isolation and cell dissociation of the zona glomerulosa was 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 resulting cell pellet was then resuspended in OPTI-MEM supplemented with 2% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Depending on experiments, cells were plated at various densities on FN- or poly-L-lysine-coated coverslips or 96-multiwell plates and cultured at 37 C in a humidified atmosphere composed of 95% air/5% CO2. The culture medium was changed every day, 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 with 100 nM for acute stimulations, as in the previous studies (8, 24). Cells were examined daily, and phase-contrast images were taken using a Leica Corp. microscope (Deerfield, IL) equipped with a x32 objective.

Proliferation assays
Cell proliferation was measured using fluorescence BrdU incorporation, as previously described (8). Cells were plated on plastic, FN, LN, or collagen IV-coated 96-well plates at a concentration of 3 x 104 cells per well. Cells were treated daily for 3 d without or with Ang II (5 nM) alone or in the presence of the appropriate inhibitors, DUP753 (1 µM) (a specific antagonist of the AT1 receptor), PD123319 (1 µM) (a specific antagonist of the AT2 receptor), echistatin (10 µM) (a snake-venom RGD-containing protein, known to be a potent antagonist of {alpha}IIbβ3, {alpha}vβ3, and {alpha}5β1 integrins), introduced 30 min before Ang II. After 24 h of culture, 10 µM BrdU was added to the culture medium 4 h before stimulation without or with Ang II and drugs. 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, 560/40 nm; emission, 645/40 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 determined by assessing tritiated phenylalanine incorporation. Cells were plated on plastic or matrix-coated 24-well plates at a concentration of 7 x 104 cells per well. After 24 h of culture, 1 µCi /ml [3H]phenylalanine was added to the media 2 h before stimulation without or with Ang II (5 nM) alone or with drugs as above. After 3 d, the medium was aspirated and cells washed three times with cold HBS solution, followed by addition of trichloroacetic acid (TCA) solution (20%) for 20 min on ice. After centrifugation (3000 x g for15 min at 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 (Beckmann counter). 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. (8).

Morphometric analysis
A total of 200 glomerulosa cells for each condition were studied. Cell diameters were measured semiautomatically using the MetaMorph Imaging System 4.5 software package (Universal Imaging Corp., West Chester, PA). Cell area was calculated by cell parameter analysis. Results are expressed as means ± SE of 200 cells for each experimental condition.

Binding studies
The analog [Sar1,Ile8] Ang II was iodinated by the Iodogen method, as previously described (27). For binding assays, 3-d cultured cells (approximately 580,000 cells per plastic petri dish and 800,000 cells per FN petri dish) were washed with 2 ml HBS buffer and binding performed as previously described (28). Briefly, cells were incubated for 15 min at 37 C with 0.07 nM 125I-labeled [Sar1,Ile8] AngII (900 Ci/mmol) and selected concentrations of [Sar1,Ile8] Ang II (10 µM, nonspecific binding, which represents less than 10% of total binding), DUP753 (10 µM), PD123319 (10 µM), or CGP42112 (10 nM). Bound radioactivity was separated from free ligand by filtration through GF/C filters (presoaked for 24 h in 2% BSA). Receptor-bound radioactivity was evaluated by {gamma}-counting. Experiments were performed three times.

Immunofluorescence
For FN immunofluorescence studies, cells were plated on poly-L-lysine-coated coverslips for 1, 3, or 5 d and cultured in PC-1 medium, a low-protein medium (BioWhittaker Specialty Media). Cells were subsequently fixed with methanol for 20 min at –20 C and washed with PBS (136.89 mM NaCl, 2.68 mM KCl, 7.18 mM Na2HPO4-7H20, 1.76 mM KHPO4, pH 7.4). After incubation with blocking buffer (45 min in PBS-0.5% BSA), cells were incubated with the anti-FN antibody (1:100) for 60 min at room temperature. After PBS washes, cells were further incubated for 60 min at room temperature with a secondary conjugated anti-IgG antibody coupled with FITC. Cells were then incubated with DAPI (1:300) for 5 min. After washes, cells were mounted in Vectashield mounting medium and examined on a Nikon Eclipse 300 microscope (Nikon Canada, Mississauga, Ontario, Canada) equipped with a CoolSnap fx digital camera (Roper Scientific, Tucson, AZ). Images were acquired using a x40 objective.

For cytoskeleton immunofluorescence studies, cells were plated on FN-coated petri dishes without or with echistatin (10 µM) (density of 1 x 105 cells after 3 d in culture) and stimulated with Ang II (100 nM) for 15–30 min. Cells were fixed with 3.7% (vol/vol) formaldehyde in HBS buffer for 15 min at 4 C and permeabilized for 10 min with 0.2% Triton X-100/HBS. 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 washes, 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 DAPI (1:300) for 5 min. After washes, cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlington, Ontario, Canada) and images acquired with an ORCA-ER digital camera (Hamamatsu, Bridgewater, NJ) mounted on a Nikon Eclipse TE-2000 inverted microscope (Nikon Canada) equipped for epiillumination. Images were acquired using a x100 objective.

Immunoprecipitation
Immunoprecipitation and Western blot analyses were performed as previously described (29). Glomerulosa cells grown on plastic or FN-coated petri dishes were incubated without or with echistatin (10 µM) or Na3VO4 (1 mM) (a phosphotyrosine phosphatase inhibitor). At a density of 1 x 106 cells after 3 d in culture, glomerulosa cells were washed once and stimulated with 100 nM Ang II for indicated times at 37 C. Cells were then washed twice with ice-cold HBS buffer and lysed in 500 µl cold Tris buffer [containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, and 1 µg/ml leupeptin). Cells were then scraped with a rubber policeman and transferred in Eppendorf tubes for 30 min on ice. The insoluble material was pelleted at 15,000 x g for 10 min at 4 C. Lysates were clarified with protein G-Sepharose for 45 min at 4 C, followed by centrifugation at 200 x g for 1 min. For immunoprecipitation with phosphotyrosine or paxillin antibodies, the lysates were incubated for 2 h at 4 C with the corresponding antibodies. Protein G-Sepharose was added and incubated 1 h at 4 C. Immunocomplexes were washed four times before electrophoresis on 10% SDS-polyacrylamide gels and analysis by immunoblotting (anti-paxillin at 1:1000 dilution and anti-phosphotyrosine at 1:500 dilution). Detection was performed by reaction with horseradish peroxidase-conjugated secondary antibody and visualization achieved by ECL according to the manufacturer’s instructions. The immunoreactive bands were scanned by laser densitometry and expressed in arbitrary units.

Determination of RhoA and Rac activation and interaction with paxillin
Measurement of Rac and RhoA/B activities was performed as previously described (24). Activity of Rho-GTPases was determined using the glutathione S-transferase (GST)-fused Rho-binding domain (RBD) of rhotekin for GTP-bound RhoA and the GST-fused Cdc42/Rac-interactive binding domain of p21-activated kinase (PAK1) (GST-PAK1) for GTP-bound Rac1. After 3 d of culture, cell density reached approximately 3 x 106 cells per petri dish. 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% 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 washing buffer [50 mM Tris-HCl (pH 7.2), 1% Triton X-100, 150 mM NaCl, and 10 mM MgCl2 with protease inhibitors]. The eluted proteins were resolved by SDS-PAGE. 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). Interaction with paxillin was analyzed by re-immunoblotting the membranes with anti-paxillin (dilution, 1:1000). Detection was performed by reaction with horseradish peroxidase-conjugated secondary antibody and visualization achieved by ECL according to the manufacturer’s instructions. The immunoreactive bands were scanned by laser densitometry and expressed in arbitrary units.

RT-PCR amplification
Rat adrenal glomerulosa cells were isolated as described above, plated at a density of 5 x 105 cells on plastic petri dishes and allowed to adhere for 2 d. Total RNA was extracted using the RNAqueous method according to the manufacturer’s user’s guide (Ambion). RNA was then dissolved in elution solution after determination of total RNA concentration by OD (260 nm). Two micrograms of RNA were reversed transcribed into first-strand cDNA. For each preparation, oligo- deoxythymidine (5 U), dNTPs (10 mM) from Amersham Pharmacia Biotech (Piscataway, NJ), Moloney murine leukemia virus, First 5x Moloney murine leukemia virus buffer, and RNasin (all from Promega, Madison, WI) were used. cDNA was used for each PCR. Primer pairs were designed based on GenBank with Beacon 2.2 software (Biosoft International, Palo Alto, CA) or are from previously published sequences for rat: FN sense, 5'-CCG GTT CTG AGT ACA CAG TC-3'3, and FN antisense, 5'-AGG GAC CAC TTC TCT GGG AGG-3' (30). Rat GAPDH was used as a control of RNA integrity, as previously published (19) (data not shown). The PCR was carried out using 4 µl cDNA, dNTPs (10 mM), Taq polymerase (Amersham), and 10x Taq polymerase PCR buffer. Amplification was performed on a PerkinElmer (Waltham, MA) amplification system for 35 cycles consisting of 45 sec at 94 C, 90 sec at 59 C, and 2 min at 72 C. For visualization, 10 µl PCR product were loaded on a 2% agarose gel, stained with ethidium bromide, and scanned using a Fluorimager (MultiImage Light Cabinet, Alpha Innotech Corp., San Leandro, CA).

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’s 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
 
Ang II interferes only with FN in regard to proliferation and protein synthesis
The effects of Ang II on proliferation and on protein synthesis were first compared in glomerulosa cells cultured on various matrices, namely FN, LN, and collagen IV. As recently shown in Otis et al. (11), all matrices increased the basal proliferation level of glomerulosa cells. However, addition of Ang II (5 nM) (for 3 d) (8) decreased proliferation of cells cultured either on plastic or on FN but did not modify the proliferation observed on LN or collagen IV (Fig. 1AGo). On the other hand, in cells cultured on FN and LN, protein content per cell was lower than in cells cultured on plastic, whereas Ang II increased phenylalanine incorporation only in cells cultured on plastic or FN (Fig. 1BGo).


Figure 1
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  		FIG. 1. Influence of matrices alone or in association with Ang II on proliferation (A) and protein synthesis (B) in rat adrenal glomerulosa cells cultured for 3 d. Cells were plated onto plastic (PL)-, FN-, LN-, or collagen IV (Col IV)-coated 96-well plates for proliferation studies (30 x 103 cells per well) and onto similarly coated 24-well plates for protein content (70 x 103 cells per well). Glomerulosa cells were stimulated for 3 d without or with Ang II (5 nM). Proliferation was measured by fluorescent BrdU incorporation, whereas protein synthesis was assessed by [3H]phenylalanine incorporation. Results are expressed as the mean ± SE of three experiments with each experimental condition containing four individual samples. One-way ANOVA showed a significant effect of extracellular matrix compared with basal condition on plastic (##, P < 0.01) and between Ang II-treated cells compared with basal condition cells (**, P < 0.01).

 
To verify whether such functional modifications were accompanied by morphological changes, phase-contrast examination and morphometric analyses were conducted. Cells cultured on plastic and on FN exhibited the same polygonal morphology as recently shown in a previous study (11). Measurement of cell area indicated a normal Gaussian-type distribution in the various experimental conditions. However, mean cell size decreased from 300 ± 4.6 to 218 ± 4.8 µm2 (P < 0.001) in cells cultured on plastic compared with cells cultured on FN (Fig. 3AGo). Furthermore, a 3-d stimulation with Ang II (5 nM) induced a significant increase in cell size on both substrates, with mean values increasing from 300 ± 4.6 to 384 ± 5.1 µm2 (P < 0.001) between basal conditions and Ang II-treated cells cultured on plastic and from 218 ± 4.8 to 260 ± 5 µm2 (P < 0.001) between basal conditions and Ang II-treated cells cultured on FN (Fig. 3CGo).


Figure 3
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  		FIG. 3. Effect of FN on cell morphometry (A) and involvement of AT1R and AT2R (B) in the effect of Ang II on proliferation in rat adrenal glomerulosa cells. Glomerulosa cells were plated at an initial concentration of 70 x 103 cells for morphometric analysis and at 30 x 103 cells per well onto FN-coated 96-well plates for proliferation studies. Cells were stimulated for 3 d without or with Ang II (5 nM), in the absence or presence of 1 µM DUP753 (a specific antagonist of the AT1R) or 1 µM PD123319 (a specific antagonist of the AT2R), introduced 30 min before Ang II. A, Phase-contrast morphometric analysis. Cell surface area was calculated by cell parameter analysis as described in Materials and Methods. Results are expressed as the mean ± SE of 200 cells from three independent experiments. One-way ANOVA showed a significant effect of FN compared with basal condition on plastic (##, P < 0.01) and Ang II-treated cells compared with basal condition cells (**, P < 0.01). Proliferation (B) was measured as described in Fig. 1Go. Results are expressed as the mean ± SE of three experiments with each experimental condition containing four individual samples. One-way ANOVA showed a significant effect of Ang II-treated cells compared with basal condition cells (**, P < 0.01) and between antagonist plus Ang II-treated cells compared with Ang II-treated cells (§, P < 0.01).

 
The above data indicate that glomerulosa cell morphology and proliferative properties are similar whether cells are grown on plastic or FN. One of the hypotheses in explaining these observations is the assumption that glomerulosa cells are able to produce FN. Using specific primers, the RT-PCR approach revealed the presence of a 768-bp band corresponding to the expected band size for FN in glomerulosa cells (Fig. 2AGo). Whole rat brain extracts were used as control, whereas reactions performed in the absence of cDNA or Taq polymerase were used as negative controls (data not shown). Immunofluorescence studies using FITC-coupled anti-FN antibody applied without cell permeabilization revealed the presence of FN protein surrounding all cells. Increasing culture time induced changes in FN labeling, which initially appeared diffuse after 1 d in culture (Fig. 2BGo), then as small dots in proximity of the cell membrane after 3 d (Fig. 2CGo), and finally as fibrillar structures after 5 d in culture (Fig. 2DGo). These results indicate that the similar properties observed when cells are plated on plastic or on FN could be due to the fact that glomerulosa cells can secrete FN into their microenvironment. Ang II is known to mediate its effect through binding with its two main receptor types, the AT1 receptor subtype (AT1R) and the type 2 receptor subtype (AT2R), both of which have been identified in rat adrenal zona glomerulosa (31, 32). We thus verified whether the inhibitory effect of Ang II could be mediated through the AT2R subtype. Cells were preincubated with either PD123319, the specific antagonist of AT2R, or with DUP753, the specific antagonist of AT1R. Pretreatment with DUP753 did not interfere with the basal level of proliferation on FN but reversed the inhibitory effect of Ang II. On the other hand, preincubation of cells with the AT2R antagonist PD123319 did not alter the inhibitory action of Ang II on proliferation (Fig. 3BGo). Ang II binding studies were also performed to further investigate whether AT1R and AT2R expression could be modulated by cells cultured on FN. After a 30-min incubation in the presence of 0.074 nM 125I-labeled [Sar1,Ile8] Ang II, glomerulosa cells retained an amount of bound radioactivity equivalent to 106.3 ± 8.3 fmol/1 x 106 cells when cultured on plastic, which decreased to 58.7 ± 16.1 fmol/1 x 106 cells when cultured on FN (0.05 < P < 0.01; n = 3). Considering that total specific binding of 125I-labeled [Sar1,Ile8] Ang II to the cells represents 100%, in the presence of DUP753 (10 µM), the amount of specific bound radioactivity decreased by 86%, whereas in the presence of PD123319 (10 µM) or CGP42112 (10 nM), the amount of bound radioactivity decreased by 18 and 16%, respectively (data not shown).


Figure 2
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  		FIG. 2. Expression of FN by rat adrenal glomerulosa cells. Cells were plated at a density of 5 x 105 cells on 60-mm plastic dishes for 3 d (RT-PCR) (A) and at a density of 75 x 103 cells on poly-L-lysine-coated coverslips for 1 (B), 3 (C), or 5 (D) days and cultured in a low-protein medium. Total RNA was extracted as described in Materials and Methods. Using specific primers, the RT-PCR (A) approach revealed the presence of a 768-bp amplicon corresponding to FN. Lane 1, size marker (base pairs); lane 2, positive control (brain); lane 3, glomerulosa cells. Whole rat brain extracts were used as controls, whereas reactions performed in the absence of cDNA or Taq polymerase were used as negative controls (data not shown). For immunofluorescence of FN (B–D), cells were subsequently fixed with methanol and processed for immunofluorescence labeling using anti-FN antibody coupled to FITC (green) as well as DAPI (blue) to label nuclei. Scale bar, 25 µm.

 
Ang II interferes with FN binding to integrins
Proliferation requires a well-established stress-fiber network (24), whereas activation of integrins by FN can be reproduced by stimulation of cells with the active peptide RGDS (Arg-Gly-Asp-Ser) (19). We therefore verified whether prevention or hindrance of FN-integrin interaction could affect physiological properties of glomerulosa cells in culture. Cells were preincubated with echistatin, a RGD-containing protein known to be a potent antagonist of {alpha}IIbβ3, {alpha}vβ3, and {alpha}5β1 integrins (33, 34). Addition of 10 µM echistatin (daily for 3 d) decreased cell proliferation on both plastic and FN by 25 ± 3.8 and 38 ± 4.2%, respectively (Fig 4Go, A and B). To investigate whether the inhibitory action of Ang II could be initiated at the level of integrin signaling, echistatin-treated cells cultured on plastic or FN were stimulated with Ang II. Results show that although the inhibitory effect of Ang II still persisted in echistatin-treated cells cultured on plastic (Fig. 4AGo), it disappeared in cells cultured on FN (Fig. 4BGo).


Figure 4
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  		FIG. 4. Involvement of FN binding (A and B) in the effect of Ang II on proliferation on plastic (A) or FN (B) in rat adrenal glomerulosa cells. Cells were plated onto plastic-coated (A) or FN-coated (B) 96-well plates for proliferation studies (30 x 103 cells per well). Cells were stimulated for 3 d without or with Ang II (5 nM), in the absence or presence of 10 µM echistatin (A and B) (a snake-venom RGD- containing protein, known to be a potent antagonist of {alpha}IIbβ3, {alpha}vβ3, and {alpha}5β1 integrins) introduced 30 min before Ang II. Proliferation was measured by fluorescent BrdU incorporation. Results are expressed as the mean ± SE of three experiments with each experimental condition containing four individual samples. One-way ANOVA showed a significant effect of echistatin compared with basal condition on plastic or FN (##, P < 0.01) and between Ang II-treated cells compared with basal condition cells (**, P < 0.01).

 
Ang II acts at the level of integrin binding and focal adhesions to disrupt F-actin organization
To further investigate how Ang II could interfere with the primary signaling events triggered by binding of FN to its integrins, the effect of echistatin was studied on Ang II-induced localization of actin and paxillin. Glomerulosa cells cultured on FN had a flattened polygonal morphology, characterized by a discrete network of thin stress fibers (red) crossing the entire cell and the presence of focal adhesion points revealed by paxillin labeling (green) (Fig. 5AGo). After 15 min, Ang II induced the formation of an intense F-actin ring at the cell membrane, the disruption of the well-organized stress fiber network and the formation of several thin filopodia in lieu of focal adhesions (Fig. 5BGo, arrowhead). These changes were accompanied by the dissociation of paxillin from the extremity of actin filaments (Fig. 5BGo, arrow). The effect of Ang II was transient, however, because it was no longer observed after 30 min, even in the continuous presence of Ang II in the medium (Fig. 5CGo). Preincubation of the cells with echistatin did not alter the well-structured actin cytoskeleton in control cells (Fig. 5Go, D vs. A) but further abolished the formation of the cortical actin ring as well as paxillin relocalization induced by Ang II (Fig. 5Go, E and F vs. B). We next investigated whether paxillin may be the target of integrin-induced cytoskeletal reorganization by Ang II. Cells were stimulated for periods ranging from 5–30 min with 100 nM Ang II. As shown in Fig. 6AGo, Ang II induced a biphasic effect on paxillin phosphorylation, characterized by a rapid decrease (during the first 5 min) and followed by a striking increase after 30 min (Fig. 6Go, A and E). This effect was observed on both plastic and FN-cultured conditions (Fig. 6Go, A, B, and E). Echistatin treatment of FN-cultured cells abolished the dephosphorylation of paxillin induced by Ang II but maintained and even accelerated the phosphorylation of paxillin visible after 15 min (Fig. 6Go, C and E). Preincubation of FN-cultured cells with orthovanadate, an inhibitor of tyrosine phosphatase activity, also induced a sustained increase in paxillin phosphorylation (Fig. 6Go, D and E), indicating the possible activation of a phosphotyrosine phosphatase activity by Ang II.


Figure 5
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  		FIG. 5. Effect of echistatin on Ang II-induced localization of actin and paxillin on FN. Glomerulosa cells were plated on FN-coated dishes (1 x 105 cells) as described in Materials and Methods. Cells were preincubated without (A–C) or with 10 µM echistatin (D–F) and thereafter stimulated with Ang II (100 nM) for 15 min (B and E) and 30 min (C and F). 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 (red) and with anti-paxillin antibody coupled to Alexa Fluor-488 nm for visualization of paxillin (green) as well as DAPI (blue) for visualization of nuclei. Images are representative illustrations of more than 50 cells originating from three different experiments. Scale bar, 13 µm.

 

Figure 6
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  		FIG. 6. Effect of Ang II on tyrosine phosphorylation of paxillin in rat adrenal glomerulosa cells. Cells (1 x 106 cells) were plated onto plastic (A) or FN-coated petri dishes (B–D) and prepared as described in Materials and Methods. Cells were incubated without or with 10 µM echistatin (C) or 1 mM Na3VO4 (phosphatase inhibitor) (D). After 3 d, cells were stimulated with Ang II (100 nM) for the indicated time periods (basal, b). Cell lysates containing equal amounts of protein were subjected to immunoprecipitation (IP) with antibodies to anti-phosphotyrosine, followed by Western blot analysis (IB) with antibody anti-paxillin (1:1000). E, Effect of plastic, FN, FN plus echistatin or FN plus Na3VO4 on the time course of phosphorylation of paxillin as analyzed by densitometry. Results are expressed as the mean ± SE of four experiments. One-way ANOVA showed a significant effect of Ang II-treated cells compared with basal condition cells (**, P < 0.01) and between inhibitor plus Ang II-treated cells compared with Ang II-treated cells on plastic (PL) (§, P < 0.01).

 
Association of paxillin with Rho-GTPase proteins
In a recent study, we had shown that Rho-GTPases, through Rho/ROCK and Rac, are involved in Ang II-induced cell growth (24). We thus verified how FN and integrin signaling may influence Rho-GTPase activation and the possible interaction with paxillin. Cells cultured on FN exhibited a higher basal level of RhoA/B activity compared with plastic (1.8 ± 0.2-fold increase) (Fig. 7AGo) but displayed similar basal levels for Rac activity (Fig. 7BGo). When cultured on FN, the effect of Ang II on both RhoA/B and Rac activation was enhanced by 67 ± 2.1% over plastic conditions, along a similar time course, which corresponded to a transient increase for RhoA/B (Fig. 7CGo) but with two peaks for Rac, one at 1 min (21 ± 1.4% increase over control) and a second peak at 10 min (37 ± 2.4% increase compared with plastic) (Fig. 7DGo). Addition of echistatin to cells cultured on FN restored Rho-GTPase activation levels to that seen on plastic conditions (Fig. 7Go, C and D).


Figure 7
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  		FIG. 7. Effect of FN on the time course of Rho-GTPase activation induced by Ang II. After 3 d of culture on plastic or FN without or with 10 µM echistatin, cells (3 x 106 cells) were stimulated without (basal, b) or with Ang II (100 nM) for the indicated time periods. The activity of RhoA/B (A and C) and Rac (B and D) was determined by measuring the amount of GTP-loaded protein bound to GST- Rhotekin or GST-Pak1 as described in Materials and Methods. Effect of plastic (bullet), FN ({circ}), and FN plus echistatin ({blacktriangledown}) on the time course of Rho A/B (C) or Rac (D) activation induced by Ang II was measured by densitometry. All data represent the mean ± SE of four experiments. One-way ANOVA showed a significant effect of FN compared with basal condition on plastic (##, P < 0.01) and between Ang II-treated cells on FN compared with Ang II-treated cells on plastic (§, P < 0.01).

 
The association of paxillin with Rho-GTPases was assessed with the GST pull-down of Rho-GTPases. Western blot analysis revealed that, under basal conditions, paxillin had greater association with RhoA/B (plastic, 42 ± 2.2%; FN, 58 ± 1.8%) than with Rac (plastic, 10 ± 0.8%; FN, 6 ± 0.9%) (Fig. 8Go). Ang II decreased RhoA/B-associated paxillin in the first 5 min of incubation (plastic, from 42 ± 2.2 to 6 ± 0.5%; FN, from 58 ± 1.8 to 11 ± 1.0%) (Fig. 8Go, A and C) but increased it thereafter at 10 and 30 min of incubation, both on plastic (16 ± 1.1 and 21 ± 0.9%, respectively) and on FN (21 ± 1.3 and 46 ± 2.5%, respectively). Ang II also increased the association of paxillin with Rac at 10 min (plastic, 41 ± 1.2%; FN, 29 ± 2.4%) and 30 min (plastic, 42 ± 1.2%; FN, 31 ± 2.6%) (Fig. 8Go, B–D).


Figure 8
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  		FIG. 8. Interaction of RhoA/B (A) and Rac (B) with paxillin in rat adrenal glomerulosa cells stimulated with Ang II. After 3 d of culture on plastic (C) or FN (D), cells (3 x 106 cells) were stimulated without (basal, b) or with Ang II (100 nM) for the indicated time periods. The activity of Rho and Rac was determined by measuring the amount of GTP-loaded protein bound to GST-Rhotekin or GST-Pak1 as described in Materials and Methods. Interaction with paxillin was analyzed by re-immunoblotting the membranes with anti-paxillin (dilution, 1:1000). Interaction of RhoA/B ({circ}) and Rac (bullet) with paxillin on plastic (C) and FN (D) induced by Ang II was analyzed by densitometry. All data represent the mean ± SE of four experiments. One-way ANOVA showed a significant effect of Ang II-treated cells compared with basal condition cells (**, P < 0.01) and between RhoA and Rac associated with paxillin ($, P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study extends our previous observations on the effects of Ang II on glomerulosa cells and explains how Ang II inhibits proliferation in glomerulosa cells while inducing increased cell protein content and steroid synthesis. The results herein reveal that Ang II decreases FN-induced proliferation (via AT1R) by acting at the level of FN binding to integrins followed by subsequent activation of actin- associated proteins. Indeed, Ang II induces dephosphorylation of paxillin and loss of association of paxillin with both F-actin at focal adhesion sites and with RhoA/B. These effects are responsible for the lack of F-actin organization in stress fibers attached to focal adhesion, which is necessary for the induction of cell proliferation (8). Furthermore, our results indicate that glomerulosa cells are able to synthesize and secrete FN, a process that explains how cells are able to stimulate their basal proliferative activity.

Ang II decreases proliferation of cells plated on both plastic and FN
The present study shows that FN alone is able to stimulate proliferation of glomerulosa cells. Fibronectin is a 500-kDa glycoprotein molecule that binds to the integrin family of receptors, namely {alpha}5β1, {alpha}vβ1, and {alpha}vβ3, all of which are expressed in rat adrenal glomerulosa cells (11, 19). In addition, compared with plastic (not shown), FN represents a better optimal underlying matrix for promoting the formation of focal adhesions (19). When glomerulosa cells are cultured in the presence of Ang II, cell proliferation decreases along with a concomitant increase in cell protein content (8). This effect of Ang II is observed when cells are cultured on either plastic or FN but not on the other matrices, collagen or LN.

Considering that glomerulosa cells readily proliferate on plastic, we hypothesized that glomerulosa cells are able to synthesize extracellular matrix components in their surrounding medium. Indeed, glomerulosa cells can produce collagens, LN (data not shown), and FN. In particular, time-course experiments indicate that FN production occurs rapidly after cell seeding. Immunofluorescence experiments performed herein using anti-FN antibody, applied without cell permeabilization and in low protein-containing medium, revealed the progressive presence of FN on the cell membrane. After 1 d in culture, FN labeling appeared diffuse, followed by a progressive organization into FN deposit and ultimately forming fibrillar structures after 5 d in culture. Studies have shown that the functions associated with FN are dependent on its assembly into a fibrillar network (18, 35), a phenomenon reproduced in culture, because after more than 3 d, cells had constructed a well-developed network of extracellular matrix, necessary for optimal proliferation. Thus, the observation that Ang II has a similar effect on plastic and on FN is most likely due to the ability of glomerulosa cells to produce FN. Such observations may also explain why glomerulosa cells are able to proliferate in culture.

The effect of Ang II is mediated through the AT1R. Indeed, pretreatment with DUP753 did not interfere with the basal level of proliferation on FN but reversed the inhibitory effect of Ang II, whereas pretreatment with PD123319, the specific antagonist of the AT2R, neither reversed the inhibitory effect of Ang II nor inhibited basal proliferation induced by FN alone. However, binding studies revealed that a small proportion (around 18%) of AT2R is present in glomerulosa cells, and surprisingly, the number of Ang II binding sites decreased in cells cultured for 3 d on FN. This observation indicates that FN attempts to oppose the inhibitory effect of Ang II on proliferation by acting on the number of binding sites. However, because the effect of Ang II still remains as potent as on plastic indicates that Ang II is very effective in inhibiting proliferation on FN. Furthermore, even though cells express a small proportion of AT2R, the pharmacological studies indicate that they are not involved in the inhibition of proliferation mediated by Ang II, even though inhibition of cell proliferation is considered one of the most established functions of the AT2R (36).

Disruption of cytoskeletal organization by Ang II occurs at focal adhesions
Basal proliferation requires a well-structured actin filament organization into stress fibers (8). Clustering of integrin receptors upon attachment to the ECM and the organization of microfilaments as stress fibers is promoted by the recruitment and activation of cytoskeletal regulatory proteins, such as paxillin (37, 38). Indeed, tyrosine phosphorylation of paxillin has been identified as an early event in the action of diverse hormones and growth factors that mediate cell growth and differentiation, including Ang II (4, 39). Under basal conditions on FN, thin stress fibers crossing the entire cell and focal adhesion points were observed in glomerulosa cells as evidenced herein by paxillin labeling. Phosphorylation and immunoprecipitation studies show that paxillin was phosphorylated and associated with RhoA. These observations corroborate previous results demonstrating that activation of p44mapk (ERK1) and basal proliferation requires a Rho/ROCK-dependent activation. Moreover, active RhoA localizes to the plasma membrane to regulate cytoskeletal rearrangement (40, 41) and stimulate actin polymerization (42).

Our results also show that stimulation with Ang II first decreases paxillin phosphorylation and induces its transfer from focal adhesions to the cytoplasm, suggesting a loss or decrease in focal adhesions, as previously shown by our group (19). Loss of paxillin labeling from the membrane is consistent with the disruption of focal adhesion complexes, loss of stress fibers and formation of an intense F-actin cortical ring. This reorganization is accompanied by a rapid activation of Rac, known to be involved in the depolymerization of F-actin (43). In a second phase (maximal level after 30 min of stimulation), Ang II markedly increases paxillin phosphorylation, an effect accompanied by a relocalization of paxillin from the cytoplasm to focal adhesion sites and the recovery of stress fibers, as confirmed by immunofluorescence observations. This rearrangement is accompanied by the activation of Rac and its association with paxillin. In tracheal smooth muscle, the phosphorylation of paxillin on tyrosine catalyzes the activation of Rac and Cdc42 (44). More specifically, phosphorylation of paxillin Y31 and/or Y118 is thought to be involved in the activation of Rac (45) or the inhibition of Rho-ROCK (46). In the present study, orthovanadate, an inhibitor of tyrosine phosphatase activity, stimulated paxillin phosphorylation and enhanced focal adhesion assembly. From this observation and as outlined by Burridge and Chrzanowska-Wodnicka (47), the conclusion can be drawn that dephosphorylation of paxillin mediates the loss of adhesion and its transfer from focal adhesions to the cytoplasm, inducing actin reorganization. Similar observations have also been reported in vascular smooth muscle cells where the expression of specific phosphatase proteins prevented the increase in stress fiber formation induced by Ang II and brought about a dephosphorylation of several focal adhesion-associated proteins including paxillin (48). Furthermore, these observations and the present results are consistent with the notion that disruption of actin stress fibers and focal adhesions are mediated by the dephosphorylation of focal adhesion-associated proteins.

Echistatin, a tool to investigate the effects of Ang II at focal adhesions
Echistatin (49, 50) and other snake venom disintegrins have been extensively studied for their ability to inhibit cell-matrix and cell-cell interactions and to inhibit proliferation (51, 52). At concentrations lower than 1 nM, echistatin inhibits {alpha}vβ3 binding, whereas concentrations higher than 1 µM inhibit {alpha}5β1 and {alpha}vβ1 (33, 34, 53). Herein, echistatin, at 10 µM, inhibited FN binding to {alpha}5β1 and {alpha}vβ1 integrins, decreasing cell proliferation on both plastic and on FN and abolishing the effect of Ang II. This indicates that Ang II as well as echistatin have similar effects on FN binding to {alpha}5β1 and {alpha}vβ1 integrins. In human intestinal smooth muscle cells, basal proliferation is increased by FN and inhibited by echistatin (54). In fetal cardiac myocytes, {alpha}5β1 and {alpha}3β1 have been shown to be implicated in proliferation (55). In vascular smooth muscle cells, the acquisition of a proliferative capacity is accompanied by a reorganization of the cytoskeleton, which is blocked by echistatin (56). Herein, echistatin abolished the dephosphorylation of paxillin induced by Ang II, but restored and even increased phosphorylation of paxillin. This increase was visible 10 min after stimulation with Ang II, similarly to that observed with orthovanadate. These results implicate phosphatase proteins in the effect of Ang II. Moreover, the blocking of {alpha}5β1 and {alpha}vβ3 integrins with echistatin restored the level of Rho-GTPases activation to that observed in plastic conditions. Thus, our results support the hypothesis that activation of integrins {alpha}vβ3 and {alpha}5β1 by FN, together with the activation of the AT1R, decreases RhoA activity but favors the activation of Rac necessary for the relocalization of actin under the cell membrane.

Altogether, the present study outlines two important features. First, we provide evidence that Ang II decreases FN-stimulated proliferation of glomerulosa cells by acting at the level of integrin binding, by altering phosphorylation of paxillin, inducing disruption of integrin-paxillin complexes, and consequently disrupting the stress organization of actin filaments. Second, the study documents for the first time that cultured glomerulosa cells synthesize and secrete FN and that such deposits around the cell membrane provide the integrin-cytoskeletal architecture essential for promoting basal levels of cellular proliferation, an inherent characteristic of glomerulosa cells. The actin reorganization induced by Ang II is responsible for the shift in glomerulosa cell properties from a proliferative state to a steroidogenic phenotype. Thus, the above data suggest that the specific FN component of the extracellular matrix interacts with Ang II to modify cell behavior from proliferation to other cell functions involving protein synthesis, such as steroid secretion.


   Acknowledgments
 
We deeply thank Lyne Bilodeau and Lucie Chouinard for experimental assistance.


   Footnotes
 
This work was supported by grants from the Fondation des Maladies du Cœur du Québec and the Canadian Institute for Heath Research to N.G.-P. (MOP 10998) and M.D.P. (MT-6813). N.G.P. is a recipient of a Canada Research Chair in Endocrinology of the Adrenal Gland.

Present address for S.C.: Department of Pharmacology, University of Vermont, Burlington,Vermont.

Disclosure Statement: M.O., S.C., M.D.P., and N.G.P. have nothing to declare.

First Published Online April 3, 2008

Abbreviations: Ang II, Angiotensin II; AT1R, AT1 receptor subtype; BrdU, 5-bromo-2-deoxyuridine; DAPI, 4',6'-diamino-2-phenylindole; ECL, enhanced chemiluminescence detection system; FITC, fluorescein isothiocyanate; FN, fibronectin; GST, glutathione S-transferase; HBS, Hanks’ buffered saline; LN, laminin; TCA, trichloroacetic acid.

Received February 28, 2008.

Accepted for publication March 24, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals