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Department of Physiology and Biophysics (S.C., M.C., N.G.-P., M.D.P.), Service of Endocrinology (N.G.-P., M.O.), Faculty of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
Address all correspondence and requests for reprints to: Dr. Marcel Daniel Payet, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail: marcel.payet{at}usherbrooke.ca.
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Abstract |
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5 and V as well as their fibronectin receptor partners ß1 and ß3, were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the 5ß1, vß1, or vß3 integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44mapk cascade, leading to cell proliferation and aldosterone secretion. |
Introduction |
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One of the characteristic properties of the adrenal gland, and particularly of the zona glomerulosa, is the high degree of plasticity, in which cell proliferation, migration and steroidogenesis interplay to control homeostasis and adaptive responses to metabolic disorders, such as stress (5) or a sodium-deficient situation (6). Several studies have shown that the extracellular microenvironment can orchestrate some of these functions (7, 8, 9). However, there are few data concerning cellular functions associated with expression of extracellular matrix (ECM) components and integrins in the adrenal gland. The only available studies involve expression of some ECM proteins and thrombospondins in the bovine adult adrenal cortex (10). As reviewed recently (11), differential expression of fibronectin and laminin, which are associated with cell specific activities, is observed from the periphery to the center of the gland. For example, laminin stimulates chemotaxis and haptotaxis of adrenocortical cells (10). In the human fetal adrenal gland, we recently found that laminin, collagen, and fibronectin have a specific tendency to favor proliferation, steroid secretion, or cell death (12).
Interactions between ECM and integrins occur at the focal adhesions. At these sites, integrins mediate links between extracellular components and the cytoskeleton and can regulate the generation of specific second messengers, some of which are shared with Ang II (7, 8). Indeed, both induce cytoskeletal organization (13) and activate the MAPK cascade, p42/p44mapk (14). This cascade is involved in proliferation, one important property of glomerulosa cells, evidenced by the observation that the highest expression of p42/p44mapk in the cortex is found in zona glomerulosa (15). For several ligands, the integrin recognition site consists of small amino acids sequences such as the Arg-Gly-Asp (RGD) sequence found, for example, in the fibronectin type III subunits (16). Several members of the integrin family, including integrins 5ß1, IIbß3, and vß3, bind to this sequence (17). In general, integrins that bind the RGD sequence play a role in calcium signaling (7).
To date, there are no data on the integrin population that could be expressed in the adrenal gland of adult rat. Recent studies from our group have identified integrins and matrix composition in human fetal adrenal glands (18), which suggest that cell-specific expression is associated with cell-specific functions (12).
The aim of the present study was to investigate, in rat adrenal glomerulosa cells, whether the interaction between integrins/ECM could be involved in adhesion, secretion, and proliferation in initiating two important signaling events, increase in intracellular calcium and activation of the p42/p44mapk cascade, and finally determine the identity of these integrins.
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Materials and Methods |
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5 and anti-ß1 integrins antibodies from Chemicon (Temecula, CA); antiphospho p42/44mapk and anti-p42/44mapk, MAPK kinase (MEK) inhibitor (PD98059) from New England Biolabs, Inc. (Mississauga, Ontario, Canada); phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) and protein kinase C (PKC) inhibitor (GF-109203X), Xestospongin C, thapsigargin (TG) from Calbiochem-Novabiochem Corp. (San Diego, CA); DC protein assay from Bio-Rad Laboratories, Inc. (Hercules, CA); and [H3]aldosterone (78 Ci/mmol) from NEN Life Science Products (Boston, MA). All chemical products were of grade-A purity.
Preparation of glomerulosa cells
Rat adrenal glomerulosa cells were obtained as previously described (19). Female Long Evans rats (200–250 g) were decapitated and adrenal glands excised. The successive steps in glomerulosa cells isolation were performed in MEM Eagle’s medium. After incubation (20 min) at 37 C in collagenase (2 mg/ml, 4 glomerulosa/ml) plus deoxyribonuclease (25 µg/ml), cells were disrupted by aspiration with a sterile 10-ml pipette, filtered (22 µm), and centrifuged for 10 min at 100 x g. The cell pellet was resuspended in OPTI-MEM medium supplemented with 2% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The cells were plated at different densities, depending on experiments, in 35-mm Petri dishes, coverslips, or multiwell (96 wells) plates for proliferation assays. The cells were maintained at 37 C in a humidified atmosphere of 95% air/5% CO2 and were used 60 min after isolation or after 3 d of culture (19). Cells were examined daily, and phase contrast photographs were taken using a microscope (Leica Corp., Deerfield, IL) equipped with a x32 objective.
Adhesion and inhibition assays
Rat glomerulosa cells were used 60 min after isolation and plated (1 x 105) on a 24-well plate coated with poly-L-lysine (1 mg/ml), fibronectin (10 µg/ml), or collagen I (10 µg/ml). All experiments were performed in triplicate. Cells were allowed to adhere for 60 min at 37 C in 5% CO2 atmosphere in MEM containing 2% antibiotics. The medium was then replaced by MEM containing 5 µM fluorescent probe Cell Tracker, and the plates were incubated for 30 min at 37 C. Final washings were done using 1% BSA/Hanks’ buffer saline (HBS), and the cells were bathed in MEM without probe and incubated at 37 C for counting. Three pictures were taken for each condition tested to determine the number of cells/well by image analysis with the Scion Imaging software (Scion Corp., Frederick, MD). Adhesion assays were performed by pulsing the plates 10 times at 200 rpm. After this period, detached cells were removed by gentle aspiration, and the number of adhering cells was determined. Results are shown as the number of cells after the assay/number of initial cells. For adhesion inhibition assays, cells were preincubated in a medium containing 1 mM peptides (RGDS or RGES) for 15 min at 37 C.
Immunofluorescence
For immunofluorescence studies, cells were plated on plastic Petri dishes coated with poly-L-lysine or fibronectin (density of 1 x 105 cells after 3 d of culture). 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) or antivinculin (1:1000) and Alexa Fluor-594 Phalloidin (1:60) 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 FITC. After washing, cells were mounted in Vectashield mounting medium and examined on an Eclipse 300 microscope (Nikon, Mississauga, Ontario, Canada) equipped with a G2A-rhodamin filter and CoolSnap fx digital camera (Roper Scientific, Tucson, AZ). Images were taken using a x100 objective.
Proliferation assays
The method used to evaluate cell proliferation was adapted from that described by Gillies et al. (20). Cells were plated on fibronectin- or poly-L-lysine-coated 96-well plate. After 3 d, cells were fixed with 4% formaldehyde in HBS for 10 min at room temperature. After HBS washing, cells were incubated for 10 min at room temperature with 0.1% crystal violet/H2O. After extensive washing with water, cells were lysed for 10 min in 100 µl 1% sodium dodecyl sulfate solution. OD at 595 nm was then read with a MicroQuant spectrophotometer (Bio-Tek Instruments, Inc., Winooski, VT). For each experiment a standard curve was performed using serial dilutions of a cell solution in which the exact cell number was evaluated using a hemacytometer.
Intracellular calcium measurements
Glomerulosa cells, plated on fibronectin-coated (10 µg/ml) coverslips in OPTI-MEM (2% fetal bovine serum), were used between 1 and 3 d of culture. For loading purposes with the fluorescent calcium sensitive dye Fura-2AM (5 µM), cells were incubated for 30 min at 37 C in a physiological medium containing (mM): NaCl, 140; KCl, 5.4; CaCl2, 2; MgCl2·6H2O, 1; HEPES, 10; pH 7.35. A free-Ca2+ medium with the composition [(mM): NaCl, 140; KCl, 5.4; MgCl2·6H2O, 1; HEPES, 10; EGTA, 1; pH 7.35] was used. For hydrolysis of the Fura-2 AM, cells were washed with HBS/1% BSA and incubated for 30 min at 37 C in medium without dye. Coverslips containing the loaded cells were placed in a chamber and mounted on an Eclipse 300 microscope (Nikon). The experiments were done at room temperature. Excitation wavelengths were set at 340 and 380 nm with two filters (Chroma Technology Corp., Brattleboro, VT) placed in a filter wheel (Sutter Instrument Co., Novato, CA), and emission was set at 420 nm. The images were acquired with a CoolSnap 
x camera and analyzed with the MetaFluor software (Universal Imaging Corp., Downington, PA). Ratio (340:380) was converted in intracellular calcium concentration ([Ca2+]i) by the MetaFluor software with an external high-low calibration technique (21, 22).
Aldosterone secretion measurements
For aldosterone measurements, cells were used after a resting period of 2 h or after 3 d in culture. Before each experiment, the medium was removed, and the cells were washed twice with cold HBS (130 mM NaCl, 3.5 mM KCl, 1.8 mM CaCl2, 0.5 mM MgCl2, 2.5 mM NaHCO3, and 5 mM HEPES) supplemented with 1 g/liter glucose and 0.5% BSA. The cells were incubated in 1 ml medium consisting of 0.9 ml HBS-glucose supplemented with 0.5% BSA, 0.1 mg/ml bacitracin, and 0.1 ml stimulus. After a 2-h incubation at 37 C in an atmosphere of 95% air/5% CO2, the incubation medium was removed by aspiration and stored at -20 C until RIA determinations of aldosterone, using specific antisera and [H3]aldosterone.
Protein immunoblotting
Rat glomerulosa cells were plated (4 x 105) on poly-L-lysine coated coverslips (1 mg/ml) in OPTI-MEM for 8 h. Media were discarded and cells were incubated in a serum-free medium (MEM) overnight. Cells were stimulated with 1 mM RGDS peptide for different times. MEM was removed from dishes and replaced by 1 ml stabilization solution containing 100 nM staurosporine, 1 mM sodium orthovanadate in 10 ml HBS-glucose, and (mM): NaCl, 130; KCl, 3.5; CaCl2·2H2O, 2.3; MgCl2·6H2O, 0.98; HEPES, 5; EGTA, 0.5; glucose, 0.55; NaHCO3, 0.23; BSA 5%) for 10 min on ice. The solution was removed and cells were lysed with 30 µl 50 mM HEPES (pH 7.8), 100 nM staurosporine, 1 mM sodium orthovanadate, 1% Triton X-100, 0.04 U/ml aprotinin, and 1 mM benzamidin. The insoluble material was pelleted at 12,000 x g for 15 min at 4 C.
For detection of integrins by immunoblotting, rat adrenal glomerulosa cells were plated at a density of 3 x 105 cells on fibronectin-coated dishes. After 3 d in culture, cells were washed with PBS buffer, lysed in denaturing, but nonreducing conditions, in 2x electrophoresis sample buffer (1.0 ml glycerol; 3.0 ml 10% sodium dodecyl sulfate; 1.25 ml of 1.0 M Tris-HCl, pH 6.7; 1–2 mg bromophenol blue), and boiled for 10 min.
For detection of proteins, cell lysates were separated by SDS-PAGE, transferred to polyvinyl difluoride membranes, and probed with 1:1000 antiphospho p42/p44mapk, 1:1000 anti-p42/p44mapk, 1:1000 anti-5 antibody, or 1:500 anti-ß1 antibody. Detection was performed by reaction with horseradish peroxidase-conjugated secondary antibody and visualized by enhanced chemiluminescence according to the manufacturer’s instructions. For inhibition experiments, the following inhibiting reagents were preincubated for 30 min: PD98059, 50 µM; LY294002, 10 µM; GF-109203X, 10 µM.
RT-PCR amplification
Rat adrenal glomerulosa cells were isolated as described and plated at a density of 5 x 105 cells on plastic dishes and were allowed to adhere for 3 d. Total RNA was extracted using the RNAqueous method according to the manufacture user’s guide (Ambion, Inc., Austin, TX). RNA was then dissolved in elution solution before determining the concentration. The total RNA concentration was determined by OD (260 nm), and 5 µg RNA was reversed transcribed into first-strand cDNA for each preparation, homo-oligomerase DNA deoxythymidine12–18, deoxynucleotide triphosphates (10 mM) from Amersham Pharmacia Biotech (Piscataway, NJ), dithiothreitol (0.1 M), Muloney murine leukemia virus, first 5x Muloney murine leukemia virus buffer, and RNasine (all from Promega Corp., Madison, WI). cDNA was used for each PCR. Primer pairs were designed based on GenBank or previously published sequences for rat. The primer sequences used are as follows:
1) V-sense: 5'TAT TGG GGA TGA CAA CCC TCT GAC C'3; anti: 5'CTC ATA GAT GTG CTG AAC AGG C'3
2) ß3-sense: 5'CAC TAC TAT GGA TTA CCC ATC TCT GG'3; anti: 5'GTT GTT GAG GCA GGT GGC ATT GAA GG'3
3) ß1-sense: 5'GAA TGT AAC ACG ACT GCT GGT'3; anti: 5'CAT TCT TGC AGT AGG ACT TGT'3
4) glyceraldehyde-3-phosphate dehydrogenase: sense: 5'CGC TGA GTA CGT CGT GGA GTC'3; anti: 5' TTG GTG GTG CAG GAG GCA TTG C'3.
Glyceraldehyde-3-phosphate dehydrogenase was used as a control of the integrity of RNA. The PCR was carried out using 4 µl cDNA, deoxynucleotide triphosphates (10 mM), Taq polymerase (Amersham) and 10x Taq polymerase PCR buffer. Amplification was performed on a amplification system (Perkin-Elmer Corp., Norwalk, CT) for 34 cycles consisting of 30 sec at 94 C, 60 sec at 58 C, and 2 min at 72 C for most of the samples. For visualization, 10 µl PCR product was loaded on a 2% agarose gel stained with ethidium bromide and scanned using a Fluorimager (MultiImage Light Cabinet, Alpha Innotech Corp., San Leandro, CA).
Statistical analyses
The data are presented as mean ± SE of the number of experiments indicated in the text. Statistical analyses of the data were performed using the t test.
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). Adhesion on fibronectin and collagen I was significantly different from adhesion on poly-L-lysine-covered plastic, suggesting the involvement of specific binding sites. To assess the importance and nature of these binding sites, adhesion experiments were performed on cells preincubated with 1 mM active (RGDS) or inactive (RGES) peptides. Preincubation with the RGDS peptide decreased adhesion of cells plated on fibronectin by a factor of 2.8 ± 0.24-fold (Fig. 1B). The RGES peptide, used as a negative control, did not interfere with adhesion on the fibronectin matrix (Fig. 1B). Preincubation of cells with RGDS peptide (1 mM) had no effect on the adhesion of glomerulosa cells plated on poly-L-lysine or collagen I (Fig. 1C).
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) and fibronectin (Fig. 2B) had a similar polygonal morphology. Actin filament organization was revealed with rhodamine-phalloidin staining; cells on poly-L-lysine or fibronectin had a flattened polygonal shape, characterized by a discrete network of thin stress fibers crossing the entire cell, and in some cells, a discrete ring of cortical actin was also observed (Fig. 3, A and B). Marked differences were found in regard to the presence of focal adhesions revealed by FITC immunostaining of vinculin and paxillin. Indeed, vinculin labeling was clearly observed on poly-L-lysine and fibronectin (Fig. 3, C and D), but paxillin labeling was apparent on fibronectin only (Fig. 3, E and F). We then compared the proliferative properties of glomerulosa cells, grown on poly-L-lysine and fibronectin. As shown in Fig. 4, proliferation was stimulated by fibronectin matrix (1.92 ± 0.05-fold increase, n = 3), compared with poly-L-lysine. PD98059 (10 µM), an MEK inhibitor, blocked the proliferation of cells grown on fibronectin and poly-L-lysine.
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Addition of the RGDS peptide (1 mM) to glomerulosa cells loaded with a fluorescent Ca2+ indicator triggered a sustained increase in [Ca2+]i of 263.7 ± 17.3 nM (n = 15). If the [Ca2+] of the bath was decreased by addition of EGTA, the level of [Ca2+]i rapidly shut down. On readmission of the medium with 2 mM Ca2+, [Ca2+]i was restored to the values obtained in the presence of RGDS (Fig. 5A, n = 6). In a Ca2+-free medium, the Ca2+ response was completely blocked (data not shown). To characterize the nature of the Ca2+ influx, several types of channels inhibitors were used. The ionic blockers of Ca2+ channels, Ni2+ (50 µM to 1 mM), and La3+ (1 mM) did not affect the Ca2+ response to RGDS peptide as well as nifedipine, a specific L-type Ca2+ channel blocker (1 µM) (Fig. 5B). The presence of a Ca2+ influx was assessed by addition of EGTA (10 mM) in the medium.
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[Ca2+]i increase of 78.8 ± 8.3 nM (n = 8) in the presence of external Ca2+ (Fig. 5C
), whereas a value of 63.7 ± 4.0 nM (n = 20) was found in a free-Ca2+ medium (Fig. 5D); these two values were not significantly different. To determine the nature of the stores involved, the cells were first treated with TG, a known inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (25). Application of TG (5 µM) induced a [Ca2+]i increase followed by a plateau as previously described (26). A further application of fibronectin did not trigger any Ca2+ increase, indicating that the Ca2+ pools mobilized by fibronectin have been emptied (Fig. 5E). InsP3-responsive pools were described in glomerulosa cells to release Ca2+ after activation of the AT1 receptor of Ang II, coupled to phospholipase C (26). Release of Ca2+ from these pools was blocked by Xestospongin C (XeC), a potent specific blocker of the InsP3-sensitive Ca2+ channels (27). Figure 5F shows that the Ca2+ response to fibronectin, after preincubation of the cells with XeC (20 µM, 15 min), was significantly reduced from 63.7 ± 4.0 (n = 20) to 49.5 ± 5.3 (n = 7) nM. The presence of caffeine/ryanodine-sensitive Ca2+ stores was challenged by using caffeine (10–20 mM). It came out that there is no caffeine-sensitive stores in rat glomerulosa cells (data not shown) as recently reported in bovine glomerulosa cells (28).
Modulation of aldosterone secretion by integrin binding
ACTH and Ang II, the main stimuli of aldosterone secretion in rat glomerulosa cells, use two different signaling pathways, i.e. adenylyl cyclase/cAMP and phospholipase C/inositol phosphate, respectively (29). Cells were plated for a period of 2 h or 3 d on poly-L-lysine or fibronectin. The stimuli ACTH, Ang II, and RGDS were added in the medium for a period of 2 h and aldosterone measured as described in Materials and Methods. In cells cultured for 2 h, the nature of the matrix did not influence significantly the basal level of secretion but enhanced significantly the secretion induced by ACTH, Ang II, or RGDS (Fig. 6A). After 3 d of culture, the basal level of aldosterone secretion was higher on fibronectin matrix, with a 3.8-fold increase, compared with cells on poly-L-lysine. However, in such conditions, the stimulation ratio between ACTH, Ang II, or RGDS vs. their respective control was similar (Fig. 6B).
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, activation of p42/p44mapk phosphorylation was maximal at 5 min, followed thereafter by a decrease to basal levels (n = 3).
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shows that both inhibitors of PI3K and PKC had no effect on the activation of p42/44mapk by the RGDS peptide. This indicated that in glomerulosa cells, these two kinases were not directly involved in the activation of p42/44mapk by RGD-binding integrins. As a control, cells were preincubated with PD98059, an MEK inhibitor (50 µM for 30 min), which resulted in a near total inhibition p42/p44mapk activation by RGDS (Fig. 7B).
v, 5, ß1, and ß3 integrin subunits are expressed in rat glomerulosa cells
The RGD sequence, identified as an integrin-binding motif in fibronectin (17), can interact with several members of the integrin family, including integrins 5ß1, IIbß3, vß1, and vß3. However, the expression of integrins in glomerulosa cells has not yet been demonstrated. We performed two types of experiments to reveal the putative presence of RGD-binding integrins in rat glomerulosa cells. First, RT-PCR experiments were performed on total RNA extracted from cells cultured on plastic dishes covered with poly-L-lysine. Figure 8A shows that amplification of the v subunit was detected as a band of the correct expected size (490 bp). Expression of integrins ß3 and ß1 mRNA was also detected (Fig. 8, B and C) with bands of the expected sizes (ß1, 407 bp, and ß3, 280 bp). Total RNA extracts of adult rat brain were used as positive controls. The PCR were also carried out with RNA instead of DNA or without Taq polymerase as negative controls (data not shown). Second, Western blots analyses were performed on glomerulosa cells cultured on plastic dishes covered with poly-L-lysine for 3 d. The anti-5 integrin subunit antibody detected a band at 155 kDa in nonreducing conditions (Fig. 8D), whereas the integrin ß1-subunit was detected as a band at 110 kDa (Fig. 8E). The ß1-subunit migrated as a doublet indicating a fully glycosylated form of the ß1-subunit (upper band) and the intracellular precursor of the ß1-subunit that is not fully glycosylated (faster migrating form).
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5ß1, vß1, and Vß3, which were detected in rat adrenal glomerulosa cells.
Integrins form the major family of proteins that induce cell-cell and cell-matrix interactions (33, 34) and have been found in all tissues studied to date (17). They are formed by two subunits, and ß, held together by noncovalent bonds. It has been shown that 22 -subunits can combine with nine ß-subunits to make more than 22 integrin isoform pairs able to recognize several different ECM components.
We first performed adhesion assays on fibronectin and collagen I as substratum, and poly-L-lysine as a control. Our data clearly show that fibronectin was the preferred ECM component on which glomerulosa cells bound most strongly, as previously shown for other cell types (35, 36). Collagen I also allowed a good adhesion for cells unlike poly-L-lysine, on which cells are lightly bound. Soluble synthetic peptides RGDS and RGES were used during adhesion tests to determine the role of the integrin-binding motif, the RGD. The soluble RGDS peptide almost completely abolished adhesion of glomerulosa cells to fibronectin, pointing out the essential role of the RGD sequence (16, 37, 38). This effect is highly specific as the RGES peptide, in which aspartic acid is replaced by glutamic acid, had no effect on cell adhesion (16, 37, 38). The RGDS peptide did not inhibit the attachment of glomerulosa cells on poly-L-lysine and collagen I (37). Furthermore, by using two different markers of focal adhesion formation, we confirmed that binding of glomerulosa cells to a fibronectin matrix involved a ligand/integrin process. Indeed, focal adhesions, which included vinculin, were found on poly-L-lysine as well as on fibronectin, whereas focal adhesions containing paxillin were found only on fibronectin. Similar results were described in Chinese hamster ovary cells in which focal adhesions containing paxillin-GFP were absent on poly-L-lysine substrate (39). Interaction of integrins with EMC proteins triggers the localization of paxillin and focal adhesion kinase to focal adhesions and autophosphorylation of focal adhesion kinase, which in turn recruits and phosphorylates several proteins to initiate various signaling cascades (40). Vinculin, on the other hand, is not essential for focal adhesion formation (41), and can localize to adhesion points on nonspecific substrates, without integrin activation.
Apart from their role in adhesion, integrins can induce an increase in intracellular calcium concentration following their activation (23, 42). In glomerulosa cells, experiments performed in solutions containing external Ca2+ showed that RGDS induced a sustained Ca2+ increase, abolished in a Ca2+-free medium. In contrast, the Ca2+ response to fibronectin is transient and not affected in a Ca2+-free medium. These results can be respectively interpreted as a Ca2+ influx and a Ca2+ release from intracellular Ca2+ stores. Several studies report that integrin-dependent Ca2+ increase occur only by Ca2+ influx through Ca2+-permeable channels (23, 42), but a Ca2+ influx and release from intracellular Ca2+ pools (43) or only internal Ca2+ release (44) have also been reported. The nature of the Ca2+ channel(s) involved in the Ca2+ influx is not yet firmly established and could depend both on cell type and integrins activated. It has been shown that nonsoluble 5ß1-integrin ligands as well as fibronectin-coated beads increased the amplitude of the L-type Ca2+ current in vascular smooth muscle cells, whereas soluble and nonsoluble vß3-integrin ligands caused it to decrease (45, 46).
In filopodia from growth cones, RGDS induced Ca2+ transients in part because of Ca2+ influx through channels insensitive to voltage-dependent Ca2+ channel blockers (47). In zona glomerulosa cells, we found similar results showing that the RGDS-induced Ca2+ influx is insensitive to Ni2+, nifedipine, and La3+. This raises the intriguing question of the nature of the protein entity involved (23). InsP3-sensitive intracellular Ca2+ pools have been suggested to be mobilized by vß3- and Lß2-integrins (44). In support of this, it was shown that integrins are capable of activating PI-5 kinase to regulate PtnsP2 levels (48) and PI3K to generate InsP3 (49). In glomerulosa cells, application of TG abolished the fibronectin-induced [Ca2+]i increase. Moreover, the fibronectin-induced response was blunted by pretreatment of the cells with XeC, indicating that fibronectin mobilized, in part, InsP3-sensitive Ca2+ stores. Cellular calcium signaling play a crucial role in aldosterone production by glomerulosa cells (29, 50). First, we demonstrated that the RGDS peptide was able to induce the secretion of aldosterone, an effect that could be attributed to the Ca2+ influx triggered by RGDS. In confirmation, secretion of aldosterone by the RGDS peptide is abolished in a free-Ca2+ medium (data not shown). Secretion of aldosterone induced by ACTH and Ang II is higher for cells plated for 2 h on fibronectin. After 3 d in culture, the basal level of steroid secretion is also increased. Such results indicate that the signaling cascade induces by integrin participate in activation of acute secretion, and, after 3 d in culture, fibronectin could increase expression of the enzymes of the steroidogenic pathway as observed on Matrigel substrate (51).
As already shown in many studies, p42/p44mapk may be phosphorylated following integrin-ligand recognition (52, 53, 54). Phosphorylation of p42/p44mapk was noted when glomerulosa cells were stimulated with RGDS peptide, an effect observed in the absence of growth factors. The maximal effect was found to occur after 5 min of stimulation, which thereafter decreased at the basal level. A similar time course was observed in several cell types stimulated either by growth factor such as epithelial growth factor or hormones known to promote proliferation such as Ang II (55, 56). As previously described in other systems (53, 54, 57), the adhesion of glomerulosa cells on poly-L-lysine did not activate p42/p44mapk, indicating a specific effect for fibronectin-recognizing integrins. Accordingly, glomerulosa cells show a higher proliferative activity on fibronectin matrix, which is blocked by PD98059. Several hypotheses exist regarding the nature of the proteins involved in the activation pathway of p42/p44mapk by integrins (32). A role for the PI3K has been put forward and, to verify its participation in our system, we used the PI3K inhibitor LY 294002. No reduction of p42/p44mapk phosphorylation was observed, indicating that PI3K is not involved in activation of the MAPK pathway in rat glomerulosa cells by RGD-dependent integrins. It was also suggested that PKC could activate the p42/p44mapk by association with the protein Raf (8, 32). Use of the PKC inhibitor GF-109203X to determine the involvement of this kinase in glomerulosa cells showed that p42/p44mapk phosphorylation was not affected, ruling out PKC as a participant in the signaling cascade between integrins and p42/p44mapk.
Our results (RT-PCR and Western blots analyses) indicate that V-, 5ß1-, and ß3-subunits, which form integrins that can bind the RGD peptide, were expressed in rat adrenal glomerulosa cells. The 5- and ß1-subunits associate to form the classical fibronectin receptor (8). Moreover, the integrin vß3 plays a significant role in angiogenesis (58) and migration (59). Our observations corroborate recent data indicating that 5ß1 and Vß3 mediate activation of p42/p44mapk and increase of intracellular calcium (60, 61, 62).
Together, our results indicate that a specific RGD sequence is involved in the fibronectin/integrin interaction of glomerulosa cells. This binding favored adhesion and triggered increases in intracellular calcium, activation of the MAPK pathway, p42/p44mapk, aldosterone secretion, and proliferation. These observations outline the role of integrins in mediating two important functions of glomerulosa cells, steroid secretion, and proliferation.
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Acknowledgments |
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Footnotes |
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ur du Québec Québec and Canadian Institute for Heath Research (to N.G.-P. and M.D.P.). N.G.-P. is holder of the Canadian Research Chair in Endocrinology of the Adrenal Gland. Abbreviations: Ang II, Angiotensin II; ECM, extracellular matrix; FITC, fluorescein isothiocyanate; HBS, Hanks’ buffer saline; MEK, MAPK kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; RGD, Arg-Gly-Asp; RGDS, Arg-Gly-Asp-Ser; RGES, Arg-Gly-Glu-Ser; TG, thapsigargin; XeC, Xestospongin C.
Received August 28, 2002.
Accepted for publication December 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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q/a11-subunit with cytoskeleton in adrenal glomerulosa cells: role in receptor-effector coupling. Endocrinology 138:3299–33076ß4 integrin promote carcinoma invasion. Cell 91:949–960[CrossRef][Medline]
4ß1 and 5ß1 integrin-mediated adhesion and migration of B16F1 cells. Biochem Cell Biol 77:409–420[CrossRef][Medline]
5 integrin, paxillin, and -actinin during formation and disassembly of adhesions in migrating cells. J Cell Biol 153:1427–1440Vß3-integrin ligands raise Ca2+ in rat osteoclasts and mouse-derived osteoclast-like cells. Am J Physiol 266:C376–C381
vß3 and 5ß1 integrin ligands. J Cell Biol 143:241–2525ß1 integrin requires signaling between focal adhesion proteins. J Biol Chem 276:30285–30292vß3 by osteopontin stimulates phosphatidylinositol 3-hydroxyl kinase activity. Endocrinology 136:2984–2992[Abstract]
6ß4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation. EMBO J 16:2365–2375[CrossRef][Medline]
vß3 in vascular biology. Thromb Haemost 80:726–734[Medline]
5ß1 integrin/fibronectin interaction in vitro by the serine/threonine protein phosphatase calcineurin. Biochemistry 34:5104–5112[CrossRef][Medline]
5ß1 integrin via mitogen-activated protein kinase pathway. J Clin Endocrinol Metab 86:2484–2493(v)ß(3)-integrin activates cytosolic PLA(2) in bovine pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 280:L1049–L1056This article has been cited by other articles:
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, Number of cells at time 0;
, cells cultured in control medium; 
, cells cultured in the presence of PD 98059 (10 µM). Results are means ± SE of three experiments, each experimental condition containing six individual samples. **, P < 0.01.
) in the absence or presence of ACTH (1 x 10-9 M), Ang II (1 x 10-7) or RGDS (1 mM). B, Cells were cultured for 3 d on poly-L-lysine (
