Submitochondrial Distribution of Three Key Steroidogenic Proteins (Steroidogenic Acute Regulatory Protein and Cytochrome P450scc and 3beta -Hydroxysteroid Dehydrogenase Isomerase Enzymes) upon Stimulation by Intracellular Calcium in Adrenal Glomerulosa Cells*

(Received for publication, September 18, 1996, and in revised form, December 2, 1996)

Nadia Cherradi §, Michel F. Rossier , Michel B. Vallotton , Rina Timberg par , Iddo Friedberg par , Joseph Orly par , Xing Jia Wang **, Douglas M. Stocco ** and Alessandro M. Capponi

From the  Division of Endocrinology and Diabetology, Department of Internal Medicine, Faculty of Medicine, University Hospital, CH-1211 Geneva 14, Switzerland, the par  Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel, and the ** Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

In adrenal glomerulosa cells, angiotensin II (Ang II) and potassium stimulate aldosterone synthesis through activation of the calcium messenger system. The rate-limiting step in steroidogenesis is the transfer of cholesterol to the inner mitochondrial membrane. This transfer is believed to depend upon the presence of the steroidogenic acute regulatory (StAR) protein. The aim of this study was 1) to examine the effect of changes in cytosolic free calcium concentration and of Ang II on intramitochondrial cholesterol and 2) to study the distribution of StAR protein in submitochondrial fractions during activation by Ca2+ and Ang II. To this end, freshly prepared bovine zona glomerulosa cells were submitted to a high cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM) for 2 h. Mitochondria were isolated and subfractionated into outer membranes, inner membranes (IM), and contact sites (CS). Stimulation of intact cells with Ca2+ or Ang II led to a marked, cycloheximide-sensitive increase in cholesterol in CS (to 143 ± 3.2 and 151.1 ± 18.1% of controls, respectively) and in IM (to 119 ± 5.1 and 124.5 ± 6.5% of controls, respectively). Western blot analysis revealed a cycloheximide-sensitive increase in StAR protein in mitochondrial extracts of Ca2+-clamped glomerulosa cells (to 159 ± 23% of controls). In submitochondrial fractions, there was a selective accumulation of StAR protein in IM following stimulation with Ca2+ (228 ± 50%). Similarly, Ang II increased StAR protein in IM, and this effect was prevented by cycloheximide. In contrast, neither Ca2+ nor Ang II had any effect on the submitochondrial distribution of cytochrome P450scc and 3beta -hydroxysteroid dehydrogenase isomerase. The intramitochondrial presence of the latter enzyme was further confirmed by immunogold staining in rat adrenal fasciculata cells and by immunoblot analysis in MA-10 mouse testicular Leydig cells. These findings demonstrate that under acute stimulation with Ca2+-mobilizing agents, newly synthesized StAR protein accumulates in IM after transiting through CS. Moreover, our results suggest that the import of StAR protein into IM may be associated with cholesterol transfer, thus promoting precursor supply to the two first enzymes of the steroidogenic cascade within the mitochondria and thereby activating mineralocorticoid synthesis.


INTRODUCTION

The Ca2+-mobilizing agonists angiotensin II (Ang II)1 and K+ act as regulators of aldosterone synthesis and secretion in adrenal glomerulosa cells. The crucial role of the Ca2+ messenger in the acute regulation of aldosterone production is firmly established (1-5). Indeed, the steroidogenic response of isolated adrenal cells to Ang II and K+ is highly dependent upon extracellular Ca2+ concentration (6) and can be blocked by inhibitors of Ca2+ influx across the plasma membrane (4). Moreover, calmodulin antagonists have been shown to inhibit Ang II-stimulated aldosterone production in zona glomerulosa cells (7).

Traditionally, aldosterone biosynthesis is functionally divided into three consecutive phases. (i) In the early mitochondrial steps, cholesterol is transported from intracellular lipid droplets into the outer mitochondrial membrane (OM) and then to the inner mitochondrial membrane (IM). The latter step represents the rate-limiting process in all steroidogenic pathways (8) and is followed by the conversion of cholesterol to pregnenolone by the cytochrome P450scc enzyme. (ii) The intermediate steps take place on the endoplasmic reticulum and involve the conversion of pregnenolone to progesterone by 3beta -hydroxysteroid dehydrogenase isomerase and then to 11-deoxycorticosterone. (iii) The late steroidogenic steps are localized back in the mitochondria and include the formation of corticosterone and its conversion to aldosterone by cytochrome P45011beta .

The regulation of intramitochondrial cholesterol transfer by cAMP-dependent mechanisms has been extensively studied (9). While the transport of cholesterol from lipid droplets to the outer mitochondrial membrane was found not to be affected by inhibitors of protein synthesis in ACTH-stimulated adrenal cells, by contrast, the subsequent delivery of cholesterol to the inner mitochondrial membrane has been shown to be blocked by cycloheximide, with a concomitant inhibition of steroid synthesis (10, 11). As a consequence, newly synthesized "labile" proteins, among others, have been proposed to be significant mediators of the acute steroidogenic response. In primary adrenal cell cultures (12, 13) and in MA-10 mouse Leydig tumor cells (14, 15), a family of 30-kDa mitochondrial phosphoproteins are synthesized in response to stimulation with both trophic hormone and cAMP analogs. These studies have demonstrated that the appearance and the amounts of these proteins are highly correlated with the rate of steroidogenesis. Recently, the steroidogenic acute regulatory (StAR) protein has been identified, and its complementary DNA cloned (16-19). A model has been proposed according to which cholesterol transfer from the outer to the inner membrane might occur via intermembrane contact sites (CS) during the import and proteolytic processing of the 37-kDa precursor of StAR protein into mitochondria (18, 19). The most striking evidence for the essential role of StAR protein in the acute regulation of steroidogenesis was provided by studies of a lethal disease, lipoid congenital adrenal hyperplasia, which manifests itself by a complete inability of the newborn infant to synthesize steroids (20). Mitochondria from affected adrenal glands and gonads fail to convert cholesterol to pregnenolone, and as a consequence, cholesterol accumulates within the cells. The defects responsible for this disease are mutations in the StAR gene that generate truncated and nonfunctional proteins (20). In addition to the first report on StAR gene mutations causing lipoid congenital adrenal hyperplasia (20), many additional examples of mutations in StAR gene resulting in this disease are being reported (21-23) and perhaps it is not as rare as previously thought. To date, mutations in the StAR gene are the only known causes of this potentially lethal disease and, in a most dramatic manner, have demonstrated the indispensable role of StAR protein in the production of steroids.

Our laboratory has recently provided a direct demonstration of the involvement of physiological rises in cytosolic free Ca2+ concentration ([Ca2+]i) in the activation of the early steps of steroidogenesis, namely the stimulation of pregnenolone synthesis in bovine adrenal glomerulosa cells (24). Both Ang II and K+ elicit sustained changes in mitochondrial free calcium concentration (25). However, the precise site(s) of action of Ca2+ responsible for this increased pregnenolone formation remained to be determined. We therefore undertook this work to investigate the effect of rises in [Ca2+]i on the intramitochondrial distribution of both cholesterol and StAR protein in bovine adrenal glomerulosa cells. Our data show that physiological rises in cytosolic Ca2+, produced either with a Ca2+ inonophore or with Ang II, are indeed effective in stimulating a specific StAR protein accumulation in the inner membranes as well as a concomitant cholesterol transfer from the outer membranes to the contact sites and inner membranes, where P450scc and 3beta -HSD are present to initiate the steroidogenic response.


EXPERIMENTAL PROCEDURES

Materials

Ionomycin was purchased from Calbiochem (Lucerne, Switzerland) and [Ile5]Ang II from Bachem (Bubendorf, Switzerland). Cholesterol oxidase, peroxidase, p-hydroxyphenylacetic acid, aminoglutethimide, cycloheximide, and all other chemicals were purchased from Sigma or from Fluka (Buchs, Switzerland). Antisera against 3beta -HSD and P450scc were kindly provided by Dr. G. Defaye (INSERM U244, CENG, Grenoble, France). The cytochrome c oxidase antibody was an anti-bovine cytochrome c oxidase subunit IV mouse monoclonal antibody supplied by Molecular Probes, Inc. (Eugene, OR).

Bovine Adrenal Zona Glomerulosa Cell Preparation

Bovine adrenal glands were obtained from a local slaughterhouse. Zona glomerulosa cells were prepared by enzymatic dispersion with dispase and purified on Percoll density gradients as described in detail (26). Purified glomerulosa cells were resuspended at a density of 106 cells/ml in a modified Krebs-Ringer buffer (136 mM NaCl, 5 mM NaHCO3, 1.2 mM KH2PO4, 1.2 mM MgSO4, 1.8 mM KCl, 1.2 mM CaCl2, 5.5 mM D-glucose, and 20 mM Hepes, pH 7.4) and preincubated at 37 °C for 1 h before being used in the subsequent experiments.

Calcium Clamping of Bovine Adrenal Glomerulosa Cells

After having been washed in Krebs-Ringer buffer, glomerulosa cells were Ca2+-clamped as described (24), in the presence of 2 µM ionomycin and 1 mM total extracellular Ca2+, in order to achieve [Ca2+]i = 600 nM (high Ca2+ clamp). CHX was used at a final concentration of 1 mM. Control cells were Ca2+-clamped in Krebs-Ringer buffer without Ca2+ in the presence of 0.2 mM EGTA (low Ca2+ clamp; [Ca2+]i < 100 nM). Aminoglutethimide (500 µM) was included in the incubation medium to inhibit cholesterol side chain cleavage. At the end of a 2-h incubation period at 37 °C, the cells were sedimented at 200 × g for 15 min. All subsequent operations were conducted at 4 °C in buffers containing 500 µM aminoglutethimide.

Isolation of Mitochondria and Preparation of Submitochondrial Fractions

Glomerulosa cells were homogenized with a Potter-Elvejhem homogenizer (1200 rpm, 35 strokes) in 5 mM Tris-HCl buffer, pH 7.4, containing 275 mM sucrose. The homogenate was centrifuged at 200 × g for 15 min to remove large debris and nuclei. Further centrifugation of the supernatant at 10,000 × g for 10 min yielded the mitochondria. The mitochondrial pellet was washed twice at 8000 × g with the same buffer.

We have previously shown that sucrose density gradient fractionation of osmotically shocked adrenocortical mitochondria leads to the separation of three distinct membrane populations containing specific marker enzymes (27). Submitochondrial particles were prepared and separated by sucrose density gradient (15-50%, density = 1.06-1.23) centrifugation. Subsequently, the gradients were divided into 20 fractions of 500 µl that were assayed for marker enzyme activities. Pooled fractions 4-7 (corresponding to the monoamine oxidase activity peak; density = 1.08-1.11, specific to the outer mitochondrial membranes), 9-11 (corresponding to the nucleoside-diphosphate kinase activity peak; density = 1.13-1.15, specific to contact sites), and 13-15 (corresponding to the cytochrome c oxidase activity peak; density = 1.17-1.18, specific to the inner mitochondrial membranes) from the original gradient were dialyzed against 5 mM potassium phosphate buffer, pH 7.4, and stored at -20 °C until use. Protein was quantified using the Bio-Rad protein microassay and bovine serum albumin as a standard.

For some experiments, mitoplasts and outer membranes were prepared as follows. The washed mitochondria were exposed to a first swelling by incubation in 20 mM sodium phosphate buffer, pH 7.4, for 20 min at 4 °C. Centrifugation at 10,000 × g yielded the outer membranes (supernatant) and a pellet that was subjected to a second swelling in the same buffer (15 min, 4 °C). Mitoplasts were pelleted by centrifugation at 10,000 × g, washed, and resuspended in 5 mM Tris-HCl and 250 mM sucrose buffer (pH 7.4).

Marker Enzyme Assays

Cytochrome c oxidase (EC 1.9.3.1) and monoamine oxidase (EC 1.4.3.4) activities were determined according to Appelmans et al. (28) and Otsuka and Kobayashi (29), respectively. Nucleoside-diphosphate kinase (EC 2.7.4.6) was assayed as reported previously (27), and NADPH-cytochrome c reductase (EC 1.6.99.3) activity was determined as described by Sottocasa et al. (30).

Cholesterol Determination

The cholesterol content of submitochondrial fractions was determined by a coupled cholesterol oxidase-peroxidase assay with cholesterol as a standard (31). Aliquots of the fractions (200 µl) were transferred to glass tubes. To each sample were added 20 µl of 20 mM cholate and 1% Triton X-100 in 100 mM potassium phosphate buffer, pH 7.4, followed by the addition of 25 µl of 95% ethanol. The reaction mixture containing potassium phosphate buffer (100 mM), pH 7.4, cholesterol oxidase, peroxidase, and p-hydroxyphenylacetic acid was then added to each fraction in a final volume of 1 ml. Assay tubes were incubated for 1 h at 37 °C. Cholesterol oxidase generates H2O2, and peroxidase catalyzes the reaction of H2O2 with p-hydroxyphenylacetic acid to yield a stable fluorescent product. The fluorescence was measured in a Jasco CAF-110 fluorometer (excitation, 325 nm; emission, 405 nm).

SDS-Polyacrylamide Gel Electrophoresis

SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (32). Mitochondrial proteins (5-25 µg/lane) were solubilized in sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 5% beta -mercaptoethanol, 10% glycerol, and 0.01% bromphenol blue) and loaded onto a 12% SDS-polyacrylamide minigel (Mini-Protean II system, Bio-Rad). Electrophoresis was performed at 150 V for 1 h.

Blotting Method and Immunodetection

SDS-polyacrylamide gel electrophoresis-resolved proteins were electrophoretically transferred onto a nitrocellulose membrane (Schleicher & Schuell) according to Towbin et al. (33). Following transfer, the membrane was incubated in blocking buffer (phosphate-buffered saline containing 0.4% Tween 20 and 5% nonfat dry milk) for 1 h at room temperature and then incubated either with antibodies raised by one of us (17) against a peptide fragment (amino acids 88-98) of the 30-kDa StAR protein or with antisera specific for the steroidogenic enzymes 3beta -HSD and cytochrome P450scc or for cytochrome c oxidase (1 h in phosphate-buffered saline containing 0.4% Tween). The membrane was thoroughly washed with the same buffer (3 × 10 min) and then incubated for 1 h with horseradish peroxidase-labeled goat anti-rabbit IgG (Bio-Rad). The nitrocellulose sheet was washed as described above, and the antigen-antibody complex was revealed by enhanced chemiluminescence using the Western blotting detection kit and Hyper-ECL film (Amersham, Zürich, Switzerland). Quantitation of fluorograms was performed using a Molecular Dynamics computing densitometer.

Electron Microscopy

Rat adrenal glands were removed, and small fragments were fixed in 1% glutaraldehyde solution in phosphate-buffered saline for 1 h at room temperature (34). Preparation of the tissue blocks in LR white resin (London Resin Co., Bassingstoke, United Kingdom) was performed according to Newman et al. (35). Briefly, after fixation, the tissue was washed in phosphate-buffered saline and dehydrated in a graded series of up to 70% ethanol. The non-osmicated fragments were infiltrated with LR white resin and then placed in gelatin capsules for polymerization at 50 °C for 24 h (34). Thin sections were cut with a diamond knife and collected on nickel grids. Prior to incubation with antiserum, nonspecific antigenic sites were blocked by incubation for 5 min at room temperature with normal goat serum (1:100 dilution) in Tris-HCl/Tween buffer containing 0.9% NaCl, 10 mM Tris-HCl, pH 8.2, and 0.1% Tween 20. The sections were incubated overnight (4 °C) with antisera (1:20 dilution in Tris-HCl/Tween), followed by a 1-h incubation with a 1:10 dilution of 12-nm gold-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in -TrisHCl/Tween. Finally, the sections were stained with 1% aqueous uranyl acetate and lead citrate. The sections were observed and photographed using a Philips 300 electron microscope. Cytochemical controls (data not shown) included omission of the first antibody incubation step. Additional controls included incubation of preimmune serum with the adrenal sections (data not shown).

Analysis of Data

Results are expressed as means ± S.E. The mean values were compared by analysis of variance using Fisher's test. A value of p < 0.05 was considered as statistically significant. Image analysis of electron microscope micrographs was performed as follows.

Acquisition

Low power electron microscope micrographs (see Fig. 8, A and B) were scanned with a Vista-S8 scanner (UMAX Systems, Taipei, Taiwan) using a Power Macintosh 6100/60 computer (Apple Computer Corp.) at a resolution of 300 dpi and 256 gray-levels. Digitized images were analyzed on a Compaq Prosignia 300 PC using the IPWIN 1.3 software package (Media Cybernetics, Silver Spring, MD).


Fig. 8. Intramitochondrial localization of 3beta -HSD antigenic sites in rat adrenal gland. A, low power micrograph depicting immunogold staining of 3beta -HSD distribution in giant mitochondria (m) and the endoplasmic reticulum (ER). Note the lack of colloidal gold particles in the clear lipid area (L) and intercellular spaces (ICS). Arrows depict well preserved segments of the outer mitochondrial membrane adjacent to the inner membrane. Magnification × 34,125. B, a duplicate section as shown in A immunostained with P450scc antiserum. Note heavy mitochondrial (m) decoration with gold particles, in contrast to the very low level of antigen labeling in other intracellular compartments. Magnification × 27,125. C, high power micrograph depicting a mitochondrion (m) wrapped with multiple layers of smooth endoplasmic membranes (arrows) heavily labeled with 3beta -HSD antiserum. L, coalesced lipid droplets. Magnification × 50,575. D, intramitochondrial localization of 3beta -HSD. Note the decoration of immunoreactive 3beta -HSD in the mitochondrion intermembrane space proximal to the outer membrane (arrows). The inset shows a higher magnification (×123,600) of the boxed area in D, defining an additional four categories by which the distribution of the gold particles can be described (see Fig. 9): gold particles localized in the gray area (thick arrows) of the matrix (M), membrane-bound gold particles facing the matrix (thin arrows), membrane-bound particles (small arrowheads) facing the white intermembrane spaces (IMS), and gold particles (large arrowhead) localized in the midst of the inner membrane space.
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Processing and Analysis

Regions of interest were marked manually. Analysis of gold particle density was performed within regions of interest that delineated appropriate organelles and areas of interest within the cell. Pixel values within regions of interest were modified using a gamma -correction curve of 9.7. Images were then subjected to a 5 × 5 horizontal edge detection filter. This accentuated the particles while preserving a uniform background. Data loss was negligible (data not shown). Segmentation of particles was performed using particle size, roundness, and gray-level criteria. After segmentation, particles were counted, and their density within regions of interest was determined.


RESULTS

Characterization of the Submitochondrial Fractions of Bovine Glomerulosa Cells

Fig. 1 illustrates the monoamine oxidase, nucleoside-diphosphate kinase, and cytochrome c oxidase activities measured in the pooled fractions of OM, CS, and IM isolated from mitochondria of control cells (low Ca2+ clamp). As expected, the OM fraction contained the highest monoamine oxidase activity; the IM fraction was characterized by the highest cytochrome c oxidase activity; and CS displayed monoamine oxidase and cytochrome c oxidase activities in addition to nucleoside-diphosphate kinase activity. IM contained most of the mitochondrial membrane proteins (57.9 ± 7.6% of the total, n = 3), while CS and OM contained 36.3 ± 4.3 and 5.7 ± 1.7% of the total membrane proteins, respectively. Similar profiles of mitochondrial membrane marker enzymes and protein content were obtained following fractionation of mitochondria from high Ca2+-clamped cells (data not shown).


Fig. 1. Characterization of submitochondrial membranes of bovine adrenal glomerulosa cells. Glomerulosa cells were submitted for 2 h to a high Ca2+ clamp in the presence of 500 µM aminoglutethimide as described under "Experimental Procedures." Submitochondrial particles were isolated by sucrose density gradient centrifugation as described under "Experimental Procedures." The activities of mitochondrial marker enzymes were determined in each fraction of the gradient (data not shown). The fractions containing the peak of activity of each marker enzyme were then pooled and used as OM, CS, and IM. These activities are representative of four independent experiments. Ordinate units are nmol of deaminated tryptamine/min/mg of protein for monoamine oxidase (MAO), µmol of oxidized cytochrome c/min/mg of protein for cytochrome c oxidase (COX), and nmol of phosphorylated ADP/min/mg of protein for nucleoside-diphosphate kinase (NDP-K).
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Calcium and Ang II Induce a CHX-sensitive Intramitochondrial Cholesterol Transfer

We have previously shown in bovine adrenal zona glomerulosa cells that the calcium ionophore ionomycin can be effectively used at low concentration to clamp the cytosolic free Ca2+ concentration at various physiological levels (50-1000 nM) (24). These submicromolar [Ca2+]i levels stimulate in a concentration-dependent manner the early mitochondrial steps of steroidogenesis as well as aldosterone synthesis (24).

To examine the effect of Ca2+ on intramitochondrial cholesterol distribution, cholesterol content was determined in OM, CS, and IM from control (low Ca2+-clamped) cells and from high Ca2+-clamped cells in the presence or absence of CHX. Fig. 2A shows that the stimulation of ionomycin-treated glomerulosa cells with Ca2+ for 2 h led to a marked decrease in cholesterol content in OM (to 65.2 ± 0.2% of controls, n = 4; p < 0.001), with a concomitant increase in CS (to 143 ± 3.2% of controls; p < 0.01) and a less pronounced but significant augmentation in IM (to 119 ± 5.1% of controls; p < 0.05).


Fig. 2. Effect of cytosolic Ca2+ clamp and Ang II on cholesterol content of submitochondrial fractions of bovine glomerulosa cells. Stimulation with a cytosolic high Ca2+ clamp or Ang II and submitochondrial fractionation were carried out as described under "Experimental Procedures." The cholesterol content of pooled OM, CS, and IM fractions was determined and expressed for submitochondrial fractions of high Ca2+-clamped (A) or Ang II-stimulated (B) cells (in the absence or presence of 1 mM CHX) as a percentage of that measured in the respective pooled submitochondrial fractions of control cells (mean ± S.E., n = 4 for Ca2+, n = 4 for Ang II, and n = 3 for Ca2+ or Ang II + CHX). Mass unit values for cholesterol in OM, CS, and IM were 3.73 ± 0.21, 1.56 ± 0.20, and 0.86 ± 0.22 µg/mg of mitochondrial protein for controls; 1.98 ± 0.36 (***), 2.10 ± 0.16 (**), and 1.05 ± 0.08 (*) µg for CaCl2; and 2.9 ± 0.34, 1.54 ± 0.16, and 0.83 ± 0.15 µg for CaCl2 + CHX, respectively. Similar values were recorded in experiments with Ang II. *, **, and ***, significantly different from the respective control with p < 0.05, p < 0.01, and p < 0.001, respectively.
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When CHX was added concomitantly with Ca2+, the decrease in cholesterol content in the OM fraction was almost entirely prevented (92.5 ± 5.4% of the cholesterol content in the respective control fraction, n = 3). Simultaneously, CHX reduced significantly the Ca2+-induced cholesterol increase in CS and IM (Fig. 2A).

Fig. 2B illustrates the effect of Ang II on the cholesterol content of submitochondrial fractions of intact glomerulosa cells. No significant changes were detected in the outer mitochondrial membranes (93.4 ± 1.0% of controls, n = 4). However, the hormone induced a pronounced increase in cholesterol content in the contact site-enriched fractions (151.1 ± 18.1% of controls, n = 4; p < 0.05). Finally, a less pronounced increase in cholesterol content was observed in the inner membrane fractions (124.5 ± 6.5% of controls, n = 4; p < 0.05).

The accumulation of cholesterol in mitochondrial contact sites that occurred during Ang II stimulation was entirely prevented by cycloheximide (Fig. 2B). Furthermore, there was a tendency toward an increase in cholesterol content in the outer mitochondrial membranes (110.3 ± 20.3% of controls with Ang II alone, n = 3).

Ca2+ Stimulates StAR Protein Expression in Bovine Glomerulosa Cells

The 30-kDa mitochondrial StAR protein has been recently shown to be required in the acute regulation of steroid synthesis (18, 19). To determine whether an increase in [Ca2+]i affected StAR protein expression in bovine glomerulosa cells, mitochondrial proteins from Ca2+-clamped cells were analyzed by immunoblotting. Fig. 3A shows the results obtained with one representative experiment. The anti-StAR protein antibodies recognized a protein doublet of ~30 kDa in mitochondria from both control cells and high Ca2+-clamped cells. Densitometric analysis of this particular Western blot revealed that Ca2+ increased StAR protein content to 210% of controls. CHX significantly inhibited the Ca2+-induced increase in StAR protein by 62%. In Fig. 3B, the mean results from the quantitation of the StAR protein signal in mitochondria from five separate experiments are represented. Ca2+ increased significantly mitochondrial StAR protein content to 159 ± 23% of controls (p < 0.05). This effect of Ca2+ was prevented by CHX (116 ± 8% of controls).


Fig. 3. Immunodetection of StAR protein in mitochondria of Ca2+-clamped glomerulosa cells. A, shown is a Western blot from one representative experiment. Mitochondria were isolated from control cells or high Ca2+-clamped cells incubated in the absence or presence of 1 mM CHX. For each sample, 10 µg of mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis as described under "Experimental Procedures." B, the immunospecific bands for the 30-kDa StAR proteins were quantitated by densitometry in five independent experiments. C, control (low Ca2+ clamp); Ca2+, high calcium clamp (600 nM); Ca2+ + CHX, high calcium clamp in the presence of cycloheximide; IOD, integrated optical density.
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Ca2+ and Ang II Selectively Increase StAR Protein Content in Inner Mitochondrial Membranes

We next examined the submitochondrial distribution of StAR protein in adrenal glomerulosa cells. Interestingly, no StAR protein was detected in the outer membrane (data not shown). By contrast, as shown in Fig. 4A, StAR proteins were detected by the antibody in mitochondrial CS and IM from both control (low Ca2+-clamped) and high Ca2+-clamped cells. Densitometric analysis of the immunospecific bands (Fig. 4B) revealed that no significant changes were observed in StAR protein content in mitochondrial CS from high Ca2+-clamped cells as compared with controls. By contrast, Ca2+ increased StAR protein content by 4-fold in IM, indicating that the increase in StAR protein induced by Ca2+ was specifically targeted to the inner mitochondrial membranes. The average Ca2+-induced StAR protein content in IM from five separate experiment amounted to 228 ± 50% (p < 0.01) (Fig. 5B).


Fig. 4. Immunodetection of StAR protein in submitochondrial fractions of Ca2+-clamped glomerulosa cells. A, 25 µg of protein from CS and IM from one representative experiment were analyzed by immunoblotting for their StAR protein content after incubation of glomerulosa cells under a low or a high Ca2+ clamp. B, shown is the densitometric analysis of the Western blot. IOD, integrated optical density.
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Fig. 5. Effect of CHX on Ca2+- and Ang II-induced increase in StAR protein in IM. Cells were stimulated for 2 h with Ca2+ or Ang II in the presence or absence of CHX, and submitochondrial fractions were prepared as described under "Experimental Procedures." A, 10 µg of inner membrane protein were analyzed by immunoblotting for StAR protein content. B, shown is the densitometric analysis of five separate experiments. C, control (low Ca2+ clamp); Ca2+, high calcium clamp (600 nM); Ca2+ + CHX, high calcium clamp in the presence of cycloheximide. *, p < 0.05. IOD, integrated optical density.
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Qualitatively similar results were obtained with Ang II (Fig. 5B). The hormone induced a significant increase in IM StAR protein to 150 ± 23% of controls (n = 3, p < 0.05), while having no effect on StAR protein content in CS.

The Ca2+- and Ang II-induced Increase in StAR Protein Content in Inner Mitochondrial Membranes Is Sensitive to CHX

When bovine adrenal glomerulosa cells were subjected to a high Ca2+ clamp in the presence of CHX, immunoblot analysis of inner mitochondrial membrane and contact site proteins revealed that CHX completely prevented the Ca2+-induced increase in StAR protein in IM (Fig. 5, A and B), while no significant changes occurred in contact sites (data not shown). Similarly, the increase in IM StAR protein content observed after Ang II challenge was not observed in the presence of CHX (Fig. 5B).

Cytochrome P450scc and 3beta -HSD Are Found in Mitochondrial Contact Sites and Inner Membranes

To determine whether StAR protein induction by Ca2+ and Ang II was specific, submitochondrial fractions were analyzed by immunoblotting using anti-3beta -HSD or anti-P450scc antibodies since we had shown previously that these two enzymes are co-localized within the mitochondria of bovine fasciculata cells (27). The increase in the synthesis of these two enzymes is known to require long-term exposure to steroidogenic hormones (9). Fig. 6 shows that mitochondrial contact sites and inner membranes from bovine glomerulosa cells contain significant amounts of 3beta -HSD and P450scc. In addition, no changes in 3beta -HSD and P450scc content occurred in these submitochondrial fractions with Ang II stimulation. Finally, CHX did not affect the level of these proteins in CS and IM. Identical observations were made in bovine glomerulosa cells subjected to a high Ca2+ clamp (data not shown).


Fig. 6. Immunodetection of 3beta -HSD and cytochrome P450scc in submitochondrial fractions of Ang II-stimulated glomerulosa cells. Submitochondrial fractions were prepared after stimulation for 2 h with Ang II, and proteins were separated by SDS-polyacrylamide gel electrophoresis as described under "Experimental Procedures." 10 µg of protein from CS and IM were analyzed by immunoblotting for their 3beta -HSD and cytochrome P450scc content. This Western blot is representative of three similar experiments. C, control.
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The presence of 3beta -HSD in inner mitochondrial membranes from bovine glomerulosa cells was further confirmed by preparing mitoplasts. The determination of enzyme marker activities confirmed that mitoplasts were practically devoid of any contamination with microsomes. Indeed, as can be seen in Table I, while cytochrome c oxidase activity was 10-fold higher in mitoplasts than in outer membranes, NADPH-cytochrome c reductase activity, a marker of the endoplasmic reticulum, was undetectable in mitoplasts and high in microsomes. Upon immunoblot analysis (Fig. 7A), a strong 3beta -HSD signal was detected in mitoplasts as well as in microsomes, thus confirming the presence of the enzyme inside the mitochondria. In fact, the 3beta -HSD signal in mitoplasts was considerably higher than in outer membranes in spite of the fact that the latter displayed a higher NADPH-cytochrome c reductase activity.

Table I.

Enzyme marker activities in mitoplasts, outer mitochondrial membranes, and microsomes

Cytochrome c oxidase and NADPH-cytochrome c reductase activities were determined after preparation of mitoplasts as described under "Experimental Procedures." Each value is the mean ± S.E. of duplicate determinations from two separate experiments.
COXa NADPH-cytochrome c reductase

nmol/min/mg protein nmol/min/mg protein
Outer membranes 183.5  ± 57.5 2.9  ± 0.2
Mitoplasts 1746  ± 136 ND
Microsomes ND 16.3  ± 1.6

a COX, cytochrome c oxidase; ND, not detectable.


Fig. 7. Intramitochondrial localization of 3beta -HSD in bovine glomerulosa cells and in MA-10 mouse Leydig cells. A, mitoplasts were prepared from bovine glomerulosa cells as described under "Experimental Procedures," and 5 µg of protein of outer membranes (lane 1), mitoplasts (lane 2), or microsomes (lane 3) were analyzed by immunoblotting for their 3beta -HSD content. B, shown is a Western blot of 3beta -HSD and cytochrome c oxidase (COX) in submitochondrial fractions of MA-10 mouse Leydig cells. Mitochondria were fractionated on a sucrose density gradient as described under "Experimental Procedures," and 10 µg of protein from each fraction (lanes 1-18) were analyzed by immunoblotting.
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To examine whether the intramitochondrial localization of 3beta -HSD is a common characteristic of steroidogenic cells, mitochondria from MA-10 mouse Leydig cells were fractionated on a sucrose gradient as described above for adrenal mitochondria. As shown in Fig. 7B, immunoblot analysis revealed the presence of the 3beta -HSD protein in CS and IM, but not in OM.

Immunocytochemical Localization of 3beta -HSD

As an additional proof of the presence of 3beta -HSD within steroidogenic mitochondria, the ultrastructural localization of 3beta -HSD was studied by the immunogold staining technique in rat adrenal fasciculata cells (17, 34). Fig. 8 shows that 3beta -HSD antigenic sites are clearly abundant in the mitochondria of these cells. The immunovisualization technique also confirms that, as expected, >60% of this enzyme is anchored to the membranes of the endoplasmic reticulum (Fig. 8C and Table II), where the apparent density of immunoreactive 3beta -HSD is 56% higher than that observed in the mitochondria (Table II). Higher enrichment of membrane-bound 3beta -HSD was observed in stacking of smooth endoplasmic reticulum shown in Fig. 8C. Quantitation of P450scc labeling, which serves as a classical mitochondrial marker, shows a dramatically reciprocated ratio, with the density of this enzyme per mitochondrial surface being 10 times higher than that observed in the cytoplasm (Table II).

Table II.

Distribution of colloidal gold-labeled 3beta -HSD in endoplasmic reticulum versus intramitochondrial sections

The low power photomicrographs shown in Fig. 8 (A and B) were scanned and analyzed as described under "Experimental Procedures." The localization of the gold particles was categorized as shown, and the relative area of each cellular compartment was defined. The particle number (percent in parentheses) and the density per unit area are shown.
Localization Relative area No. particles Particles/area

3beta -HSD (Fig. 8A)
  Mitochondria 0.43 1218 (39.9%) 2800
  ERa 0.42 1836 (60.1%) 4371
  Cytoplasm
  Fat, ICS 0.15 1 0
P450scc (Fig. 8B)
  Mitochondria 0.35 5.036 (86%) 14,388
  ER
  Cytoplasm 0.53 790 (14%) 1490
  Fat, ICS 0.1 5 0

a ER, endoplasmic reticulum; cytoplasm, defined as such when ultrastructural preservation did not allow a clear definition of the endoplasmic reticulum; fat, lipid droplets as shown in Fig. 8 (A and B); ICS, intercellular spaces.

The intramitochondrial pattern of 3beta -HSD localization (Fig. 8D) was monitored using an analytical approach similar to that previously applied to P450scc and StAR protein patterns (34, 36). Fig. 9 depicts the compartmentalization patterns of the three key steroidogenic proteins at a submitochondrial resolution. Clearly, whereas P450scc antigenic sites were exclusively anchored to the crista membranes protruding into the mitochondrial matrix (34), the labeling of 3beta -HSD sites partitioned in all five compartments of the organelle. Nevertheless, nearly 60% of 3beta -HSD labeling was associated with the crista membranes, whereas the rest of the antigenic sites were equally distributed between the outer membranes, the mitochondrial matrix, and the intermembrane spaces. To some extent, this profile of 3beta -HSD sites also differed from that obtained for StAR protein, the majority of which was confined to the intermembrane spaces of the vesicular cristae (Fig. 9).


Fig. 9. Intramitochondrial distribution of colloidal gold-labeled 3beta -HSD, P450scc, and StAR protein. Colloidal gold particles were manually counted in 6-10 different mitochondria of rat adrenal fasciculata cells stained with rabbit antisera to bovine 3beta -HSD, rat P450scc (based on previously published data (34)), and mouse StAR (based on previously published data (17)) (787, 587, and 443 particles, respectively). The localization of the particles in five different intramitochondrial compartments was categorized using high power photomicrographs (magnification × 50,575) as described for Fig. 8D. Data are presented as percentage (mean ± S.E.) of gold particles in each of the submitochondrial compartments. IMS, intermembrane spaces.
[View Larger Version of this Image (27K GIF file)]



DISCUSSION

In a previous study on the mechanisms of activation of aldosterone biosynthesis by Ca2+, we have narrowed the potential target domain for this messenger to the early steps of the steroidogenic pathway (24). Using the ionomycin-mediated Ca2+ clamp and Ang II as activators of steroid synthesis in bovine glomerulosa cells, we have recently shown that physiological [Ca2+]i changes acutely stimulate cholesterol redistribution across mitochondrial membranes (37). We now demonstrate that this cholesterol transfer from the outer mitochondrial membranes to both contact sites and inner membranes is associated with a selective increase in StAR protein content in inner mitochondrial membranes and that both these responses require protein synthesis.

The major finding of this work is the clear-cut and specific increase in the mature form(s) of the 30-kDa StAR protein in the inner mitochondrial membranes of bovine glomerulosa cells under stimulation of the calcium messenger system. To our knowledge, this is the first demonstration of a submitochondrial modulation of StAR protein by Ca2+. Indeed, most studies that have investigated the regulation of the 30-kDa proteins involved in the acute steroidogenic response have used total cellular or mitochondrial protein extracts and have focused on trophic agents activating adenylyl cyclase and cAMP production (16, 38, 39). Recently, Clark et al. (40) have shown that calcium-mobilizing agonists such as Ang II, K+, and the dihydropyridine agonist Bay K8644 produce significant increases in the cellular levels of StAR protein in H295R human adenocarcinoma cells within 4-6 h. Our results extend this observation by confining the StAR protein increase to a submitochondrial compartment and by showing that it can be observed after shorter stimulation times (2 h), thus confirming the acute role of StAR protein in the initiation of the steroidogenic response.

In MA-10 Leydig tumor cells and in the human H295R adrenocarcinoma cell line, StAR protein appears as a single band upon immunoblot analysis (16, 40), whereas in bovine adrenal glomerulosa cells, we detected a StAR protein doublet around 30 kDa. While the nature of this protein doublet remains to be elucidated, it is worth mentioning that a similar pattern has been observed in bovine fasciculata cells (41). In MA-10 mouse Leydig tumor cells, phosphorylation of StAR protein on a threonine residue is required for the acute induction of steroidogenesis (42). The doublet we observed may therefore represent phosphorylated and non-phosphorylated forms of StAR protein. These proteins could also be related to the 28.5-30-kDa proteins shown by Elliott et al. (43) to be induced by Ang II in bovine glomerulosa cells.

Remarkably, Ca2+ and Ang II induced StAR protein accumulation exclusively in the inner mitochondrial membranes. Interestingly, although StAR protein was present in contact sites, the increase in cholesterol induced by [Ca2+]i rises was not associated with a concomitant increase in StAR protein content, indicating that StAR protein is only transiting via those structures. Moreover, we could not detect the 37-kDa precursor of StAR protein in any of the submitochondrial fractions. These observations confirm the short half-life of the StAR protein precursor, which is known to be rapidly processed by mitochondrial proteases (17), and indicate that the inner mitochondrial membranes constitute the final destination of mature StAR protein. The present biochemical evidence is therefore in agreement with our visualization of StAR protein localization in adrenal mitochondria labeled by immunogold staining (17).

Early studies in adrenocortical cells have demonstrated that the inhibitor of protein translation, cycloheximide, prevents the increase in the rate of steroid synthesis induced by ACTH (10, 11). The cycloheximide-sensitive step in steroidogenesis has been located to the ACTH-activated cholesterol transport to the inner mitochondrial membrane (10). In adrenal glomerulosa cells, Elliott and Goodfriend (44) have reported that the cycloheximide-sensitive step in Ang II-stimulated aldosterone synthesis is mitochondrial pregnenolone synthesis, the first event in steroidogenesis following cholesterol supply to cytochrome P450scc. Activators of steroidogenesis mobilizing either the cAMP or the Ca2+ messenger system have been shown to induce a net accumulation of total cellular StAR protein (19, 40), and we have obtained similar data in bovine glomerulosa cells (data not shown). These findings indicate that cycloheximide is most likely to act at a translational level. Indeed, our data show that both StAR protein accumulation and intramitochondrial cholesterol transfer induced by [Ca2+]i rises are highly sensitive to cycloheximide, strongly suggesting that the blockade of StAR protein synthesis prevents cholesterol mobilization to the inner membranes. Taken together, these results provide strong correlative evidence that the increase in StAR protein expression and its targeting to the mitochondrial membranes are linked to cholesterol redistribution from the outer to the inner membranes induced by calcium-mobilizing agents in adrenal glomerulosa cells.

Our current model of the mechanisms of calcium-induced activation of steroidogenesis would favor a dual site of action for Ca2+: in addition to an obligatory role for Ca2+ influx into the mitochondria, as demonstrated previously in permeabilized glomerulosa cells (45) and in glomerulosa cells treated with a blocker of the mitochondrial Na+/Ca2+ exchanger (25), the present cycloheximide results imply an effect of cytosolic Ca2+ on StAR protein expression.

The effect of increased [Ca2+]i is shown to be specific for StAR protein since the levels of two additional key steroidogenic enzymes in the mitochondria, cytochrome P450scc and 3beta -HSD, were not affected under similar experimental manipulations. It should be noted that whereas P450scc is a typical nuclearly encoded mitochondrial protein, 3beta -HSD has been considered as an enzyme anchored in the endoplasmic reticulum (46), with a few exceptions (47). Yet, we have previously provided evidence showing that P450scc and 3beta -HSD co-localize in the inner membranes and contact sites of bovine adrenocortical zona fasciculata mitochondria (27), and their association into a macromolecular complex retains the catalytic activities of both enzymes (48). We report here that cytochrome P450scc and 3beta -HSD also coexist in the contact sites and inner membranes of zona glomerulosa mitochondria. In addition, immunogold staining of rat adrenal fasciculata mitochondria, as well as immunoblot analysis of MA-10 mouse Leydig submitochondrial fractions, clearly confirmed the intramitochondrial localization of 3beta -HSD. The consistency of this finding, observed with different techniques in various steroidogenic cell types from three different species, speaks in favor of a common feature of 3beta -HSD distribution in all steroid-producing cells. Although the N terminus of the 3beta -HSD protein does not appear to include a mitochondrial targeting sequence (49), various proteins of the inner mitochondrial membrane, such as the ADP/ATP carrier (50), porin (51), and BCS1 (52), appear to contain important targeting information either internally or at their C terminus, rather than at the N terminus (53). The intramitochondrial presence of 3beta -HSD could add a facilitating step of important functional significance to the Ca2+-mediated cholesterol transfer: channeling pregnenolone to progesterone should be highly favored by 3beta -HSD located in close proximity to P450scc and may actually represent a major pathway, in line with the fact that pregnenolone is the preferential substrate of mitochondrial 3beta -HSD (27). In this regard, it is worth mentioning that the existence of multihormonal enzyme complexes ("hormonads") has already been proposed some years ago by Lieberman and Prasad (54) and Prasad et al. (55).

Collectively, our biochemical approaches, together with the ultrastructural analysis showing that >85% of P450scc and 60% of mitochondrial 3beta -HSD are anchored to the inner crista membranes of this organelle, raise the possibility of a supramolecular organization of P450scc and 3beta -HSD into a functional unit directly catalyzing the metabolism of cholesterol to pregnenolone and to progesterone following StAR protein-mediated cholesterol supply via contact sites.


FOOTNOTES

*   This work was supported in part by Swiss National Science Foundation Grants 31.42178-94 (to A. M. C.) and 32.39277-93 (to M. F. R.), by National Institutes of Health Grant HD 17841 (to D. M. S.), and by United States-Israel Binational Science Foundation Grant 95-350 (to J. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Div. of Endocrinology and Diabetology, University Hospital, 24, rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. Tel.: 4122-372-93-21; Fax: 4122-372-93-29; E-mail: cherradi-nadia{at}diogenes.hcuge.ch.
   Recipient of a grant from the Prof. Max Cloëtta Foundation.
1   The abbreviations used are: Ang II, angiotensin II; ACTH, adrenocorticotropic hormone; OM, outer mitochondrial membrane(s); IM, inner mitochondrial membrane(s); CS, intermembrane contact sites; StAR, steroidogenic acute regulatory; [Ca2+]i, cytosolic free calcium concentration; 3beta -HSD, 3beta -hydroxysteroid dehydrogenase isomerase; CHX, cycloheximide.

Acknowledgments

We are grateful to Liliane Bockhorn, Walda Dimeck, Gisèle Dorenter, Marcella Klein, and Maria Lopez for excellent technical assistance.


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