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Endocrinology Vol. 141, No. 5 1599-1607
Copyright © 2000 by The Endocrine Society

ARTICLES

Regulation by Adrenocorticotropin (ACTH), Angiotensin II, Transforming Growth Factor-ß, and Insulin-Like Growth Factor I of Bovine Adrenal Cell Steroidogenic Capacity and Expression of ACTH Receptor, Steroidogenic Acute Regulatory Protein, Cytochrome P450c17, and 3ß-Hydroxysteroid Dehydrogenase1

Christine Le Roy2, J. Yuan Li, Douglas M. Stocco, Dominique Langlois and José M. Saez

INSERM, U-369 and U-418, Institut Fédératif Recherches en Endocrinologie de Lyon, and Université Claude Bernard Lyon 1, Faculté de Medecine Laennec (C.L.R., J.Y.L., D.L., J.M.S.), 69372 Lyon, France; and Department of Cell Biology and Biochemistry, Texas University (D.M.S.), Lubbock, Texas 79430

Address all correspondence and requests for reprints to: Dr. José M. Saez, INSERM, U-369, and IFREL, Faculté de Medecine Laennec, rue Guillaume Paradin, 69372 Lyon Cedex 07, France. E-mail: saez{at}lyon151.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The purpose of this study was to evaluate the time-course effect of a 36-h treatment with ACTH (10-8 M), transforming growth factor-ß1 (TGFß1; 10-10 M), angiotensin II (AngII; 10-7 M), and insulin-like growth factor I (IGF-I; 10-8 M) on the steroidogenic capacity of bovine adrenocortical cells (BAC) and on messenger RNA (mRNA) levels of ACTH receptor, cytochrome P450c17, 3ß-hydroxysteroid dehydrogenase (3ßHSD), steroidogenic acute regulatory protein (StAR), and StAR protein. ACTH and IGF-I enhanced, in a time-dependent manner, the acute 2-h ACTH-induced cortisol production, whereas TGFß1 and AngII markedly reduced it. ACTH, IGF-I, and AngII increased ACTH receptor mRNA, but the opposite was observed after TGFß1 treatment. ACTH and IGF-I increased P450c17 and 3ßHSD mRNAs, whereas AngII and TGFß1 had the opposite effects. However, the effects of the four peptides on ACTH-induced cortisol production appeared before any significant alterations of the mRNA levels occurred. The most marked and rapid effect of the four peptides was on StAR mRNA. The stimulatory effect of ACTH was seen within 1.5 h, peaked at 4–6 h, and declined thereafter, but at the end of the 36-h pretreatment, the levels of StAR mRNA and protein were higher than those in control cells. IGF-I also enhanced StAR mRNA levels within 1.5 h, and these levels remained fairly constant. The effects of AngII on StAR mRNA expression were biphasic, with a peak within 1.5–3 h, followed by a rapid decline to almost undetectable levels of both mRNA and protein. TGFß1 had no significant effect during the first 3 h, but thereafter StAR mRNA declined, and at the end of the experiment the StAR mRNA and protein were almost undetectable. Similar results were observed when cells were treated with ACTH plus TGFß1. A 2-h acute ACTH stimulation at the end of the 36-h pretreatment caused a higher increase in StAR mRNA and protein in ACTH- or IGF-I-pretreated cells than in control cells, which, in turn, had higher levels than cells pretreated with TGFß1, ACTH plus TGFß1, or AngII.

These results and the fact that the stimulatory (IGF-I) or inhibitory (AngII and TGFß1) effects on ACTH-induced cortisol production were more pronounced than those on the ability of cells to transform pregnenolone into cortisol strongly suggest that regulation of StAR expression is one of the main factors, but not the only one, involved in the positive (IGF-I) or negative (TGFß1 and AngII) regulation of BAC for ACTH steroidogenic responsiveness. A high correlation between steady state mRNA level and acute ACTH-induced cortisol production favors this conclusion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BIOSYNTHESIS OF corticosteroids involves the participation of various steroidogenic enzymes (1). The first step in steroidogenesis is the conversion of cholesterol to pregnenolone catalyzed by the cytochrome P450 side-chain cleavage (P450scc) enzymes, which reside in the inner mitochondrial membrane (2). However, many studies have shown that the limiting step was not due to P450scc activity, but, rather, to the translation of cholesterol from the outer to the inner mitochondrial membrane. Several proteins, including the sterol carrier protein-2 (3), the steroidogenesis activator polypeptide (4), and the peripheral benzodiazepine receptor (5), are able to promote steroidogenesis under some experimental conditions and therefore become candidates for mediators of cholesterol transport between the mitochondrial membranes. Although data supporting an important role for these proteins in cholesterol transport are convincing, the time course and regulation of their expression as well as the magnitude and specificity of their responses indicate that they are unlikely to be the primary determinant of the acute steroidogenic response in adrenals. More recently, a protein, namely StAR (steroidogenic acute regulatory protein), expressed exclusively in adrenals and gonads, the synthesis of which is rapidly induced by the steroidogenic hormones, has been purified and cloned (6). The biochemical and genetic evidence for the key role of this protein in the hormonally induced acute steroidogenic response of steroidogenic cells has been reviewed (7, 8, 9).

In both human and bovine zona fasciculata cells, ACTH and angiotensin II (AngII) acutely stimulate, in a similar manner, cortisol production (10, 11, 12), and, at least in bovine adrenal cells and the H295R human tumor cell line, this is associated with a rapid increase in StAR messenger RNA (mRNA) and protein (13, 14, 15, 16). In addition to this well established acute steroidogenic action, both hormones have a long term effect on steroidogenic responsiveness by regulating the expression of the genes encoding their own receptor (17, 18, 19, 20) and several enzymes involved in the steroidogenic pathway (21, 22, 23, 24). However, many other factors, such as cytokines (25) and growth factors (26), have been shown to participate in the fine tuning of adrenocortical function. In particular, two growth factors, insulin-like growth factor I (IGF-I) (27, 28) and transforming growth factor-ß1 (TGFß) (29, 30, 31, 32, 33, 34, 35, 36, 37), have long term opposite effects on the steroidogenic responsiveness of both human and bovine adrenocortical cells.

The purpose of this study was to examine the time-course effects of ACTH, AngII, IGF-I, and TGFß on the acute steroidogenic responsiveness to ACTH of bovine zona fasciculata-reticularis cells (BAC) and to examine the effects of these peptides on the expression of four key genes, ACTH receptor (ACTHR), StAR, cytochrome P450c17, and 3ß-hydroxysteroid dehydrogenase (3ßHSD), involved in the response to ACTH.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Synthetic AngII was obtained from Bachem (Bubendorf, Switzerland); synthetic ACTH [ACTH-(1–24)] was obtained from Ciba (Rueil-Malmaison, France); porcine TGFß1 and human recombinant IGF-I were purchased from R&D Systems (Minneapolis, MN). Ham’s F-12 medium-DMEM (F12-DMEM), insulin, transferrin, and FCS were obtained from Life Technologies, Inc. (Paris, France). All other products were obtained from Sigma (St. Louis, MO). Bovine P450c17 mRNA was donated by Dr. M. R. Waterman (38), bovine StAR complementary DNA (cDNA) was donated by Dr. R. Ivell (39), human 3ßHSD was donated by Drs. F. Labrie and V. Luu The (40), and a 0.9-kb fragment of bovine ACTH receptor cDNA was donated by Dr. R. Cone (41).

Isolation and culture of BAC
BAC were prepared by sequential treatment of adrenal cortical slices with trypsin (0.15%) (20). The cells were cultured in a chemically defined medium of DMEM/F12 supplemented with NaHCO3 (14 mM) and HEPES (10 mM) and containing gentamicin (20 µg/ml), penicillin (100 U/ml), streptomycin (0.1 mg/ml), nystatin (100 U/ml), transferrin (10 µg/ml), insulin (10 µg/ml), and FCS (1%). On the second day of culture, the medium was removed and replaced with serum-free medium. Treatment was started at the end of the second day of culture. In the time-course experiments, the protocol was designed in such a way that all of the treatments finished at the same time. In these experiments, the medium with the corresponding factors was renewed after 24 h.

RNA preparation and Northern blot analysis
Total RNA was isolated from cells by the method of Chomczynski and Sacchi (42). Samples (20–25 µg RNA) were separated by electrophoresis through a 1% agarose gel containing 10% formaldehyde. RNA was then transferred on Hybond-N membrane. The prehybridization and hybridization solutions used were described previously (11, 18). Labeling of these probes in the presence of [{alpha}-32P]deoxy-CTP was performed with the Megaprime DNA labeling system (Amersham Pharmacia Biotech, Arlington Heights, IL). The blots were washed with more or less stringency depending on the probes used and then exposed to photographic film. The relative intensity of hybridization signals was quantified using a scanning densitometer (Preference Sebia, Paris, France). Loading of RNA samples was verified by scanning the 28S RNA negatives. The same blots were successively hybridized with bovine StAR cDNA, bovine ACTH receptor cDNA, bovine P450c17 cDNA, and human 3ßHSD cDNA (~2 x 106 dpm/ml). The 28S RNA signal was used to normalize data for the above mRNA.

Isolation of mitochondria, SDS-PAGE, and immunodetection of StAR protein
At the end of each treatment, BAC cells were washed with cold 50 mM NaCl and scraped off using a rubber policeman into a buffer containing 10 mM Tris-HCl (pH 7.2), 250 mM sucrose, 0.1 mM EDTA, and 10 mM phenylmethylsulfonylfluoride. The cells were homogenized at 4 C. Mitochondrial preparations were obtained by differential centrifugation. The homogenate was centrifuged at 800 x g for 10 min to remove broken cell debris and nuclei, and the resulting supernatant was further centrifuged at 10,000 x g for 25 min. The pellet containing mitochondria was washed twice at 10,000 x g for 15 min each time in the same buffer.

The mitochondrial proteins (15–20 µg/lane) were solubilized in sample buffer (25 mM Tris-HCl (pH 6.8), 1% SDS, 5% ß-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue) and loaded onto a 10% SDS-polyacrylamide gel as described by Laemmli (43), with minor modifications. Electrophoresis was performed at 100 V for 4 h, and the proteins were electrophoretically transferred onto a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech). The membranes were incubated in a blocking buffer (Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk) for 2 h at room temperature, followed by incubation with antipeptide antibodies to the StAR protein generated in rabbit against amino acids 88–98 (6). The incubation with the antibodies was carried out overnight at 4 C. The membranes were washed three times (10 min each time) in Tris-buffered saline buffer and incubated for 1 h at room temperature with horseradish peroxidase-labeled donkey antirabbit IgG (Amersham Pharmacia Biotech). The membranes were washed as stated above, the immunodetection of StAR protein was revealed using the enhanced chemiluminescence Western blotting detection kit (Amersham Pharmacia Biotech), and the membranes were exposed for 1–3 min to x-ray films (Fuji Photo Film Co. Ltd., Tokyo, Japan). The immunospecific bands were quantitated by densitometry.

Steroids and cAMP determination
To test the hormonal activity of the adrenal cells, cortisol, corticosterone, pregnenolone and cAMP in the medium were measured by a specific RIA (29, 44, 45).

Statistics
For each cell preparation, 20–25 adrenals were used. Most of the experiments were performed at least three times, and each experimental point was determined in triplicate or quadruplicate. For StAR mRNA, the two major transcripts of 3.0 and 1.8 kb were taken into consideration for all calculations. Student’s t test and one-way ANOVA, as appropriate, were used for statistical evaluation of the data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time-course effects of ACTH, TGFß1, AngII, and IGF-I pretreatment on BAC steroidogenic responsiveness to ACTH
To investigate the effects of these factors on ACTH-induced cortisol production, cells were pretreated without (control) or with ACTH (10-8 M), TGFß1 (10-10 M), AngII (10-7 M), or IGF-I (10-8 M) for 1.5–48 h. The concentrations used for each peptide were those previously shown to produce maximal effects on BAC. At the end of each period, cells were washed with acidic buffer to remove the bound peptide and stimulated with ACTH (10-8 M), and the cortisol production was measured after 2 h. None of the peptides significantly modified the response to ACTH within the first 1.5 h of treatment (Fig. 1Go). Thereafter, marked changes were observed. ACTH progressively enhanced the response to further stimulation with ACTH and reached a maximum (6-fold compared with control cells) at 36 h. Similarly, IGF-I pretreatment enhanced ACTH responsiveness after 3 h and reached a maximum at 36 h (3-fold increase). In contrast, pretreatment with either AngII or TGFß1 markedly reduced the steroidogenic responsiveness to ACTH within 6 h, but the inhibitory effect of TGFß1 was always more marked than that produced by AngII. Interestingly, the ACTH responsiveness of cells pretreated with ACTH plus TGFß1 was significantly lower than that of cells pretreated with ACTH alone, but higher than that of cells pretreated with TGFß1, indicating that TGFß1 blocked the enhanced steroidogenic responsiveness induced by ACTH pretreatment.



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  		Figure 1. Time-course effects of several peptides on ACTH-induced cortisol production by BAC. Cells were cultured for the indicated periods in the absence (control) or presence of ACTH (10-8 M), TGFß1 (10-10 M), ACTH plus TGFß1, AngII (10-7 M), or IGF-I (10-8 M). After an acidic washing, cells were acutely stimulated with ACTH (10-8 M) for 2 h, and cortisol production was measured. The results, expressed as percentage of the response of control cells, are the mean ± SEM of three or four independent experiments. a, P < 0.01 compared with control; b, P < 0.001 compared with ACTH or TGFß1 stimulation.

 
To elucidate which step(s) in the mechanism of action of ACTH could be involved in the changes induced by the peptides, cells were treated for 36 h and after an acidic wash were incubated in the absence (basal) or presence of ACTH; cAMP, cortisol, and corticosterone were measured after 2 h. The results presented in Table 1Go show that the ACTH-induced cAMP production was significantly stimulated by three of these peptides (ACTH>IGF-I>AngII), whereas TGFß1 alone reduced it by 70% and blocked the stimulatory effect of ACTH. Concerning the acute effects of ACTH on cortisol production, the results confirm those shown in Fig. 1Go. Moreover, ACTH and IGF-I increased total glucocorticoid secretion (cortisol and corticosterone) and the cortisol/corticosterone ratio, but the effects of ACTH were significantly higher than those of IGF-I. On the contrary, AngII and TGFß1 significantly reduced cortisol and total glucocorticoid secretion and the cortisol/corticosterone ratio, but the effects of TGFß1 on all of these parameters were more pronounced than those caused by AngII.


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  		Table 1. Effects of pretreatment with several peptides on cAMP and steroid production by BAC in response to ACTH

 
To further analyze which step(s) of the steroidogenic pathway was modified by the peptides, we studied the conversion of 25-hydroxycholesterol to pregnenolone in the presence of inhibitors of P450c17 and 3ßHSD activities and the conversion of pregnenolone to cortisol and corticosterone. The results show (Table 2Go) that only ACTH alone, and to a lesser extent in combination with TGFß1, increased P450scc activity. Moreover, pretreatment with ACTH or IGF-I increased the conversion of pregnenolone to cortisol, the total glucocorticoid (cortisol and corticosterone) secretion, and the cortisol/corticosterone ratio, whereas AngII and TGFß1 pretreatment caused the opposite effects. However, compared with control cells, the above-mentioned stimulatory action of ACTH and IGF-I and the inhibitory effects of AngII and TGFß1 were less marked than those after acute ACTH stimulation. These results suggest that the peptides modified the ACTH response at least at three levels: formation of cAMP and before and after pregnenolone formation.


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  		Table 2. Effects of pretreatment with several peptides on the conversion of 25OH-cholesterol to pregnenolone and of pregnenolone to cortisol (F) and corticosterone (B)

 
Effects of ACTH, TGFß1, AngII, and IGF-I on BAC-specific gene expression
To identify more precisely the effects of the peptides on BAC-specific functions, the mRNA levels of four key genes involved in the steroidogenic action of ACTH were analyzed after 36 h of treatment. The results presented in Fig. 2Go demonstrate that ACTHR, IGF-I, and AngII increased ACTHR mRNA, whereas it was decreased by TGFß1, and this peptide blunted the stimulatory effect of ACTH. Similarly, P450c17 mRNA was increased by ACTH and to a lesser extent by IGF-I, whereas TGFß1 and AngII markedly reduced it. Again, TGFß1 blocked the stimulatory effect of ACTH. The effects of these peptides on 3ßHSD mRNA levels were less marked. Thus, they were increased by two peptides (ACTH>IGF-I), whereas AngII and TGFß1 alone slightly reduced them, but TGFß completely blunted the stimulatory action of ACTH.



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  		Figure 2. Effects of several peptides on ACTHR, StAR, P450c17, and 3ßHSD mRNA levels. Cells were treated as indicated in Fig. 1Go for 36 h, and the mRNA levels were evaluated by Northern blot. The results, expressed as percentage of the response of control cells, are the mean ± SEM of three independent experiments. a, Significantly higher (P < 0.05) than control; b, significantly lower (P < 0.01) than control value. Bottom panel, Representative Northern blot.

 
However, the most pronounced effects, stimulatory or inhibitory, of the four peptides were observed with StAR mRNA levels. As previously reported (14, 39), BAC express two major transcripts of 3 and 1.8 kb and a minor one of 1.3 kb. The two main transcripts were increased by ACTH and IGF-I, whereas AngII and TGFß1 markedly reduced them to very low levels, detectable only after long exposures (data not shown). Interestingly, pretreatment with ACTH plus TGFß1 reduced StAR mRNA to levels similar to those observed after treatment by TGFß1 alone.

Time-course effects of ACTH, TGFß, AngII, and IGF-I on StAR mRNA
As it has been reported that in steroidogenic cells the effects of hormones acting through cAMP on StAR mRNA are transient (7), we studied the time-course effects of the four peptides on this parameter. ACTH induced a rapid and sustained increase in both StAR transcripts, with a zenith at 6 h (Fig. 3Go). Thereafter, StAR mRNA decreased, but at 36 h the level remained higher than that in control cells. AngII also induced a rapid but transient increase, with a zenith at 3 h. Thereafter, the level declined progressively, reaching very low levels (12 ± 4% of controls) at 36 h. IGF-I induced a rapid 2-fold increase within 1.5 h and remained at this level until the end of the experiment. In contrast, TGFß1 had no significant effect on StAR mRNA within 3 h, but progressively reduced it to undetectable levels after 12 h. Moreover, TGFß1 reduced the stimulatory effects of ACTH during the first 6 h and blocked it thereafter.



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  		Figure 3. Time-course effects of several peptides on StAR mRNA levels. Cells were treated for the times indicated without (control) or with ACTH (10-8 M), TGFß1 (10-10 M), ACTH plus TGFß1, AngII (10-7 M), or IGF-I (10-8 M). At the end of each time period, StAR mRNA was measured as described in Materials and Methods. The results, expressed as a percentage of the response of control cells, are the mean ± SEM of three or four independent experiments. a, Significantly higher (P < 0.05) than control value; b, significantly lower (P < 0.01) than control value. Bottom panel, Representative Northern blot.

 
To determine the time-course effects of the four peptides on the other specific mRNAs regulated by the peptides, the blot in Fig. 3Go was rehybridized successively with the ACTHR, P450c17, and 3ßHSD cDNA probes. The results (Fig. 4Go) show several differences by comparison with the effects of the peptides on StAR mRNA. First, the effects on these mRNAs appeared only after 6 h of treatment and were less pronounced, except in the case of ACTH on P450c17. Secondly, the inhibitory effects of TGFß1, either alone or together with ACTH, were less marked than those on StAR mRNA. Thirdly, AngII increased ACTHR mRNA levels and reduced those of P450c17 and, to a lesser extent, those of 3ßHSD.



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  		Figure 4. Effects of ACTH, TGFß1, AngII, and IGF-I on ACTHR, P450c17, and 3ßHSD mRNA. The blot in the lower panel of Fig. 3Go was rehybridized successively with ACTHR, P450c17, and 3BHSD cDNA probes.

 
Effects of ACTH on StAR mRNA and protein on BAC cells pretreated with the various peptides
As StAR appears to be the most precocious gene regulated by the peptides tested, we wondered whether the intensity of the acute steroidogenic response to ACTH at the end of a 36-h pretreatment (Fig. 1Go) might correlate with the levels of StAR mRNA and protein induced by ACTH during the acute stimulation. Therefore, we analyzed StAR mRNA levels at the end of the 36-h pretreatment with several peptides and after the 2-h acute stimulation with ACTH (Fig. 5Go). The results after the 36-h treatment (basal) confirm those presented in Fig. 2Go, i.e. stimulation by IGF-I and ACTH and inhibition by TGFß1, alone or together with ACTH, and AngII. After the 2-h stimulation with ACTH, the StAR mRNA levels of cells pretreated with IGF-I and ACTH were significantly higher than those of control cells, which, in turn, were higher than those of cells pretreated with TGFß1 alone or together with ACTH and AngII.



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  		Figure 5. StAR mRNA levels in either pretreated cells (basal) or pretreated and ACTH-stimulated cells (ACTH). Cells were treated for 36 h as described in Fig. 3Go. The RNA was extracted immediately (basal) or after 2-h treatment with ACTH (10-8 M; ACTH). The results, expressed as a percentage of the values in cells pretreated in control medium (control-basal), are the mean ± SEM of three experiments. a, P < 0.05 compared with basal value in the same group; b, P < 0.05 compared with control basal value; c, significantly higher (P < 0.05) than value in control ACTH-treated cells; d, significantly lower (P < 0.05) than value in control ACTH-treated cells. Bottom panel, Representative Northern blot.

 
The StAR protein levels of cells submitted to the same protocol are presented in Fig. 6Go. At the end of the 36-h treatment, StAR protein levels were higher in cells treated with IGF-I or ACTH than in control cells, whereas the levels in cells treated with TGFß1, TGFß1 and ACTH, and AngII were almost undetectable. After the 2-h stimulation with ACTH, StAR protein levels were significantly higher in cells treated with IGF-I and ACTH than those in controls, which, in turn, were higher than those in cells treated with TGFß1 alone or together with ACTH and AngII.



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  		Figure 6. StAR protein levels in either pretreated cells (basal) or pretreated and ACTH-stimulated cells (ACTH). Cells were treated exactly as described in Fig. 5Go. Then mitochondria were isolated, and StAR protein was determined by Western blot. The results, expressed as percentage of the response of control-basal cells, are the mean ± SEM of three determinations. a, P < 0.05 compared with basal value in the same group; b, P < 0.05 compared with control basal value; c, significantly higher (P < 0.05) than ACTH basal value; d, significantly lower (P < 0.05) than ACTH basal value. Bottom panel, Representative Western blot.

 
Correlation between StAR mRNA and the acute steroidogenic response to ACTH
To investigate whether the steady state StAR mRNA levels correlated with the steroidogenic capacity of BAC, we analyzed, in cells cultured under several conditions, the StAR mRNA levels and the cortisol response to a 2-h ACTH stimulation. The results (Fig. 7Go) demonstrated a high correlation between these two parameters.



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  		Figure 7. Correlation between StAR mRNA levels and ACTH-induced cortisol production. Cells were cultured in the absence (control) or presence of the peptides indicated in Fig. 1Go for 12–36 h. At the end of this pretreatment, some dishes were used to evaluate StAR mRNA levels, whereas others were treated with ACTH (10-8 M), and after 2 h, the cortisol produced was measured. The results for both parameters are expressed as fold stimulation over the control value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results confirm and extend previous work showing that the steroidogenic response to ACTH of adrenocortical cells can be regulated positively by ACTH (11, 17, 18, 19, 21) and IGF-I (27, 28, 46) and negatively by AngII (20, 22, 23, 24, 30, 47) and TGFß (14, 29, 30, 31, 32, 33, 34, 35, 36, 37). Analysis of the effects of these peptides on ACTH responsiveness on BAC and on their capacity to transform several precursors into cortisol indicated that these peptides modified the ACTH response at least at three levels: formation of cAMP and before and after pregnenolone formation. However, the availability of cholesterol to P450scc enzyme appears to be the main factor responsible for the stimulatory action of IGF-I and the inhibitory effects of AngII and TGFß, because none of these peptides modified P450scc activity and the changes were more pronounced on ACTH-induced cortisol production than on the conversion of pregnenolone to cortisol. On the other hand, ACTH pretreatment appears to have comparable effects on the three steps.

The stimulatory effects of ACTH, IGF-I, and AngII on ACTH-induced cAMP production are in agreement with the positive action of these peptides on ACTHR mRNA and binding sites (17, 18, 19, 48, 49). In addition, ACTH and IGF-I, but not AngII, enhance the coupling of ACTHR to adenylate cyclase by increasing Gs{alpha} protein (50, 51), and this might explain why stimulation of ACTH-induced cAMP production is higher with ACTH and IGF-I than with AngII. In contrast, TGFß1 treatment inhibits ACTH-induced cAMP production, and this is in agreement with its inhibitory effects on ACTHR mRNA and binding sites (52). Moreover, TGFß1 blocked the positive effect of ACTH on ACTHR mRNA, binding sites (49, 52), and responsiveness.

Works from many laboratories (reviews in Refs. 2, 21, 53) have shown that ACTH stimulates the gene expression of most steroidogenic enzymes in a time-dependent manner, and this was confirmed by the present results for P450c17 and 3ßHSD. Similarly, in BAC, IGF-I also increases P450c17 and 3ßHSD mRNAs, protein, and activities (Ref. 49 and the present work) as well as P450c21 and P450c11ß mRNAs (54, 55) and activities (27). The effects of AngII on P450c17 appear to be species specific. Thus, in human adrenal cells (11) and in the H295R human tumor cell line (56, 57, 58), AngII stimulates the expression of P450c17. In fetal bovine adrenal cells, AngII alone slightly increased P450c17 mRNA and activity, but inhibited the stimulatory action of ACTH (23). Similarly, in ovine fetal adrenal cells (30) and in ovine and bovine adult adrenal cells (22, 24, 45), AngII alone had limited effects, but inhibited the stimulatory action of ACTH on P450c17. Our results clearly demonstrate that 36 h of AngII treatment significantly decreased P450c17 mRNA and activity. In contrast, TGFß1 in all species studied inhibited the expression of P450c17 (29, 30, 31, 32, 33, 34, 35). However, the changes in the expression of the steroidogenic enzymes that these peptides induce cannot explain the rapid modifications observed in the steroidogenic response to ACTH for at least two reasons. First, the changes in mRNA levels do not appear before 6 h of treatment, whereas the changes in the response to ACTH were observed earlier. Second, the peptide-induced changes in ACTH-induced cortisol production were more pronounced than those observed for the capacity to transform pregnenolone into cortisol. These two observations suggest an alteration of an early step of the steroidogenic pathway.

The first step in steroid hormone biosynthesis is the conversion of cholesterol to pregnenolone. Many studies (reviewed in Refs. 7, 8, 9) show that the rate-limiting step is not due to P450scc activity but, rather, to the transfer of cholesterol from the outer to the inner mitochondria membrane, which in the adrenals and gonads is mainly mediated by the StAR protein. In both adrenals and gonads, StAR mRNA and protein are induced by factors acting through cAMP (7, 59). Our results confirm and extend previous work (14, 15, 16) showing that in ACTH-treated cells StAR mRNA increases within 1.5 h, peaks at 4–6 h, and declines thereafter, but at any time during the time course its levels are higher than those in control cells. Moreover, after 36 h of treatment, StAR protein levels were higher than those in control cells. More important, when ACTH-pretreated cells were acutely stimulated with ACTH for 2 h, the increase in StAR mRNA and protein was significantly higher than that in control cells.

IGF-I caused a 2-fold increase in StAR mRNA levels within 1.5 h, and these levels remained fairly constant for at least 36 h, at which time the StAR protein level was 2-fold higher than that in control cells. Moreover, as in the case of ACTH-treated cells, acute stimulation with ACTH of IGF-I treated cells significantly enhanced StAR mRNA and protein levels compared with those in control cells. These positive effects of IGF-I alone on StAR expression in BAC differ from those observed in rat Leydig cells (60) and porcine granulosa cells (61), in which IGF-I alone had no or very limited effects on StAR mRNA and protein. However, in both cell types, IGF-I potentiated the stimulatory action of hCG and FSH, respectively. It is of interest that despite the fact that ACTH and IGF-I pretreatment for 36 h caused similar effects on StAR mRNA and protein levels, the cortisol response to the acute 2-h ACTH stimulation was higher in ACTH- than IGF-I-pretreated cells. This apparent discrepancy might be due to the fact that ACTH, but not IGF-I, increased P450scc mRNA, protein, and activity (reviewed in Ref. 21), and that the stimulatory action of ACTH on p450c17 and 3ßHSD was higher than that produced by IGF-I.

AngII has been shown to acutely stimulate the StAR protein in bovine zona glomerulosa cells (14) and in the human H295R adrenocortical cell line (16). Our results show, however, that the effects of AngII on StAR mRNA are biphasic; the rapid increase with a peak at 1.5–3 h is followed by a rapid decline to very low levels after 12 h. Moreover, after 36 h of AngII treatment, the levels of StAR mRNA and protein, before and after the 2-h ACTH stimulation, were significantly lower in AngII-treated cells than in control cells. These results suggest that the strong inhibition of StAR expression by AngII may contribute to the homologous and heterologous desensitization induced by this hormone (22, 23, 24, 45, 47). In favor of this hypothesis is the fact that AngII, which has no effect on P450scc activity, has a stronger inhibitory action on ACTH-induced cortisol production than on the conversion of pregnenolone to cortisol. However, the inhibitory effect of AngII on ACTH-induced cortisol production within the first 6 h appears to be StAR independent, as StAR mRNA levels were increased during this period.

TGFß1 treatment of both adrenal cells (29, 30, 31, 32, 33, 34, 35) and Leydig cells (62, 63) has been shown to result in the inhibition of their steroidogenic capacity, which was attributable to a decreased expression of P450c17 in both cell types (29, 31, 33, 35, 63) and to a decreased expression of ACTHR in adrenals (49, 52) and of LHR in Leydig cells (63). However, the fact that the inhibitory action of TGFß1 on the expression of these genes occurred later and was less pronounced than its inhibitory action on ACTH-induced cortisol production suggests that TGFß1 inhibits upstream steps. Recently, it has been shown that TGFß1 alone, in a time-dependent manner, caused a rapid reduction of StAR mRNA and was able to block the stimulatory action of ACTH on StAR mRNA when both peptides were added together (14). Our findings confirm and extend these results. In particular, they show that the low StAR mRNA level was associated with a very low level of StAR protein, and that the ACTH response (StAR mRNA and protein) in TGFß1-treated cells was markedly reduced compared with that in control cells.

Taken together, the present results suggest that regulation of StAR mRNA levels is one of the main mechanisms, but not the only one, by which AngII, TGFß1, IGF-I, and, to a lesser extent, ACTH regulate the steroidogenic responsiveness of BAC to ACTH. In favor of this hypothesis was the significant correlation between the steady state StAR mRNA levels in cells cultured under different conditions and their cortisol production in response to acute ACTH stimulation.

The molecular mechanisms by which the four peptides positively (ACTH and IGF-I) or negatively (AngII and TGFß) regulate StAR expression are largely unknown. The promoters of the mouse (64), rat (65), human (66), porcine (61), and bovine (67) StAR genes do not contain any cAMP response element, but all of them contain several putative binding sites for the steroidogenic factor-1 (SF-1), although their number and localization vary from one species to another. Heterologous transfection of StAR promoter-reporter constructs has shown that some of these SF-1-binding sites are crucial for basal and/or cAMP-dependent expression. More recent studies have shown, however, that the transcriptional activity of SF-1 on the promoters of the StAR (68, 69), LHß, and anti-Mullerian hormone (70) murine genes is potentiated by CCAAT/enhancer-binding protein-ß and a bicoid-related homeobox transcription factor, Ptx1, respectively. Although these cis-elements and trans-activating factors might be involved in the effect of ACTH, their implication in the mechanisms of the three other factors tested is completely unknown.

In conclusion, the present results show that the inhibitory effects of TGFß on adrenal cell steroid hormone biosynthesis and the AngII-induced homologous and heterologous desensitization are mainly due to the negative effects of these peptides on StAR expression, whereas the stimulatory effects of IGF-I on BAC steroidogenic capacity are mainly due to its positive action on StAR expression.


   Acknowledgments
 
We thank Dr. M. Waterman for providing P450c17 cDNA, Drs. F. Labrie and V. Luu-The for 3ßHSD cDNA, and Dr. R. Ivell for bovine StAR cDNA. We also thank J. Bois and M. A. Di Carlo for their secretarial help, Dr. J. Carew for editorial assistance, and M. C. Berthelon for technical assistance.


   Footnotes
 
1 This work was supported by grants from INSERM and University Claude Bernard Lyon 1. Back

2 Supported by a predoctoral fellowship from Ministère de l’Education et de la Recherche et de l’Education National. Back

Received September 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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