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Endocrinology Vol. 147, No. 7 3462-3471
Copyright © 2006 by The Endocrine Society

Inhibins Differentially Antagonize Activin and Bone Morphogenetic Protein Action in a Mouse Adrenocortical Cell Line

Paul G. Farnworth, Peter G. Stanton, Yao Wang, Ruth Escalona, Jock K. Findlay and Guck T. Ooi

Prince Henry’s Institute, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Paul G. Farnworth, Prince Henry’s Institute, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: paul.farnworth{at}princehenrys.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibin, a member of the TGF-ß superfamily, has been proposed to act as an inhibitor of activin and bone morphogenetic protein (BMP) by sequestering their type II receptors in nonsignaling complexes with betaglycan. This mechanism of inhibin action was tested in a mouse adrenocortical (AC) cell line by examining the effects of inhibins A and B on cytochrome P450 17{alpha}-hydroxylase 17,20-lyase (Cyp17) expression and 17{alpha}-hydroxylase activity, measured by progesterone 17{alpha}-hydroxylation, in the absence and presence of activin or BMP isoforms. Cyp17 mRNA endogenously expressed by AC cells was suppressed by activins A and B and BMP-2, -6, and -7, and each ligand accordingly inhibited 17{alpha}-hydroxyprogesterone production (IC50 of 0.24, 0.27, 0.4, 0.51, and 2.2 nM, respectively). Neither inhibin A nor inhibin B alone affected Cyp17 expression or 17{alpha}-hydroxyprogesterone production. Both inhibin A and inhibin B blocked the inhibitory actions of activins A and B in AC cells, supporting the antiactivin model of inhibin action. Inhibin A provided more potent and effective antagonism of both activins than did inhibin B, and activin A was less subject to antagonism by either inhibin than was activin B. In contrast to the major antagonism of activin by both inhibins, only inhibin A antagonized the actions of BMP-2, BMP-6, and BMP-7, whereas inhibin B was ineffective against all tested BMP isoforms except BMP-7 at high concentrations. These results provide limited support for the anti-BMP model of inhibin action and reveal that, relative to inhibin A, inhibin B essentially behaves as a selective activin antagonist in AC cells. In conclusion, inhibins A and B differentially antagonize the actions of activins and BMPs to control adrenocortical C19 steroid production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INHIBINS AND ACTIVINS are members of the TGF-ß superfamily of pleiotropic growth and differentiation factors that also includes bone morphogenetic proteins (BMPs), and which display a wide range of local actions within diverse tissues (1, 2, 3, 4). Activins, BMPs and TGF-ßs generally signal by bringing together distinct combinations of type I and type II serine/threonine kinase receptors (2, 5), which leads to the phosphorylation of specific intracellular receptor-activated Smads (R-Smads). Phosphorylated R-Smads combine with the common signaling Smad4 and other transcription factors to modify gene expression in a ligand-selective manner (1, 2, 5). Two forms of type II receptor, activin receptor type II (ActRII) and ActRIIB, are bound by activins, and BMPs use BMP receptor type II (BMPRII) or activin type II receptors, whereas TGF-ßs exclusively signal via TßRII (2).

Inhibins antagonize activins (and some BMPs) by binding to ActRII and ActRIIB with moderate affinity (6, 7, 8), but such binding does not explain the dominant antiactivin and more recently identified anti-BMP actions of inhibin (9, 10). A model for inhibin action has been deduced, primarily from cell systems made to overexpress the critical binding proteins, in which inhibin promotes the formation of a high-affinity ternary complex involving ActRII/IIB or BMPRII and the inhibin/TGF-ß type III coreceptor, betaglycan (9, 10). Sequestration of these type II receptors in this way is believed to prevent activin and BMP from accessing their respective signaling type I receptors and to account for the functional antagonism of these agonists by inhibin without the need for an independent inhibin signaling pathway. Our previous finding that the antiactivin action of inhibin on pituitary gonadotrophs can be relieved by TGF-ß, which blocks inhibin access to betaglycan on the gonadotroph surface (11), provides additional support for this model, at least for the classical, endocrine action of inhibin.

In addition to its endocrine action, inhibin can have local actions in tissues where it is made, most notably the gonads and adrenal gland (1, 12). We have previously shown that a mouse adrenocortical (AC) cell line expresses the requisite ligands and associated binding/signaling proteins to allow antagonism of locally produced activin and BMP by inhibin to occur (13). In the present study, we tested two hypotheses: first, that the antiactivin and anti-BMP actions of inhibin A observed in cells made to overexpress betaglycan and activin/BMP type II receptors (9, 10) also occur in the physiological context of C19 steroid production by an adrenocortical cell line that binds inhibin A in the same way as normal adrenocortical cells (13), and second, that inhibin B exhibits similar antiactivin and anti-BMP actions to inhibin A, with similar potency. To do this, we first confirmed that the AC cells express Cyp17, the gene that encodes the enzyme P450 17{alpha}-hydroxylase 17,20-lyase, and also display 17{alpha}-hydroxylase activity. We then established that both activin and BMP inhibit expression of Cyp17 and 17{alpha}-hydroxylation of progesterone in AC cells. Finally, we investigated whether inhibin A or inhibin B modifies Cyp17 expression and steroid 17{alpha}-hydroxylation by AC cells when acting alone or in the presence of exogenous activin and BMP. The results support the antiactivin model of inhibin action, with no evidence for an agonist-independent action of inhibin on steroid 17{alpha}-hydroxylation, but provide only limited support for the anti-BMP model of inhibin action. Indeed, inhibin B at low nanomolar concentrations is unable to block BMP action and therefore is a selective activin antagonist in AC cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The AC cell line was derived from the C{alpha}-1 mouse adrenocortical cell line kindly provided by Drs. Ilpo Huhtaniemi and Nafis Rahman (13, 14). Rats were obtained from Central Animal House, Monash University (Clayton, Victoria, Australia). Adult males were maintained under standard conditions, with free access to food and water, whereas 3-d-old rats were killed by cervical dislocation on the day they were obtained. The studies were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, and procedures were approved by the Monash Medical Centre Animal Experimentation Ethics Committee. Recombinant human 31-kDa inhibin A and 25-kDa activin A (obtained in crude form from Biotech Australia, Sydney, Australia) were purified by HPLC and then stored at –70 C in 0.1% trifluoroacetic acid/acetonitrile. Recombinant human 31-kDa inhibin B (obtained in partly purified form from DSL, Webster, TX) was similarly purified and then stored at –70 C in 0.1% trifluoroacetic acid/acetonitrile. Additional inhibin B, ACTH, human chorionic gonadotropin, and all-trans retinoic acid were obtained from Sigma-Aldrich Co. (St. Louis, MO). Additional activin A, activin B, BMP-6, and BMP-7 were obtained from R&D Systems (Minneapolis, MN), BMP-2 was from Research Diagnostics Inc. (Flanders, NJ), TGF-ß1 and -ß2 were from PeproTech Inc. (Rocky Hill, NJ), insulin (Actrapid, 100 IU/ml) was from Novo Nordisk Pharmaceuticals (North Rocks, Australia), IGF-I was obtained from GroPep (Adelaide, Australia), and UltraSpec RNA total isolation reagent was from Fisher Biotec (West Perth, Australia). DNA-free was from Ambion (Austin, TX), and Primer p(dT)15, Expand reverse transcriptase, FastStart DNA Master SYBR Green I, and LightCycler capillaries were obtained from Roche Diagnostics Australia (Castle Hill, Australia). Primers for gene amplification were made commercially (Sigma-Genosys, Castle Hill, Australia). ELISA kits for measuring 17{alpha}-hydroxyprogesterone were obtained from Immuno Biological Laboratories (IBL GmbH, Hamburg, Germany).

Culture and treatment of mouse AC and rat adrenal cells
Adult (60- to 90-d-old) rats were killed, and primary cultures of adrenal cells were prepared by trypsin/deoxyribonuclease digestion of the diced tissue from 20–60 whole glands, as previously described (4, 13). Primary adrenal and AC cells were cultured at 37 C in a humidified atmosphere of 5% CO2 in air using a 1:1 (vol/vol) mixture of DMEM and Ham’s F12 medium (F12) supplemented with 10% fetal bovine serum (FBS), buffered with bicarbonate, and containing antibiotics, nonessential amino acids for MEM, HEPES (2 mM), and supplementary glutamine (2 mM final). Before its use in experiments involving retinoic acid, the serum was stripped of steroids using dextran-coated charcoal (15).

After preincubation of AC and primary adrenal cell cultures for 1 d in medium containing FBS, the medium was changed to a chemically defined serum-free DMEM/F12 medium containing 10% artificial serum (AS) that also included fatty acid-free BSA (60 g/liter; final concentration, 0.6%), transferrin (1.0 mg/liter final), and insulin (0.5 mg/liter final, equivalent to 100 nM), as previously described (13). In the absence of insulin, the condition of the AC cells under phase-contrast microscopy was poor, proliferation was minimal, and many cells became detached from the plate (data not shown). Unless otherwise indicated, AC and rat adrenal cells were routinely cultured with insulin as a component of the chemically defined medium. Treatments or matching vehicles were applied to cells overnight (18 h) under standard culture conditions in this chemically defined medium before the analyses. When single concentrations were used to compare inhibin’s antagonism of various TGF-ß superfamily members, activin and BMP were tested at concentrations that were just maximally active in initial assays (1 or 2 nM), and TGF-ß1, TGF-ß2, and inhibin A were tested at concentrations that were maximally active in other assays in our laboratory (≥0.2, ≥0.4, and ≥1 nM, respectively). Because of its limited availability during most of the study, inhibin B was tested at 1 nM throughout, until improved supplies made concentration-response studies possible.

mRNA purification and real-time PCR analyses
Total RNA extracted from whole dissected adrenal glands, a portion of dissected testis, or cell monolayers (2 x 106 cells/5 ml medium per well in six-well, 3.5-cm-diameter cluster dishes) using UltraSpec RNA reagent was purified using standard procedures. RNA concentration was determined by spectrophotometry at 260 nm, then a 0.5-µg aliquot was fractionated on a 1.7% agarose gel and stained with ethidium bromide to assess rRNA integrity, and the 18S ribosomal band was quantitated by densitometry within the linear response range of the instrument. A 2-µg aliquot of each RNA sample was reverse transcribed with 50 U of Expand reverse transcriptase in a volume of 20 µl using 250 ng oligo-dT primers. Desalted primers for amplification of specific cDNA products were as follows: mouse Cyp17 (forward primer, 5'-TGACCAGTATGTAGGCTTCAGTCG-3'; reverse primer, 5'-TTCTCCGGGATGGCAAACTCTC-3'), product size of 171 bp; mouse TßRII (forward primer, 5'-CGGGTCATCATCAGAAACTGGAA-3'; reverse primer, 5'-TGTAGAGTAGTGAACAAGAGTCAAC-3'), product size of 146 bp.

Real-time PCR amplification assays were performed in a LightCycler (Roche Diagnostics Australia), using denaturation for 10 min to activate the Taq polymerase, followed by 40 cycles of amplification and quantification. Amplification specificity was assessed by consistent melting point analyses and the occurrence of a single product by ethidium bromide-stained agarose gel analysis. The identity of each AC cell product was confirmed by nucleotide sequencing. For quantitative analyses, levels of Cyp17 were normalized against 18S rRNA content.

In addition to reversing morphological changes brought about by the removal of serum from AC cells, insulin increased the AC cell level of Cyp17 mRNA relative to 18S RNA content in a concentration-dependent manner, and replacement of insulin with IGF-I had similar effects (Table 1Go). The level of Cyp17 mRNA in AC cells was subject to regulation by factors (human chorionic gonadotropin, ACTH, and 8-bromo-cAMP, all-trans-retinoic acid) known to regulate its expression in other systems (16, 17) (data not shown).


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  		TABLE 1. Effects of 18 h of treatment with insulin or IGF-I on the relative level of Cyp17 mRNA in mouse AC cells

 
Steroid 17{alpha}-hydroxylation studies
To determine the 17{alpha}-hydroxylase activity of AC cells, near-confluent monolayers of cells (0.25 x 106 cells/0.50 ml per well in 24-well cluster plates) were cultured for 18 h either without treatment or with TGF-ß superfamily members (concentrations given in the text, table, and figure legends) alone or in combination and then the medium was removed and replaced with fresh medium (0.24 ml) containing 10% AS and progesterone (500 nM) (substrate medium). After incubation at 37 C for another 2 h in the absence of the treatments, conditioned medium was collected from each culture, diluted 1:1 with PBS containing 0.1% BSA, and subsequently assayed for 17{alpha}-hydroxyprogesterone content by ELISA according to the manufacturer’s instructions. To correct for the matrix effect of the culture medium in the ELISA, a standard curve for the conditioned medium samples was also prepared by serial dilution of the top standard (20 ng/ml) in substrate medium that had been handled in the same way as the samples. The sensitivity of the assay was 0.03 ng/ml, and cross-reactivities with 17{alpha}-hydroxypregnenolone, progesterone, and 11-deoxycortisol are 1.7, 1.4, and 1.3%, respectively, whereas cross-reactivity with other steroids is less than 0.2%. The within-assay coefficient of variation of the quality control samples was less than 5%, and the between-assay coefficient of variation for the quality control sample (1.24 ng/ml) was 8.9%.

Data analyses
Concentration-response curve analyses by nonlinear regression and statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA). Unless otherwise indicated, mean data from replicate experiments were compared using one-way ANOVA followed by post hoc Neuman-Keuls multiple comparison test. P < 0.05 was considered indicative of a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AC cells express Cyp17 mRNA
The RT-PCR assay for Cyp17 mRNA revealed that AC cells abundantly express this species (Fig. 1Go, lane 4), confirmed by sequencing of the predicted 171-bp product (data not shown). The same sized product was also amplified from total RNA of adult rat testis (Fig. 1Go, lane 5). Low levels of Cyp17 mRNA were detected in adrenal tissue from the 3-d-old male rat (Fig. 1Go, lane 3), and in dispersed cells from the adult male rat adrenal after 5 d in culture with 10% AS (Fig. 1Go, lane 6) but not in adrenal gland tissue freshly obtained from the adult male rat (Fig. 1Go, lane 2). No product was obtained from any of these samples in the absence of reverse transcriptase (Fig. 1Go, lanes 7–11).


Figure 1
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  		FIG. 1. Ethidium bromide-stained 1.7% agarose gel showing the presence of Cyp17 mRNA in mouse AC cells. Molecular weight markers were run in lane 1. +RT and -RT signify amplification yields after the inclusion and omission, respectively, of reverse transcriptase. Cyp17 products (171 bp) were obtained by RT-PCR from total RNA of whole adrenal glands from the adult rat (lanes 2 and 7), 3-d-old male rat (lanes 3 and 8), AC cells that had been cultured in the presence of 10% AS (lanes 4 and 9), rat testis (as positive control; lanes 5 and 10), and primary (1°) cultures of dispersed adrenal cells prepared from the adult male rat and maintained for 5 d in the presence of 10% AS (lanes 6 and 11). No products were detected in any of these samples in the absence of reverse transcriptase (-RT, lanes 7–11).

 
Inhibins lack direct effects on Cyp17 mRNA expression in AC cells
The levels of Cyp17 mRNA in AC cells after overnight incubation in the presence of TGF-ß superfamily members were determined by real-time PCR amplification. To test whether inhibins regulate adrenal Cyp17 independently of activin and BMP, AC cells were treated with inhibin A (2 nM) or inhibin B (1 nM) for 18 h. Inhibin A alone did not significantly alter the expression of Cyp17 (Fig. 2AGo). Direct ELISA measurement of 17{alpha}-hydroxyprogesterone production from progesterone by AC cells confirmed that Cyp17 mRNA was translated into the enzyme P450 17{alpha}-hydroxylase 17,20-lyase. Consistent with the result for Cyp17 mRNA, inhibin A (1 nM) also had no significant effect on 17{alpha}-hydroxylase activity in AC cells (Fig. 2BGo). Inhibin B (1 nM) similarly lacked effect on either the Cyp17 mRNA level (Fig. 2AGo) or 17{alpha}-hydroxylase activity (Fig. 2BGo).


Figure 2
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  		FIG. 2. Effects of treatment with TGF-ß superfamily members, either alone or in combination, on the levels of Cyp17 mRNA expressed and 17{alpha}-hydroxyprogesterone produced by AC cells. AC cells were treated with the indicated concentrations of activin (Act), TGF-ß, and/or bone morphogenetic protein (BMP) isoforms for 18 h in the absence (no added inhibin) or presence of inhibin A or B, and then the content of Cyp17 mRNA relative to 18S RNA was determined by real-time PCR (A), and 17{alpha}-hydroxyprogesterone production from progesterone (initial concentration, 500 nM) during the subsequent 2 h was determined by ELISA (B). Results (percentage of matching untreated control) from at least three replicate experiments are presented as mean ± SEM, except where there is no error bar, which indicates n < 3. a, b, and c, Results that significantly differ from the untreated control group, the matching inhibin-free treatment group, and the matching group receiving inhibin A or B alone, respectively (P < 0.05). In A, inhibin effects against TGF-ß were not determined.

 
Activins, but not TGF-ß, regulate Cyp17 expression by AC cells
Activin A and activin B suppressed the level of Cyp17 mRNA in AC cells (Fig. 3Go), with average IC50 of 0.26 ± 0.03 and 0.46 ± 0.11 nM, respectively (mean ± SEM; n = 4 independent experiments). Both activin A and activin B accordingly inhibited production of 17{alpha}-hydroxyprogesterone in a concentration-dependent manner (closed symbols in Fig. 4Go, A and B, respectively), with average IC50 of 0.24 and 0.27 nM, respectively (Table 2Go). When tested at equimolar concentrations (2 nM) that gave close to the maximal effect, both activin A and activin B significantly suppressed the level of Cyp17 mRNA in AC cells to an average 21–26% of that in untreated cells (Fig. 2AGo), and at 1 nM, both inhibited production of 17{alpha}-hydroxyprogesterone to 22–32% of that in untreated cells (Fig. 2BGo).


Figure 3
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  		FIG. 3. Concentration-dependent suppression of the AC cell Cyp17 mRNA level by activin isoforms and its antagonism by inhibins. AC cell cultures were treated overnight with the indicated concentrations of activin A (A and B) or activin B (C and D) in the absence (control) and presence of inhibin A (Inh A, 2 nM; A and C) or inhibin B (Inh B, 1 nM; B and D). Other details are as described in the legend to Fig. 2AGo. In each panel, data at each point for both curves represent the average result ± SD obtained from replicate amplifications of single samples obtained in one experiment. Similar patterns were obtained in three experiments for each agonist with inhibin A. Sigmoidal curves were fitted to the data using GraphPad Prism software, and the computed IC50 (nM) are given in each panel. Aggregate data from multiple independent experiments with a single concentration (2 nM) of agonist are presented in Fig. 2AGo.

 

Figure 4
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  		FIG. 4. Concentration-dependent suppression of AC cell 17{alpha}-hydroxyprogesterone production by activin isoforms, and its antagonism by inhibin A and inhibin B. AC cells were treated overnight with the indicated concentrations of activin A (A) and activin B (B) in the absence or presence of inhibin A or inhibin B (each 1 nM). Other details are as described in for Fig. 2BGo. Data at each point were obtained from single samples from one experiment and are representative of results from three to four independent experiments. Sigmoidal curves were fitted to the data using GraphPad Prism software, and the computed IC50 (nM are given in each panel. Aggregate data from multiple independent experiments with a single concentration of agonist are presented in Fig. 2BGo, and aggregate IC50 data, including fold changes induced by inhibin treatment, are summarized in Table 2Go.

 

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  		TABLE 2. Comparison of the IC50 for suppression of 17{alpha}-hydroxyprogesterone production by TGF-ß superfamily agonists in AC cells, and fold increases in each IC50 brought about by 1 nM inhibin A and inhibin B

 
TGF-ß isoforms were tested in the same assays because they signal via the same Smad molecules (2, 3, and 4) as activins, they share a binding site on betaglycan with inhibin, and have been shown to modify Cyp17 expression in other cell types. Neither TGF-ß1 nor TGF-ß2 altered the expression of Cyp17 mRNA or, in a single confirmatory experiment, the extent of progesterone 17{alpha}-hydroxylation in AC cells (Fig. 2Go, A and B, respectively). However, measurement of TßRII mRNA using real-time PCR analysis revealed that AC cells express little of this type II receptor (data not shown).

Differential antagonism of activins by inhibins in AC cells
To test the antiactivin model of inhibin action, AC cells were treated with activin isoforms in the absence or presence of inhibin A or inhibin B. At set concentrations, each inhibin significantly blocked the suppression of Cyp17 mRNA by activins A and B (Fig. 2AGo) and reduced, but did not abolish, the inhibitory effects of activins on 17{alpha}-hydroxyprogesterone production (Fig. 2BGo). In each case, the effect of inhibin A exceeded that of inhibin B. Indeed, inhibin B antagonism of the effects of activin A and activin B (each 1 nM) on progesterone hydroxylation was not statistically significant (Fig. 2BGo).

The concentration-response curve for the suppression of Cyp17 mRNA obtained with activin A alone was shifted to the right in a parallel fashion in the presence of inhibin A (2 nM), with the IC50 increased 3.2 ± 0.5-fold (mean ± SEM; n = 3) and the maximum effect unchanged (Fig. 3AGo; additional data not shown). In a single experiment, inhibin B antagonism of activin A showed similar characteristics, with a 2-fold increase in IC50 and little change in maximum effect (Fig. 3BGo). The AC cell response to inhibin A was amplified at the level of progesterone hydroxylation. Inhibin A (1 nM) significantly increased the activin A IC50 (reduced its potency) for suppression of 17{alpha}-hydroxyprogesterone production in three experiments by an average 9-fold (Table 2Go), with little change in the maximum level of inhibition [26 ± 5% of the level in untreated cultures with activin alone and 24 ± 3% of that level in the presence of inhibin A (mean ± SEM; n = 4 in each case); e.g. Fig. 4AGo]. Inhibin B also significantly reduced the potency of activin A but only by 2.6-fold (Table 2Go). In contrast to inhibin A, inhibin B significantly reduced the maximum effect of activin A to 47 ± 3% of the level in untreated cultures (mean ± SEM; n = 3; P < 0.001; cf. both control and inhibin A groups; e.g. Fig. 4AGo).

Activin B maximally suppressed the level of Cyp17 mRNA to less than 50% of control in the absence of inhibin A but much less (to 70–85% of control) in its presence, with no increase in the activin B IC50 (Fig. 3CGo). Inhibin B antagonism of activin B likewise could not be surmounted by increasing the agonist concentration, and the activin B IC50 was little changed in the presence of inhibin B (1 nM) (Fig. 3DGo). However, at the level of 17{alpha}-hydroxylase activity, inhibin A and inhibin B (each 1 nM) increased the activin B IC50 by an average 21-fold (P = 0.013) and 2.6-fold (P = 0.077), respectively (Table 2Go), with little change in the respective maximum effects [activin B alone, 19 ± 6% of the level in untreated cultures; with inhibin A, 23 ± 5%; with inhibin B, 25 ± 5% (mean ± SEM; n = 5 in each case); e.g. Fig. 4BGo].

Inhibin A readily abolished the inhibition of 17{alpha}-hydroxyprogesterone production by a constant concentration of activin B (inhibin A EC50 < 0.01 and 0.028 nM when activin B was 0.25 and 1 nM, respectively) but only partly prevented the suppression by activin A, even at concentrations above 1 nM (Fig. 5AGo) (inhibin A EC50 of 0.032 and 0.50 nM when activin A was 0.25 and 1 nM, respectively). Inhibin B at concentrations up to 3 nM was a poor antagonist of the inhibitory action of activin A (0.25 nM, Fig. 5BGo; 2 nM, Fig. 2BGo) but completely blocked the suppression of 17{alpha}-hydroxyprogesterone production by 0.25 nM activin B (Fig. 5BGo; inhibin B EC50 of 0.01 nM). Nevertheless, inhibin B at 1 nM poorly blocked the action of higher concentrations of activin B (e.g. Fig. 2BGo).


Figure 5
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  		FIG. 5. Activin suppression of AC cell 17{alpha}-hydroxyprogesterone production and its concentration-dependent antagonism by inhibin A and inhibin B. AC cells were treated overnight with ({blacktriangleup} and {triangledown}) or without ({circ}) activin A (Act A, {blacktriangleup}) or activin B (Act B, {triangledown}) (each 0.25 nM), in the absence or presence of graded concentrations of inhibin A (panel A) or inhibin B (panel B). Other details are as described for Fig. 2BGo. Data at each point were obtained from single samples from one experiment and are consistent with results from two to three independent experiments. Sigmoidal curves were fitted to the data using GraphPad Prism software. Aggregate data from multiple independent experiments with a single concentration of agonist are presented in Fig. 2BGo.

 
In summary, both inhibins antagonized activin suppression of Cyp17 mRNA and 17{alpha}-hydroxylase activity, but inhibin A was more effective than inhibin B in each respect, and activin B was more susceptible to antagonism than activin A in most circumstances. The interaction between inhibin A and activin A almost always showed competitive characteristics (decreased potency, little change in maximum effect). The patterns for inhibin antagonism of activin B at the level of Cyp17 mRNA, and inhibin B antagonism of activin A at the level of 17{alpha}-hydroxylase activity, showed complex, partly noncompetitive kinetics (decreased maximum effect, little change in potency) that require additional investigation.

BMP isoforms regulate Cyp17 expression by AC cells
BMP-2, -6, and -7 (each 2 nM) significantly suppressed the level of Cyp17 mRNA in AC cells to minima between 7 and 16% of the level in untreated cells (Fig. 2AGo). The actions of BMP-2, -6, and -7 were concentration dependent, with IC50 of 0.25 ± 0.05, 0.57 ± 0.06, and 0.60 ± 0.13 nM, respectively (mean ± SEM; n = 3–4) (e.g. Fig. 6Go). All tested BMPs also suppressed 17{alpha}-hydroxyprogesterone production in a concentration-dependent manner (Fig. 7Go). BMP-7 had a significantly (4- to 5-fold) higher IC50 than the activins and other BMPs (Table 2Go).


Figure 6
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  		FIG. 6. Concentration-dependent effects of BMP isoforms on AC cell Cyp17 mRNA levels in the absence and presence of inhibin or activin. AC cell cultures were treated overnight with the indicated concentrations of BMP-2 (A and C) or BMP-7 (B) in the absence (control, open symbols) and presence (closed symbols) of inhibin A (2 nM in A, 1 nM in B) or activin B (1 nM in C). Other details are as described for Fig. 2AGo. Aggregate data from multiple independent experiments with a single concentration (2 nM) of each agonist are presented in Fig. 2AGo.

 

Figure 7
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  		FIG. 7. Concentration-dependent suppression of AC cell 17{alpha}-hydroxyprogesterone production by BMP isoforms and its antagonism by inhibin A and inhibin B. AC cells were treated overnight with graded concentrations of BMP-2 (A), BMP-6 (B), or BMP-7 (C) in the absence (bullet) or presence of 1 nM inhibin A ({blacktriangleup}) or inhibin B ({square}). Other details are as described for Fig. 2BGo. Aggregate data from multiple independent experiments with a single concentration of agonist are presented in Fig. 2BGo, and aggregate IC50 data, including fold changes induced by inhibin treatment, are summarized in Table 2Go.

 
When compared on an equimolar basis, BMPs suppressed Cyp17 mRNA at least as effectively as activins (Fig. 2AGo). To gain some insight into the relationship between activin and BMP actions on AC cells, the suppression of Cyp17 mRNA by BMP-2 was examined in the absence and presence of a partly inhibiting concentration of activin B (1.0 nM). The BMP-2 IC50 was essentially unchanged by the presence of activin B (Fig. 6CGo), providing no evidence for either synergism or antagonism.

Differential antagonism of BMPs by inhibins in AC cells
When the anti-BMP model of inhibin action was tested, inhibin A and inhibin B poorly blocked the inhibitory actions of BMP-2 and -6 (each 2 nM) on the Cyp17 mRNA level in AC cells (Fig. 2AGo). The concentration-response curve for BMP-2 showed only a small increase in the IC50 with little change in the maximum effect in the presence of inhibin A (2 nM) (Fig. 6AGo).

Despite its insignificant effects against BMP-2 and -6 at the level of Cyp17 mRNA, inhibin A (1 nM) significantly blocked their suppression of 17{alpha}-hydroxyprogesterone production (Fig. 2BGo) and increased the IC50 for this effect of BMP-2 and -6 by averages of 10- and 6-fold, respectively (Table 2Go) without reducing their maximum effects (e.g. Fig. 7Go, A and B, respectively). On the other hand, inhibin B (1 nM) did not overcome the suppression of 17{alpha}-hydroxyprogesterone production by BMP-2 and BMP-6 at 2 nM (Fig. 2BGo) and had only minor effects on their concentration-response curves for inhibition of 17{alpha}-hydroxylase activity, at most doubling the IC50 (Table 2Go) without reducing the maximum effects (Fig. 7Go, A and B, respectively).

Inhibin interactions with BMP-7 differed in some respects from those with the other tested BMPs. For example, inhibin A significantly blocked the suppression of both Cyp17 mRNA and 17{alpha}-hydroxyprogesterone production by BMP-7 (2 nM), although inhibin B was again ineffective under the given conditions (Fig. 2Go, A and B). Inhibin A (1 nM) also altered the BMP-7 concentration-response curve for the suppression of Cyp17 mRNA, increasing the IC50 and reducing the maximum suppression by BMP-7 (Fig. 6BGo). Inhibin A increased the BMP-7 IC50 for suppression of 17{alpha}-hydroxyprogesterone production by 4-fold (Table 2Go), while reducing the maximum effect from an average of 95% inhibition to 80% inhibition (e.g. Fig. 7CGo). Despite its minor effects under the other assay conditions, inhibin B increased the BMP-7 IC50 for suppression of 17{alpha}-hydroxyprogesterone production by 2.3-fold (Table 2Go) and also greatly decreased the maximum effect to only 53% inhibition (e.g. Fig. 7CGo). Thus, inhibin B antagonism of BMP-7 became evident only at concentrations of this agonist above 2 nM.

In the presence of a constant inhibitory concentration (2 nM) of BMP, inhibin A progressively blocked the inhibition of 17{alpha}-hydroxyprogesterone production by BMP-7 (inhibin A EC50 of 0.10 nM) (Fig. 8AGo) but only partly prevented the suppression by BMP-6, even at concentrations above 1 nM (inhibin A EC50 of 0.12 nM) (Fig. 8AGo). Partial antagonism of BMP-2 action by inhibin A (1 nM) was also evident (Fig. 8AGo). In contrast, inhibin B at concentrations up to 3 nM did not antagonize the inhibitory action of BMP-7 (2 nM) (Fig. 8BGo).


Figure 8
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  		FIG. 8. Suppression of AC cell 17{alpha}-hydroxyprogesterone production by BMP isoforms and its concentration-dependent antagonism by inhibin A and inhibin B. AC cells were treated overnight with graded concentrations of inhibin A (panel A) or inhibin B (panel B) in the absence ({circ}) or presence of BMP-2 ({triangledown}), BMP-6 ({diamond}), or BMP-7 ({blacksquare}) (each 2 nM). Other details are as described for Fig. 2BGo. Data at each point were obtained from single samples from one experiment and are consistent with results from two to three independent experiments. Sigmoidal curves were fitted to the data using GraphPad Prism software except in the case of data for BMP-2. Aggregate data from multiple independent experiments with a single concentration of agonist are presented in Fig. 2BGo.

 
In summary, both inhibins were less effective antagonists of BMPs than of activins. As with activins, inhibin A was the more effective antagonist of BMPs, to the extent that inhibin B antagonism of BMP-2 and -6 was minimal. BMP-7 was more susceptible than BMP-2 and -6 to antagonism by inhibins in all circumstances. The interactions between inhibin A and BMP-2 and -6 showed competitive characteristics, but more complex kinetics were evident for inhibin A antagonism of BMP-7 at the level of Cyp17 mRNA and for inhibin B antagonism of BMP-7 at the level of 17{alpha}-hydroxylase activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies show that activins and BMPs suppress Cyp17 mRNA expression by AC cells with similar potencies and maximum effects, with concomitant reduction in the production of 17{alpha}-hydroxyprogesterone. Inhibins A and B alone lack effects on Cyp17 mRNA expression and 17{alpha}-hydroxylase activity in AC cells. Inhibin A and inhibin B nevertheless block activin suppression of both parameters, with inhibin A the more potent and effective isoform. Only inhibin A significantly blocks the suppression of Cyp17 mRNA expression by BMP-7 and the suppression of 17{alpha}-hydroxylase activity by BMP-2, -6, and -7, when BMP concentrations are at or less than 2 nM. Inhibin B antagonism of BMP is only evident with BMP-7 at concentrations above 2 nM. In contrast, TGF-ß lacks effect in the AC cell system. This study therefore reveals a hierarchy of interactions between activins and BMPs as agonists, and inhibins as their antagonists, to control adrenal C19 steroid production.

The AC cell model
Adult mouse adrenocortical cells normally do not display steroid 17{alpha}-hydroxylation (18, 19), but the C{alpha}-1 cell line from which the AC cells are derived produces estradiol, indicative of expression of the Cyp17 gene and production of C19 androgen precursors (20), and these characteristics were confirmed in the present study. In these respects, this cell line emulates cells from the fetal X zone of the mouse adrenal cortex that normally expresses Cyp17 and produces C19 steroids in the period before birth (21, 22). The AC cell requirement for insulin (or IGF-I) stimulation is consistent with the stimulation of fetal adrenal cell growth and proliferation (23, 24) and of Cyp17 expression in human and bovine adrenocortical cells and other steroidogenic cell types by these trophic factors (24, 25, 26, 27).

Cyp17 expression in AC cells is inhibited by isoforms of activin and BMP. We previously showed that AC cells express the requisite type I and II receptors and signaling molecules for activin and BMP (13), and the present data establish that both signaling pathways are functional. In contrast, TGF-ß was unable to suppress Cyp17 mRNA in AC cells, but this presumably reflects their deficient expression of the type II receptor for TGF-ß. AC cells also express mRNA encoding many members of the TGF-ß superfamily, including activins A, B, and AB and several BMP/growth differentiation factor species (13). The current studies indicate that these locally produced TGF-ß superfamily members can provide autocrine inhibition of adrenocortical steroid 17{alpha}-hydroxylation. The AC cells can therefore be regarded as a good model for testing the physiological influences of inhibins, activins, and BMPs on steroidogenesis.

AC cell responses to activin, TGF-ß, and BMP
The responses of AC cells to activins A and B and BMP-2, -6, and -7 resemble those described for responses to representative activin and BMP agonists by some steroidogenic cells and tissues but not others. Thus, activin A and BMP-4 selectively suppress Cyp17 expression and action in human ovarian theca-like tumor cells (28, 29), activin A inhibits androgen production by human or rat ovarian theca-interstitial and rat testicular primary cell cultures (12, 30), and BMP-4, -6, and -7 suppress Cyp17 expression and androgen production by bovine theca interna cells (31), consistent with the present results. In contrast, Cyp17 expression by human H295R adrenocortical cells and juvenile porcine Leydig cells is stimulated by activin A (32, 33). Moreover, activin A enhances ACTH-stimulated cortisol secretion in human adrenal fetal zone cells, although it lacks effects in cells of the definitive zone and the adult adrenal cortex (34). The various phenotypes observed for activin effects on Cyp17 expression in these different contexts are not easily explained but presumably reflect critical differences in the prevailing culture conditions and the ligands, receptors, and/or intracellular signaling cofactors expressed in each circumstance. It may be significant that cell types in which Cyp17 expression was not inhibited by activin were treated in the presence of 6–10 µg insulin/ml (32, 33), concentrations that are 12- to 20-fold higher than that used in the current studies (0.5 µg/ml). Our preliminary insulin concentration-response data (Table 1Go) show that the curve is bell shaped, with insulin concentrations above 0.05 µg/ml providing suboptimal stimulation of Cyp17.

The similarity in action of activin and BMP to suppress AC cell Cyp17 expression is intriguing: mutual antagonism by BMP and activin is the more common outcome in other situations, reflecting competition from the respective R-Smads (Smad1, 5, or 8 for BMPs; Smad2 or 3 for activins) for the finite pool of Smad4 (35) or competition for available activin/BMP-cross-reacting type II receptors, particularly ActRII (5, 36). Parallel suppression of Cyp17 expression by activin and BMP has also been observed in human ovarian theca-like tumor cells (28, 29), and TGF-ß acts similarly in that cell type (37), raising the possibility of a common action shared by all three families of ligands in some circumstances. Other shared actions of activins and BMPs, including enhancement of mRNA levels for steroidogenic acute regulatory protein and P450 side-chain cleavage enzyme, were recently identified in H295R cells (38). The signaling pathways used by activin and BMP to elicit common actions are yet to be determined. However, activin B neither synergized with nor antagonized BMP-2 when both were acting on AC cells, which suggests they act independently but additively to regulate Cyp17.

Lack of an independent action of inhibin
Recent findings that inhibin A stimulation of Cyp17 expression parallels the action of activin A in the juvenile porcine Leydig cell (33) and adrenocortical H295R cell (32) suggested that inhibin A regulates Cyp17 independently of activin under some circumstances. An anti-BMP action of inhibin can be invoked to resolve such anomalies but only where activin and BMP have opposite effects (10), which was not the case in AC cells. AC cells, like rat adrenocortical cells, bind inhibin A with high affinity via multiple proteins (39). However, this binding involves not only betaglycan and proteins consistent in size with ActRII/IIB and BMPRII but also several other proteins not accounted for by the current model of inhibin action (13). Thus, AC cells provided an opportunity to determine whether inhibin-binding proteins expressed by AC cells, but not required in the current model of inhibin action, subserve inhibin actions independently of activin and/or BMP receptor agonists. However, there was no evidence for an independent action of inhibin on the measured endpoints in this model, consistent with studies showing that inhibin A by itself does not influence steroidogenesis in cells from the human fetal and adult adrenal gland (24, 34). Whether the additional inhibin-binding proteins subserve other, as yet unidentified, adrenal action(s) of inhibin, or play a part in determining the observed selectivity of inhibin B antagonist actions in AC cells, remains to be determined.

Antiactivin action of inhibin
Both inhibin A and inhibin B block the suppression of Cyp17 mRNA expression by activins A and B in AC cells and concomitantly reduce activin inhibition of steroid 17{alpha}-hydroxylation. These findings are consistent with several previous findings for inhibin A in nonadrenal steroidogenic tissues, including testis cell cultures derived from hypophysectomized rats (12), purified Leydig cells from intact adult rats (40), and ovarian thecal cells (30). Thus, the adrenocortical AC cells display an antiactivin mode of inhibin action (9) that is shared by steroidogenic cells from the testis and ovary. Inhibin A stimulation of Cyp17 mRNA expression is a consistent finding across many studies, even in juvenile porcine Leydig and adrenocortical H295R cells under circumstances where activins do not suppress Cyp17 mRNA (32, 33). The high affinity of adrenal binding sites for inhibin A (13) and secretion of inhibins by AC cells, normal adrenocortical cells, adrenocortical tumors, and human adrenocortical H295R cells (13, 32, 38, 41) together suggest that a dynamic equilibrium might occur between locally produced or endocrine inhibins on the one hand and locally produced activins on the other to control C19 steroid production by adrenocortical cells.

Activin A suppresses the level of Cyp17 mRNA in AC cells with greater potency than activin B (comparison of average IC50, P < 0.05, two-tailed t test). More significantly, each activin displays a distinct pattern of interaction with inhibins, with activin B more readily antagonized than activin A and inhibin A providing more effective antagonism of each activin than inhibin B. Higher concentrations of activin A can overcome antagonism of Cyp17 suppression by either inhibin A or inhibin B in a competitive-like manner, but inhibin antagonism of activin B is noncompetitive. The different patterns of interaction may reflect differences in the affinities of activin A, activin B, inhibin A, and inhibin B for type II activin receptors and/or the selective action of activin B to recruit and activate activin receptor-like kinase 7 (42), which is expressed by AC cells (13).

Anti-BMP action of inhibin
The present results for antagonism of BMP action by inhibins qualitatively agree with, but quantitatively differ from, results obtained in an earlier study in which inhibin A virtually abolished the actions of BMP-2, -7, or -9 and growth differentiation factor 5 (10). BMP down-regulation of Cyp17 mRNA in AC cells is in most cases poorly relieved by inhibin A and not at all by inhibin B. Overall, our results suggest that the antiactivin and anti-BMP actions of inhibins differ considerably in a physiological context. We previously found that BMPs, but not activins, compete for part of inhibin A binding to AC cells (13), and others have shown regulation of betaglycan expression by BMP-2 (43). These provide possible mechanisms by which BMPs could counteract inhibin binding and action and could partly explain some of the complex kinetics observed for some ligands in the concentration-response studies. Autocrine stimulation of the AC cells by endogenously expressed TGF-ß superfamily members (13) might also contribute, although the serum-free conditions were chosen to minimize such complications.

Conclusions
We conclude from these studies that both inhibin A and inhibin B can relieve the suppression of insulin-stimulated adrenal glucocorticoid and androgen precursor production by activins A and B, and inhibin A partly blocks the same actions of BMP-2, -6, and -7, through the counterregulation of Cyp17. The effects of inhibins, activins, and BMPs on Cyp17 expression are reflected in parallel changes in the activity of 17{alpha}-hydroxylase responsible for C19 steroid production by AC cells. This spectrum of interactions between activins/BMPs and inhibins is mediated by physiological levels of their respective endogenous binding and signaling proteins and therefore more closely represents the pattern of responses that occur in steroid-producing cells, including adrenocortical cells, that make androgen and glucocorticoid precursors under various (patho)physiological circumstances. It is notable that human adrenal cells from Cushing’s adenomas and virilizing carcinomas secrete activin A and both inhibins (41) and contain progressively more inhibin {alpha}-immunopositive cells in comparison with normal adrenocortical cells, consistent with a specific role for inhibins in promoting adrenal androgen production (44). Inhibin counterregulation of Cyp17 may have broader pathophysiological significance, one example being the hyperandrogenemia associated with polycystic ovary syndrome (PCOS) (45). Inhibin mRNA and protein levels in arrested PCOS follicles are lower than in similar-sized follicles of normal women (46, 47), which, in the context of the present findings, should result in less androgen precursor generation in such follicles. However, the adrenal gland appears to be a major source of circulating androgens in women (48), and the present results reveal that inhibins could play a significant role in the PCOS adrenal gland. The adrenal cortex also expresses inhibins (13) and has binding sites of sufficiently high affinity to respond to both autocrine and endocrine inhibin signals (13). It therefore seems that inhibins, whether locally produced or originating elsewhere, differentially integrate the inputs from multiple members of the TGF-ß superfamily to control steroidogenesis. The roles of other high-affinity inhibin A-binding proteins expressed by adrenal cells, but not required for mediating the antiactivin and anti-BMP actions of inhibin, are yet to be determined.


   Acknowledgments
 
We acknowledge initial, fruitful discussions with Dr. Teresa Woodruff, critical review of the manuscript by Dr. Craig Harrison and Prof. David Robertson, and the gift of inhibin A from Biotech Australia (Sydney, Australia). Thanks also to Prof. Ilpo Huhtaniemi and Dr. Nafis Rahman (University of Turku, Finland) for providing the C{alpha}-1 cells.


   Footnotes
 
This work was supported by Program Grants (Reg Key Nos. 983212 and 241000) and a Fellowship for J.K.F. (Reg Key No. 198705) from the National Health and Medical Research Council of Australia.

Present address for G.T.O.: Sun Biomedical Technologies, Ridgecrest, California 93555.

Disclosure: None of the authors has any potential conflicts of interest to declare.

First Published Online April 6, 2006

Abbreviations: ActRII, Activin receptor type II; AS, artificial serum; BMP, bone morphogenetic protein; BMPRII, BMP receptor type II; FBS, fetal bovine serum; PCOS, polycystic ovary syndrome; R-Smads, receptor-activated Smads.

Received January 6, 2006.

Accepted for publication March 24, 2006.


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