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Department of Pediatrics (T.V., T.K., R.V.), Kuopio University and University Hospital, Kuopio FIN-70211, Finland; and Department of Pathology (J.L., R.V.), Haartman-Institute, University of Helsinki, Helsinki FIN-00014, Finland
Address all correspondence and requests for reprints to: Raimo Voutilainen, M.D., Department of Pediatrics, Kuopio University Hospital, P.O. Box 1777, FIN-70211 Kuopio, Finland. E-mail: . raimo.voutilainen{at}uku.fi
Abstract
Activins and inhibins are glycoprotein hormones produced mainly in gonads but also in other organs. They are believed to be important para/autocrine regulators of various cell functions. We investigated activin/inhibin receptor and binding protein gene expression and the regulation of activin/inhibin secretion in human adrenal cells. RT-PCR revealed inhibin/activin 
-, ßA/B-subunit, follistatin, activin type I/II receptor, and inhibin receptor (betaglycan and inhibin-binding protein) mRNA expression in fetal and adult adrenals and cultured adrenocortical cells. Cultured cells secreted activin A and inhibin A/B as determined by specific ELISAs. ACTH stimulated inhibin A/B secretion in fetal (1.8- and 1.8-fold of control, respectively) and in adult cells (3.4- and 1.7-fold of control, respectively) without significant effect on activin A. 8-bromoadenosine cAMP (protein kinase A activator) increased activin A and inhibin A/B secretion in the human adrenocortical NCI-H295R cell line (32-, 17-, and 3-fold of control, respectively). 12-O-tetradecanoyl phorbol-13-acetate (protein kinase C activator) stimulated both activin A and inhibin A secretion (764- and 32-fold of control, respectively), and activin treatment increased inhibin B secretion in these cells (25-fold of control). In conclusion, human adrenocortical cells produce dimeric activins and inhibins. ACTH stimulates inhibin secretion and decreases activin/inhibin secretion ratio, probably via the protein kinase A signal transduction pathway. This, together with the adrenocortical activin/ inhibin receptor and binding protein expression, suggests a physiological role for activins and inhibins in the human adrenal gland.
ACTIVINS AND INHIBINS are dimeric glycoproteins formed by two of three different subunits (, ßA, and ßB). Activins are dimers of ß-subunits (ßA:ßA, ßA:ßB, and ßB:ßB; activin A, activin AB, and activin B, respectively). Inhibins consist of either of the ß-subunits dimerized with a common -subunit (:ßA and :ßB; inhibin A and inhibin B, respectively). Activins are produced in many cell types and organs, whereas inhibin production is restricted mainly to the steroidogenic tissues and the hypophysis. Both activins and inhibins have been suggested to be para/autocrine regulators of cell function (1, 2, 3).
It is not known whether human adrenocortical cells secrete biologically active dimeric activins or inhibins. This is, however, likely because all three activin/inhibin subunit mRNA species and peptides are expressed in the human fetal and adult adrenal cortex (4, 5, 6, 7, 8). The mRNA (9) and peptide (10) for the activin binding protein follistatin are expressed in the fetal adrenal cortex. Human adult adrenal cells have also been shown to secrete inhibin-like immunoreactive material both in vitro and in vivo (11, 12).
The expression of the specific activin/inhibin receptors in the human adrenal gland has not been described previously. However, the presence of adrenal activin/inhibin receptors was suggested on the basis of specific activin/inhibin binding on rat adrenals (13), inhibin binding on cultured rat adrenal cells (14), and responsiveness of human fetal adrenal cells to activin treatment (5). Two different cell-membrane activin receptors have been identified (types I and II, further divided into subtypes I and IB, II and IIB). Both types I and II are needed on the cell surface to mediate activin signaling. Less is known about the inhibin signaling system. Two membrane proteins specifically binding inhibin, inhibin-binding protein (InhBP, previously called p120) and betaglycan, have been identified and suggested to be the inhibin receptors (15, 16).
It has been suggested that activins could be important para/autocrine regulators of human fetal adrenal function, whereas the role of inhibins remains more hypothetical. In cultured fetal adrenal cells, activin A decreased cell proliferation, stimulated ACTH-induced cortisol secretion, and induced apoptosis, whereas inhibin had no effect (5, 17, 18). Nevertheless, there is indirect evidence that inhibins might regulate steroidogenesis because the inhibin -subunit peptide is highly expressed in normal and neoplastic androgen-producing adrenocortical cells (7). It has also been shown that Cushing’s adenomas secrete more inhibin-like immunoreactive material into circulation than normal adrenals do (12).
Despite previous studies reporting the expression of activin/inhibin subunit mRNAs and peptides in the adrenocortical cells, the production of biologically active dimeric peptides and the activin/inhibin receptor gene expression have not been reported previously. In this study, we investigated whether adrenocortical cells produce immunoreactive activin A and inhibins A/B, and how this production is regulated by ACTH, modulators of PKA and PKC, and recombinant human activin A. To clarify further the biological function of the locally produced activins and inhibins in adrenocortical cells, the mRNA expression of all known activin/inhibin receptor components and binding proteins was investigated. Human fetal and adult adrenal cells and the human adrenocortical cell line NCI-H295R (19, 20, 21) were used as material.
Materials and Methods
Ethical considerations
The study was approved by the Research Ethics Committees of the Kuopio and Helsinki University Hospitals, and the patients gave informed written consent.
Tissue material and cell cultures
Human fetal adrenals were obtained from legal abortions performed for social or medical reasons. The gestational ages varied from 13 to 20 wk, as estimated from fetal foot lengths. Adult adrenals were obtained during surgery for adrenal or renal tumors. Fetal and adult adrenal cells were cultured as described previously (4). In brief, the fetal adrenals were first decapsulated and the adult adrenal cortical tissues separated from the surrounding adipose tissue and adrenal medulla. After dissection, the adrenals were minced and dispersed enzymatically with collagenase-dispase (Roche Molecular Biochemicals, Mannheim, Germany) and Dnase (Sigma, St. Louis, MO). The cells intended for cultures were plated on 40-mm plastic culture dishes at a density of 3 x 105 cells/dish. The medium was Ham’s F-10 medium with 10% fetal calf serum, penicillin (125 IU/ml), streptomycin sulfate (125 µg/ml), and glutamine (2 mM). NCI-H295R human adrenocortical cell line was obtained from American Type Culture Collection (Manassas, VA). The cells were plated with a density of 1 x 106 cells/well on 35-mm plastic culture dishes (Nunc, Roskilde, Denmark). The medium was DMEM-F12 containing 2% Ultroser (Life Technologies, Inc., Paisley, Scotland), ITS+1 liquid media supplement (Sigma), penicillin (100 IU/ml), streptomycin sulfate (100 µg/ml), and glutamine (0.5 mM). Both primary and NCI-H295R cell cultures were maintained at 37 C in a 95% air/5% CO2 humidified environment, and the culture media were changed every 2–3 d. The viability of the cells was assessed by phase contrast light microscopy, cortisol secretion, and trypan blue exclusion test.
Hormonal and other treatments
All experimental procedures were performed when the cell cultures had reached subconfluency. At that stage primary adrenocortical cell cultures are the most responsive to ACTH (4). The cultured cells were incubated for 24 or 48 h with or without ACTH (1–24) (S-Cortrophin; Organon, Oss, Holland), 8-bromoadenosine cAMP (8-BrcAMP) (Sigma), 12-O-tetradecanoyl phorbol-13-acetate (TPA, Sigma), or recombinant human activin A peptide (R&D Systems, Minneapolis, MN). These reagents caused morphological changes typical for each reagent. In time course experiments, 250 µl culture media were removed for analysis from every dish at 12-h intervals. After the experiments the culture media were collected and stored at -70 C.
RNA preparation
Total RNA was extracted from cultured cells using TriZol reagent (Life Technologies, Inc., Rockville, MD) according to the manufacturer’s protocol and from the in vivo tissues by ultracentrifugation through a cesium chloride cushion. Trace amounts of genomic DNA were removed by Dnase treatment. Twenty micrograms of total RNA were incubated with 10 U RQ1 Rnase-Free Dnase (Promega Corp., Madison, WI), 5 µl 10-fold reaction buffer (Promega Corp.), and 25 U human placental Rnase inhibitor (Amersham Pharmacia Biotech, Piscataway, NJ) in a 50-µl reaction volume at 37 C for 30 min. One hundred microliters of Rnase-free water were added to the reaction mixture before extraction with equal volumes of phenol and chlorophorm:isoamyl alcohol (49:1). RNA was precipitated from aqueous phase overnight with equal volume of isopropanol, washed once with 1 ml 75% ethanol, air dried, and dissolved in Rnase-free water.
RT-PCR
Two micrograms of Dnase-treated total RNA were used for cDNA synthesis. The RNA and 40 pmol oligo(dT)20 primer (Amersham) were first incubated in a 10-µl volume at 70 C for 10 min. The four deoxynucleotide triphosphates (final concentration, 2 mM for each, Amersham), 20 U avian myeloblastosis virus reverse transcriptase (Finnzymes, Espoo, Finland), and 2 µl 10-fold concentrated reverse transcription-reaction buffer (Finnzymes) were added to the RNA-primer mixture, and the reaction volume was adjusted to 20 µl with Rnase-free water. The reaction mixture was incubated at 42 C for 40 min. Control reactions were made in a similar fashion without reverse transcriptase. The amplification primers used for the PCR of human inhibin/activin subunits, activin receptors, betaglycan, InhBP and follistatin and the expected lengths of the PCR products are shown in Table 1
. PCR was performed by combining 2 µl reverse transcription mixture with: 1) 14.7 µl water; 2) 2 µl 10-fold concentrated GeneAmp PCR buffer (Roche Molecular Systems, Branchburg, NJ); 3) 0.4 µl deoxynucleotide triphosphate mixture (0.2 mM final concentration for each deoxynucleotide, Amersham); 4) 0.4 µl (4 pmol) 3'- and 5'-oligonucleotide primers (Amersham); and 5) 0.1 µl (0.5 U) AmpliTaq Gold DNA polymerase (Roche Molecular Systems). After the addition of 20 µl mineral oil (Sigma-Aldrich, Steinheim, Germany), the tubes were heated in 94 C for 10 min to activate the enzyme and then immediately cycled using Hybaid PCR Express thermal cycler (Ashford, Middlesex, UK). After 40 or 45 cycles (94 C, 20 sec for denaturation; 58–62 C, 20 sec for annealing; 72 C, 40 sec for extension) and the final extension step at 72 C for 10 min, 15-µl aliquots of the PCR reaction products (20 µl) were size fractionated in 1.5% agarose gel (Pronadisa, Alcobendas, Spain).
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The concentrations of total (free and follistatin-bound) activin A in culture media were measured with an ultrasensitive ELISA intended for use with various human and animal fluids including culture media. The method has been described in detail previously (22). The activin A assay kit (product code MCA 1426KZZ) was purchased from Serotec (Oxford, UK). The detection limit of the assay was 50 pg/ml. The intra- and interassay coefficients of variation were 6.1% and 6.5%, respectively. According to the manufacturer, there is no detectable cross-reaction with other forms of activins or inhibins.
Inhibins A and B were also measured by ultrasensitive ELISAs in a similar manner to activin A. These methods have been described in detail previously (23, 24, 25). The inhibin A and B assay kits (product codes MCA 950KZZ and MCA 1312KZZ, respectively) were purchased from Serotec. The detection limit of the inhibin A assay was reported to be less than 3.9 pg/ml and that of the inhibin B assay less than 15 pg/ml. The intra- and interassay coefficients of variation for inhibin A were 2.4% and 7.6% and for inhibin B 16.9% and 11.1%, respectively. Inhibin A assay should not have any detectable cross-reaction with inhibin B or activins. Inhibin B assay has a minimal (<1%) cross-reaction with inhibin A and no detectable cross-reaction with activins.
Cortisol was measured by a competitive enzyme immunoassay kit (product code MDKCO1, Diagnostic Products, Los Angeles, CA). The detection limit of the assay was 8.3 nmol/liter. The intra- and interassay coefficients of variation were reported to be 4.4% and 6.7%, respectively.
Statistical analyses
Single experiments consisted of several hormonal or other manipulations each on two to three separate dishes. Results are shown as arithmetic means ± SEM in relation to control adjusted to 1. Absolute hormone concentrations are presented in Tables 2 and 3. The statistical significances were estimated in two group comparisons by the Mann-Whitney test and in the multiple comparisons by the Kruskal-Wallis test. If the P value was lower than 0.05 in the Kruskal-Wallis test, the Mann-Whitney test with Bonferroni’s correction was used as a post hoc test. The level of significance chosen was P less than 0.05.
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The mRNA expression of activin/inhibin subunit, receptor, and binding protein genes in adrenocortical cells
Fetal (Fig. 1, A and D) and adult (Fig. 1B) adrenals and NCI-H295R cells (Fig. 1, C and D) expressed mRNAs for all activin/inhibin subunits, receptors, and binding proteins as determined by RT-PCR. The expression of the -subunit was high and easily detectable. Both ßA-and ßB-subunit mRNAs were expressed uniformly. Follistatin mRNA was abundantly expressed. The mRNAs for all known activin receptor types, i.e. type I/IB, and type II/IIB were detected in fetal and adult adrenals and in the NCI-H295R cells. Detection of the type I activin receptor mRNAs, especially in the fetal adrenals, required 45 PCR cycles instead of 40 cycles for all the other mRNAs. In contrast, the inhibin receptors, betaglycan and InhBP, were expressed abundantly.
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Cultured fetal and adult adrenal cells secreted abundantly activin A, which tended to increase further with the culture age. At the same time, inhibin and cortisol secretion decreased. Cushing’s adenoma cells secreted activin A and inhibins at about the same ratio as normal adult adrenal cells did, although inhibin B dominated over inhibin A. In contrast, inhibin/activin secretion ratio was quite high in a virilizing carcinoma cell culture (Table 2
). In NCI-H295R cells inhibin/activin secretion ratio was also higher than in fetal and adult primary cultures (Table 3).
Regulation of activin A and inhibin A/B secretion by ACTH and 8-BrcAMP in cultured fetal and adult adrenal cells
In fetal adrenal cells, ACTH stimulated the secretion of inhibin A/B up to 1.8 ± 0.4- and 1.8 ± 0.3-fold of control, respectively (n = 7, P < 0.05 for both) but not that of activin A (1.2 ± 0.1-fold of control, P = 0.169). In adult adrenocortical cells, ACTH stimulated inhibin A/B secretion up to 3.4 ± 0.3- and 1.7 ± 0.2-fold of control, respectively (n = 3; P < 0.05 for both), but it had no effect on activin A (1.0 ± 0.1-fold of control) (presented graphically in Fig. 2). Poor availability of fetal and adult adrenals prevented detailed studies on the dose and time dependence of ACTH stimulation of inhibin and activin secretion in primary cultures. ACTH stimulated cortisol secretion in both fetal (110.5 ± 40.3-fold of control, P < 0.01) and adult adrenal cell cultures (3.8 ± 2.5-fold of control, P < 0.05). 8-BrcAMP stimulated activin A and inhibin A/B secretion in a single fetal adrenal culture 1.7-, 1.6-, and 1.7-fold of control, respectively. In two separate adult adrenal cultures, 8-BrcAMP stimulated activin A and inhibin A/B secretion up to 2.0-, 3.6-, and 3.6-fold of control, respectively (data not shown).
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The 8-BrcAMP (1 mM, 12–48 h) stimulated activin A and inhibin A/B secretion throughout the experimental period in time-course experiments (Fig. 3, A–C). The stimulatory effect of 8-BrcAMP on the secretion of activin A and inhibin A/B was dose (0.03–3 mM) dependent (at 48 h up to 31.8 ± 4.3-, 17.2 ± 3.3-, and 3.2 ± 0.5-fold of control, respectively, n = 5; P < 0.05 for all; Fig. 3, D–F). TPA (10 ng/ml, 12–48 h) stimulated activin A secretion with the maximal effect at 24 h of incubation (Fig. 4A). Inhibin A was stimulated steadily throughout the experimental period (Fig. 4A), whereas no change in the low inhibin B secretion was detected. The effect of TPA was dose dependent (1–30 ng/ml, 48 h) on activin A and inhibin A secretion (up to 764.2 ± 258.7- and 32.2 ± 7.9-fold of control, respectively; n = 5; P < 0.05 for both; Fig. 4B). Activin A treatment stimulated inhibin B secretion up to 25.2 ± 6.0-fold of control time (12–48 h) and dose (3–100 ng/ml) dependently (n = 5, P < 0.05; Fig. 5, A and B). ACTH had no effects on activin A or inhibin A/B secretion in NCI-H295R cells (data not shown).
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In this study, we analyzed systematically the expression of activin/inhibin subunits, binding proteins, and receptors as well as the secretion of the different activin/inhibin dimers in human adrenocortical cells. We found that all known activin/inhibin receptor and follistatin mRNAs were expressed in both fetal and adult adrenals as well as in cultured adrenocortical cells. Cultured normal and neoplastic adrenal cells secreted activin A, inhibin A, and inhibin B. Inhibin secretion was stimulated by ACTH, and activin A secretion increased in culture independently of ACTH. Activin A and inhibin A secretion were also increased in response to the stimulation of both PKA and PKC signal transduction pathways. Inhibin B secretion was increased in response to PKA stimulation and activin A treatment.
Adrenocortical production of dimeric inhibins was modest, compared with that of human ovarian granulosa cells cultured in similar conditions (26). This is in line with the immunohistochemical studies showing only a restricted expression of inhibin -subunit in the fetal and especially in the adult adrenal cortex (6, 7). High inhibin/activin secretion ratio in cultured virilizing adrenal tumor cells is in line with the high inhibin -subunit expression in androgen producing tumors (7). Interestingly, cultured adrenocortical cells secreted much more activin A than inhibins despite the dominance of the -subunit expression as determined by RT-PCR in the present study or by Northern blot technique in a previous study (4). It is possible that only a small portion of the translated -subunits form dimeric inhibins because they may be secreted as monomers (27). However, the abundant -subunit expression in virilizing adrenal tumors seems to lead to increased secretion of dimeric, biologically active inhibins. Whether this could lead to increased serum inhibin concentrations in patients with virilizing adrenal tumors is not known.
ACTH stimulated inhibin A and B secretion in cultured adrenocortical cells, which indicates that these peptides could mediate some regulatory effects of ACTH in the human adrenal gland. In contrast, adrenal cell activin secretion was not stimulated in response to ACTH but increased during the dedifferentiation of the cultured cells in the absence of ACTH. The ACTH-induced decrease in adrenal cell activin/inhibin secretion ratio may be an important regulatory element in these cells because inhibins are thought to antagonize the effects of activins in the presence of inhibin receptors betaglycan and InhBP (15, 16). Our findings are in line with a previous report (4) demonstrating that ACTH increases the expression of inhibin -subunit and to some extent ßA-subunit genes in fetal and adult adrenal cells. The increase we saw in inhibin B production could be explained by an increase in -subunits because the expression of the ßB-subunit gene is probably not induced by ACTH.
The unresponsiveness of the NCI-H295R cell line to ACTH treatment is probably explained by the low or absent expression of ACTH receptors. However, the cell line responded to 8-BrcAMP treatment by increasing inhibin and activin secretion. TPA, an activator of PKC, increased activin A and inhibin A production, and activin A treatment increased inhibin B production in NCI-H295R cells. Similar regulation is seen in human ovarian granulosa cells (26, 28). The molecular basis for the effects of cAMP and TPA is well explained by the cAMP response element in the - and ßA-subunit genes and the activating protein-1 response element, also known as 12-O-tetradecanoyl phorbol-13-acetate response element, in the activin/inhibin ßA gene (29, 30). Of special interest is the response to TPA because activating protein-1 transcription factor is known to mediate signals induced by a wide variety of growth factors and cytokines and also to play a role in carcinogenesis (31).
Our findings that adrenocortical cells produce and secrete significant amounts of activin A, express activin receptor mRNAs, and respond to activin A by increasing inhibin B secretion, in line with the studies of Spencer et al. (5, 17, 18), support the hypothesis that activins are important regulators of human adrenal function. On the contrary, the relatively low inhibin secretion combined with absent effects of inhibins on steroid production or cell proliferation in a previous cell culture study (5) raises a question whether adrenal inhibins have any function in vivo. However, the expression of the inhibin -subunit mainly in zona reticularis cells (7) may indicate that only a fraction of adrenocortical cells produce inhibins. The expression of the mRNAs for the inhibin receptors InhBP and betaglycan suggests that inhibins could be important in the regulation of activin signaling in adrenocortical cells, even if they do not have any direct effects of their own. Similarly to the ligand production, inhibin receptors could also be localized on a restricted number of adrenocortical cells. The expression of inhibin receptors on adrenocortical cells in vivo has to be confirmed before definitive conclusions on the role of inhibins in the adrenal physiology can be made.
Follistatin gene expression in adrenal cells suggests that follistatins could be important adrenal regulatory peptides because they could limit the availability of free, biologically active activins. This is supported by our unpublished finding that NCI-H295R cells secrete immunoreactive follistatin in substantial excess over activin A. However, this does not exclude a physiological role for endogenous activins because follistatins may protect cells from only exogenous, but not endogenous, activins acting in an autocrine manner (32). In addition, despite high follistatin secretion, exogenous activin was able to stimulate NCI-H295R cell inhibin B secretion at concentrations likely to be physiological on the basis of activin secretion in primary adrenal cell cultures. Furthermore, the clarification of follistatin secretion requires further attention because its significance in the adrenal gland is largely unknown.
In summary, human fetal, adult, and neoplastic adrenocortical cells are capable to produce dimeric activin A, inhibin A, and inhibin B peptides. ACTH stimulates the production of inhibins A and B via the PKA signal transduction pathway and decreases the activin/inhibin secretion ratio in cultured adrenal cells. Moreover, human adrenocortical cells express mRNAs for activin/inhibin receptors, suggesting the presence of specific receptors for these peptides. The ACTH regulated inhibin secretion and the expression of activin and inhibin receptors support the hypothesis that the activin/inhibin system is a paracrine and autocrine mediator of ACTH in adrenocortical cells.
Acknowledgments
Laboratory technicians Merja Haukka and Minna Heiskanen are thanked for their skillful assistance.
Footnotes
This work was supported by the Foundation for Pediatric Research, Academy of Finland, Novo Nordisk Foundation, Sigrid Juselius Foundation, Kuopio University Hospital, and Paulo Foundation.
Abbreviations: 8-BrcAMP, 8-Bromoadenosine cAMP; InhBP, inhibin-binding protein; TPA, 12-O-tetradecanoyl phorbol-13-acetate.
Received March 25, 2002.
Accepted June 10, 2002.
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