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Home , Activin and inhibin, Avidin, Glycoprotein

479 Expression of activin/inhibin signaling components in the human and the effects of activins and inhibins on adrenocortical steroidogenesis and

TVa¨nttinen1, J Liu2, T Kuulasmaa1, P Kivinen3 and R Voutilainen1,2 1Department of Pediatrics, Kuopio University and University Hospital, Kuopio, Finland 2Department of Pathology, Haartman Institute, University of Helsinki, Helsinki, Finland 3Department of Dermatology, Kuopio University and University Hospital, Kuopio, Finland (Requests for offprints should be addressed to R Voutilainen, Department of Pediatrics, Kuopio University Hospital, PO Box 1777, FIN-70211 Kuopio, Finland)

Abstract Activins and inhibins are structurally related hydroxylase/17,20-lyase mRNA accumulation as evalu- modulating pituitary FSH and gonadal ated by the Northern blot technique, and decreased steroidogenesis. Activins and inhibins are also produced in , , and the adrenal cortex where their physiological role is poorly dehydroepiandrosterone sulfate secretion as determined known. Hormonally active human adrenocortical tumors by specific immunoassays. Activin A increased express and secrete inhibins, while in mice adrenal inhib- apoptosis as measured by a terminal deoxynucleotidyl ins may function as tumor suppressors. To clarify the in situ apoptosis detection method. Inhibins had significance of adrenal activins and inhibins we investi- no effect on steroidogenesis or apoptosis. gated the localization of activin/inhibin signaling compo- In summary, activin/inhibin signaling components are nents in the adrenal gland, and the effects of activins and coexpressed in the zona reticularis and the innermost zona inhibins on adrenocortical steroidogenesis and apoptosis. fasciculata indicating full signaling potential for adrenal Activin type II/IIB and IB, activin signal activins and inhibins in these layers. Activin inhibits transduction Smad2/3, and inhibin receptor beta- steroidogenic enzyme gene expression and secre- were expressed throughout the adrenal cortex, tion, and increases apoptosis in human adrenocortical cells. whereas Smad4 expression was seen mainly in the zona Thus, the activin–inhibin system may have a significant reticularis and the innermost zona fasciculata as evaluated role in the regulation of glucocorticoid and by . Treatment of cultured adreno- production and apoptotic cell death in the human adrenal cortical carcinoma NCI-H295R cells with activin A cortex. inhibited steroidogenic acute regulatory and 17- Journal of Endocrinology (2003) 178, 479–489

Introduction duction and apoptosis (reviewed in Woodruff 1998). Activins and inhibins are also produced in adrenal cells Activins and inhibins are members of the transforming (Voutilainen et al. 1991, Spencer et al. 1992, Voutilainen growth factor  (TGF) superfamily of growth and 1995, Vänttinen et al. 2002). differentiation factors. They are structurally related glyco- In the adrenals, inhibins were originally suggested to be proteins composed of two of the three different subunits tumor-suppressor proteins on the basis that inhibin (A:A, A:B, B:B; activin A, activin AB and activin -subunit knockout mice developed malignant adrenal B; :A, :B; inhibin A and inhibin B respectively). tumors (Matzuk et al. 1994). However, later studies are the most important sources of inhibins in showed that inhibins do not have marked tumor- humans (reviewed in Vale et al. 1988, Ying 1988). suppressor activity in human adrenals, although loss of Gonadal inhibins suppress pituitary follicle-stimulating immunopositivity was reported in a subgroup of adreno- secretion and stimulate ovarian theca cell andro- cortical carcinomas (Munro et al. 1999). In contrast, gen production (reviewed in Findlay 1993). Activins are increased inhibin -subunit mRNA and expres- widely expressed and they have been shown to regulate a sion, and increased secretion of immunoreactive inhibins, variety of cell functions, including pro- were reported in virilizing and, to some extent, in

Journal of Endocrinology (2003) 178, 479–489 Online version via http://www.endocrinology.org 0022–0795/03/0178–479  2003 Society for Endocrinology Printed in Great Britain

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cortisol-producing tumors (Nishi et al. 1995, Pelkey et al. kinase A activator, was used to mimic the effects of ACTH 1998, Arola et al. 2000, Vänttinen et al. 2002). on adrenocortical steroidogenesis as the NCI-H295R cell Adrenocorticotropin (ACTH) regulates adrenal line does not express functional ACTH receptors. inhibin/activin production suggesting that inhibins and activins may mediate or modulate the effects of ACTH in a para/autocrine manner (Voutilainen et al. 1991, Materials and Methods Vänttinen et al. 2002). ACTH regulates steroidogenesis at multiple levels. In addition to its acute effect on steroid Ethical considerations secretion, it increases the production of the steroidogenic acute regulatory protein (StAR) enabling the transport of The Research Ethics Committees of the Kuopio and cholesterol into mitochondria to be metabolized to steroid Helsinki University Hospitals approved the study proto- hormones (reviewed in Stocco 2001). ACTH stimulates cols, and the patients gave informed written consent. the synthesis of steroidogenic , including 17- hydroxylase/17,20-lyase (P450c17) essential for gluco- Immunohistochemistry corticoid and androgen production (reviewed in Miller 1988). The role of adrenal activins in the regulation of Normal adult adrenal tissues were obtained during surgery steroidogenesis is controversial; they have been shown to for renal tumors. Immunostaining was performed on stimulate ACTH-induced cortisol secretion in human fetal 5 µm sections cut from paraffin-embedded blocks of adrenal cells (Spencer et al. 1992), but to inhibit ACTH- paraformalin-fixed tissues. The sections were deparaffin- induced cortisol and dehydroepiandrosterone (DHEA) ized in xylene, rehydrated in a series of graded alcohols, secretion in bovine adrenal cells (Nishi et al. 1992). and permeabilized by microwaving at high power for Inhibins had no effects on adrenal steroidogenesis in these 5 min three times in citrate buffer (10 mM Na citrate, pH studies. 6·0). The sections were then washed in PBS and non- The signaling system of activins and inhibins in the specific was blocked with normal rabbit serum for adrenal gland is poorly characterized. Activins mediate at least 30 min at room temperature. Endogenous peroxi- their effects through specific receptors on cell membranes dase activities were blocked by 1% H2O2 for 30 min. The and via a cascade of cytosolic Smad proteins (reviewed in in the sections were detected with corresponding Itoh et al. 2000, Pangas & Woodruff 2000). In brief, both affinity-purified goat polyclonal (ActRII/IIB, type I and II receptors (ActRI/IB and ActRII/IIB re- sc-5669; ActRIB, sc-11986, Smad2/3, sc-6032; Smad4, spectively), receptor-regulated Smads (R-Smads; Smad2 sc-1908, TGFRIII (G), sc-6199; all from Santa Cruz and Smad3) and a common-mediator Smad (Co-Smad; Biotechnology, Inc., Santa Cruz, CA, USA; and another Smad4) are needed for successful activin signaling. In TGFRIII from Research Diagnostics, Inc., addition, inhibitory Smads (I-Smads; Smad6 and Smad7) Flanders, NJ, USA), which were applied at 1:100 dilution. regulate activin signaling. Inhibins, in turn, antagonize The primary antibodies were added on the adrenal sections activin signaling through binding to ActRII co-receptor in PBS, and incubation was performed overnight at 4 C. betaglycan (G, also known as TGF type III receptor; After this incubation, the slides were washed in PBS three reviewed in Gray et al. 2002). It is known that radiolabeled times. The primary antibodies were identified using bind to rat adrenals and that activin and -conjugated rabbit anti-goat IgG from an ABC-Elite inhibin receptor mRNAs are expressed in human adrenal Kit (PK-6105; Vector Laboratories, Inc., Burlingame, CA, cells (Woodruff et al. 1993, Vänttinen et al. 2002). USA), followed by incubation with ABC solution accord- Adrenocortical function is determined by both the ing to the manufacture’s instructions. Finally, light coun- number and the steroidogenic activity of the adrenocorti- terstaining was performed with hematoxylin, and the cal cells. The rate of cell proliferation and apoptosis sections were dehydrated and mounted. Each staining was contribute to the cell number. In this study we wanted to repeated with at least five different sections with similar shed more light on the role of activins and inhibins in the results. For the negative controls, the primary antibodies regulation of both adrenal steroidogenesis and apoptosis. were replaced by normal goat serum or PBS alone. To Activin and inhibin receptor and Smad exclude the effect of possible endogenous biotin, biotin peptide localizations in the adrenal cortex were studied by blocking (-biotin blocking kit; Vector Laboratories) immunohistochemistry. Human adrenocortical carcinoma was performed in two samples for each detection cell line NCI-H295R, expressing all adrenal steroidogenic before the addition of the primary antibodies. enzymes, secreting glucocorticoids and (Gazdar et al. 1990, Staels et al. 1993, Rainey et al. 1994), and Cell cultures expressing activin/inhibin receptor genes (Vänttinen et al. 2002) was used as a model for studying the effects of NCI-H295R human adrenocortical cell line was obtained activins and inhibins in adrenocortical cell steroidogenesis from American Type Culture Collection (Manassas, VA, and apoptosis. 8-Bromo cAMP (8-BrcAMP), a protein USA). For the steroid and mRNA studies the cells were

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Table 1 The sequences of the oligonucleotides used as primers in PCR for detecting Smad2-4 and 6-7 gene expression. The numbering after each sequence is according to the sequences given in the database (the corresponding GenBank accession numbers are shown after each gene). The lengths of the expected PCR products are shown PCR product 5 Sense 3 Antisense (bp)

Gene Smad2 (NM_005901) 5-TCCTACTACTCTTTCCCCTG-3 (805-824) 5-GTTTCTCCAACCCTCTGAT-3 (903-921) 117 Smad3 (NM_005902) 5-GCTGTTCCAGTGTGTCTTAG-3 (1325-1344) 5-AATGGGTTGAGTAGAGTTCC-3 (1411-1430) 106 Smad4 (NM_005359) 5-GGTGAAGGATGAATATGTGC-3 (599-618) 5ACTCAGATGGGGCTAACAG-3 (726-744) 146 Smad6 (NM_005585) 5-GCTACCAACTCCCTCATCAC-3 (1849-1868) 5-GGTCGTACACCGCATAGAG-3 (1972-1990) 142 Smad7 (NM_005904) 5-GAAGTCAAGAGGCTGTGTTG-3 (812-831) 5-CGGGTATCTGGAGTAAGGAG-3 (921-940) 129

plated at a density of 1106 cells per well on 35 mm centrifugation. DNA-pellet was then washed twice with plastic culture dishes (Nunc, Roskilde, Denmark). The 0·1 M Na citrate/10% ethanol, and once with 75% medium was DMEM-F12 containing 2% Ultroser (Life ethanol, air-dried, and dissolved in 10 mM Tris/1 mM Technologies, Paisley, Strathclyde, UK), ITS+1 liquid EDTA solution with pH adjusted to 8·0 with NaOH. media supplement (Sigma, St Louis, MO, USA), RNA and DNA were stored at – 70 C if not used (100 IU/ml), streptomycin sulfate (100 µg/ml) and immediately for further analyses. glutamine (0·5 mM). Cell cultures were grown as mono-  layers at 37 C in a 95% air–5% CO2 humidified environ- ment and the culture media were changed every 2–3 days. RT-PCR The viability of the cell cultures was monitored by phase The RT-PCR techniques were as described previously contrast light microscopy. (Vänttinen et al. 2002). Briefly, 1 µg total RNA, Experimental procedures were performed when the cell oligo(dT)20 primer (Amersham, Little Chalfont, Bucks, cultures had reached confluency. In the experiments the UK), four dNTPs (Amersham), avian myeloblastosis virus cultured cells were incubated for 48 h with or without (Finnzymes, Espoo, Finland), and RT 8-BrcAMP (Sigma), recombinant human activin A, in- reaction buffer (Finnzymes) were used for cDNA syn- hibin B (both from R&D Systems, Minneapolis, MN, thesis. The amplification primers used for the PCR of USA), inhibin A (generously provided by Dr A F Parlow human Smads and the expected lengths of the PCR from NIDDK National Hormone and Pituitary Program, products are shown in Table 1. PCR was performed by NIH, Bethesda, MD, USA), or cycloheximide (inhibitor combining one-tenth of the RT mixture with GeneAmp of protein synthesis; Sigma). After the experiments the PCR buffer (Roche Molecular Systems, Branchburg, culture media were collected and stored at – 70 C. NJ, USA), dNTP mixture (Amersham), 3- and 5- oligonucleotide primers (TAG Copenhagen, Copenhagen, Denmark) and AmpliTaq Gold DNA polymerase (Roche RNA and DNA preparation Molecular Systems). The samples were then cycled (40) Fetal adrenals were obtained from legal abortions per- using Hybaid PCR Express thermal cycler (Ashford, formed for social or medical reasons. Normal adult adrenal Middlesex, UK). Aliquots of the PCR reaction products tissues were obtained during surgery for renal tumors. were size-fractionated in 3·0% gel (Pronadisa, RNA was extracted by ultracentrifugation through a Alcobendas, Spain). PCR products were sequenced with cesium chloride cushion. an automatic DNA analysis system to confirm their The cultured cells were lysed using TriZol Reagent specificity (Abi Prism 3100 Genetic Analyzer; Applied (Life Technologies) according to the manufacturer’s pro- Biosystems, Foster City, CA, USA). tocol. Cell lysates were separated by chloroform into an aqueous phase containing RNA and an organic phase Northern blot analysis containing proteins and DNA. Trace amounts of genomic DNA were removed from the RNA-containing phase A Northern blot sample consisted of 20 µg total RNA in  by treatment with Rnase-free Dnase (Promega Corp., H2O, 10 MOPS, deionized formamide, 37% formalde- Madison, WI, USA). RNA was precipitated from the hyde and ethidium bromide. Samples were size-fractioned aqueous phase overnight with equal volume of isopropa- in denaturing 1·0% agarose gel (Pronadisa). After the nol, washed once with 75% ethanol, air-dried, and dis- electrophoresis, RNA was transferred onto Hybond-N+ solved in Rnase-free water. DNA was extracted from nylon filters (Amersham), and fixed by UV cross-linking. the organic phase by incubation with 10% ethanol and The prehybridization and hybridization techniques were www.endocrinology.org Journal of Endocrinology (2003) 178, 479–489

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used as described previously (Liu et al. 1994). The StAR cell apoptosis was studied also by the DNA fragmentation mRNA was detected with an oligonucleotide probe (Liu analysis described previously (Zhu & Wang 1997). et al. 1996), P450c17 mRNA (Chung et al. 1987) and 28S rRNA (loading control; Arnheim 1979) with cDNA probes. Hybridized filters were autoradiographed and Statistical analyses the hybridization signals quantified by densitometric scanning. Single experiments consisted of several hormonal or other manipulations each on one to three separate dishes. The steroid concentrations of the cell culture media were Enzyme immunoassays for steroid hormones normalized with the DNA content of the respective Steroid hormones were analyzed by competitive enzyme dishes, and the scanning values of StAR and P450c17 mRNA signals with the respective 28S rRNA values. immunoassays. A cortisol measurement kit was purchased  from Diagnostic Products Corporation (Los Angeles, CA, Results are shown as arithmetical means S.E.M. in relation USA; product code MDKCO1). Androstenedione, to the control adjusted to 100%. The statistical significan- DHEA and DHEA-S (DHEA sulfate) measurement kits ces were estimated using the Mann–Whitney test in were purchased from Diagnostic System Laboratories two-group comparisons or the Kruskal–Wallis test in (Webster, TX, USA; product codes DSL-10–3800, DSL- multiple comparisons followed by a Mann–Whitney test 10–9000 and DSL-10–3500 respectively). The detection with Bonferroni’s correction as a post hoc test. The level of limits of the cortisol, androstenedione, DHEA and significance was chosen as P,0·05. DHEA-S assays were 8·3, 0·1, 0·3 and 40·7 nM respect- ively. The intra- and interassay coefficients of variation were respectively 4·4 and 6·7%, 8·8 and 7·5%, 9·9 and Results 5·4%, and 5·1 and 7·2%. Activin/inhibin receptors and intracellular activin signaling and mRNAs are expressed in the human adrenal Apoptosis analysis cortex A TACS terminal deoxynucleotidyl transferase (TdT) in Human adrenal cortex showed differential staining pattern situ apoptosis kit (R&D Systems) was used for detecting for the activin/inhibin signaling system components. apoptosis in cultured NCI-H295R cells. Briefly, the ActRII/IIB was expressed throughout the adrenal cortex NCI-H295R cells were plated on chamber glass slides and medulla, but the staining was most intense in the zona  5 2 (Nunc) at a density of 0·2 10 per each 0·8 cm well and reticularis (ZR) and the innermost zona fasciculata (ZF). treated with activin A for 48 h. The slides were fixed in ActRIB and Smad2,3 were coexpressed throughout the cold methanol for 5 min and permeabilized in Cytonin adrenal cortex with the most intense staining in the zona solution (R&D Systems) for 60 min. Then TdT labeling glomerulosa (ZG), and very faint staining in the medulla. ff 2+ bu er containing TdT-dNTPs, Mn and TdT enzyme Smad4 showed minimal or no expression in the ZG,  was added on slides for 60 min incubation at 37 C. The moderate expression in the medulla and strong expression ff labeling was stopped after incubation by TdT stop bu er in the ZR and the innermost ZF. G was expressed for 5 min. The slides were washed twice in deionized throughout the adrenal cortex (Fig. 1). Its expression was water (dH20), incubated in -horseradish per- relatively weak but consistent with two different anti- oxidase for 10 min, washed again twice in dH20, and bodies. Human fetal and adult adrenal, and NCI-H295R finally incubated in TACS Blue Label for 2–5 min. The carcinoma cells expressed Smad2–4 and Smad6–7 mR- slides were processed through increasing concentrations of NAs as determined by RT-PCR, although Smad2 ethanol, then xylene and finally covered with Depex expression in fetal cells was very weak (Fig. 2). (BDH Laboratory Supplies, Poole, Dorset, UK). Positive and negative cell control slides provided by R&D Systems, and a properly stained slide without TdT enzyme were used. The cells were considered to be apoptotic when the 8-BrcAMP stimulates whereas activin A inhibits StAR and bluish color for nuclear apoptosis was seen and the P450c17 gene expression morphological criteria for apoptosis were fulfilled StAR and P450c17 specific hybridizations of Northern (Gorczyca et al. 1993). The apoptotic index (%) was blot filters revealed single mRNA bands with relatively defined as a ratio between the apoptotic and apoptotic plus strong signals. 8-BrcAMP (0·1 mM) induced parallel non-apoptotic cells determined from six randomly counted StAR and P450c17 mRNA accumulation in NCI- non-overlapping microscope fields per well with400 H295R cells (at 48 h up to 120% of control, P,0·05; Fig. magnification with an Olympus BH-2 microscope 3). Activin A inhibited time- and dose-dependently StAR equipped with a 0·20·2 mm ocular grid (Olympus and P450c17 mRNA accumulation. This inhibitory effect Optical Company, Hamburg, Germany). NCI-H295R was relatively slow and significant for both genes after

Journal of Endocrinology (2003) 178, 479–489 www.endocrinology.org

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Figure 1 Immunohistochemical localization of activin/inhibin signaling components in the adrenal cortex. Human adrenal sections were stained with antibodies detecting both type II and IIB (ActRII/IIB), activin receptor type IB (ActRIB), both Smad2 and Smad3 (Smad2,3), and Smad4 and betaglycan (G). Each panel shows both cortex and medulla (magnification100), except the G staining on the right bottom showing only the adrenal cortical layers (magnification200). Positive staining for each antigen is seen in red. Nuclei are counterstained with hematoxylin, producing a light blue color. ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis; M, medulla.

48 h incubation at activin concentrations of 30–100 ng/ no effects on StAR or P450c17 expression (1–100 ng/ml, ml. Activin A decreased StAR and P450c17 mRNA 48 h for both; Fig. 3; inhibin B data not shown). Cyclo- accumulation maximally down to 60–80 and 40–70% of heximide (an inhibitor of protein synthesis) inhibited control respectively (P,0·05 for both, n=11; Figs 3–5). StAR but increased P450c17 mRNA accumulation Activin A inhibited totally the stimulatory effect of (P,0·05 for both). Cycloheximide enhanced the inhibi- 8-BrcAMP on P450c17, and partially that on StAR gene tory effect of activin A on StAR expression but inhibited expression (P,0·05; Fig. 3). Inhibin A or inhibin B had it on P450c17 expression (Fig. 5). www.endocrinology.org Journal of Endocrinology (2003) 178, 479–489

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when the cultures became confluent. At that stage the basal secretion rates of cortisol, androstenedione, DHEA and DHEA-S were 72·27·0, 23·43·9, 0·80·2 and 33·72·2 nmol/mg DNA per 48 h respectively. 8-BrcAMP (0·1 mM, 48 h) stimulated cortisol, andros- tenedione, DHEA and DHEA-S secretion up to 240, 210, 150 and 110% of control (P,0·05 for all, n=4; Fig. 6). Activin A inhibited the secretion of all measured dose- and time-dependently (1–100 ng/ml, 6–48 h; Fig. 7). The inhibition became statistically significant at 24–48 h. At 48 h activin A inhibited cortisol, androsten- edione, DHEA and DHEA-S production in different experiments down to 60–80% of control (P,0·05, n=12; Figs 6 and 7). Inhibin A (Fig. 6) or inhibin B (0·1–100 ng/ Figure 2 Smad2–4 and 6–7 gene expression in cultured ml both, 48 h) had no significant effect on steroidogenesis adrenocortical NCI-H295R carcinoma cells, and in human fetal (inhibin B data not shown). and adult adrenals as determined by RT-PCR analysis. Forty PCR When NCI-H295R cells were incubated for 48 h with amplification cycles were used for each cDNA. M: 200, 100 and a combination of 8-BrcAMP and activin A, the secretion 80 bp molecular mass markers. A no-template control was also used in each reaction (no PCR product, data not shown). of cortisol, androstenedione and DHEA-S decreased down to 76, 45, and 66% of the levels secreted by merely 8-BrcAMP-treated cells (P,0·05 for all, n=4; Fig. 6). Activin A inhibited 8-BrcAMP-induced DHEA secretion only transiently (6–36 h, not shown), but at 48 h it increased it up to 116% (P,0·05, n=4). Inhibin A (Fig. 6) or inhibin B (40 ng/ml for both) did not interfere with the effects of 8-BrcAMP (data not shown for inhibin B).

Activin A increases apoptosis Phase contrast light microscopy revealed that activin A treatment induced apoptotic morphology in NCI-H295R cells. TdT labeling showed that activin A induced apop- tosis dose-dependently and this effect was observable after 24 h of treatment. Activin A treatment (30 ng/ml, 48 h) increased the apoptotic index of NCI-H295R cells up to 290% of control (P,0·05, n=5; Fig. 8). DNA fragmen- tation analysis confirmed the apoptotic effect of activin A (data not shown). Inhibins had no effect on NCI-H295R apoptosis (data not shown).

Figure 3 The effect of 8-BrcAMP (0·1 mM; cAMP), activin A (40 ng/ ml; Act), inhibin A (40 ng/ml; Inh), and their combinations on StAR Discussion and P450c17 (c17) mRNA expression in cultured NCI-H295R cells. 28S ribosomal RNA was used as a loading control. The bars represent StAR/28S and P450c17/28S mRNA expression We have shown that: (i) activin/inhibin receptors and (meansS.E.M.) in three separate experiments when control (C) Smad signal transduction peptides are coexpressed in the is adjusted to 100%. Different letters above the bars indicate innermost ZF and the ZR of the adult adrenal gland; (ii) statistically significant differences (P,0·05) between the treatments. Smad genes are expressed in fetal and adult adrenal cells; Please note that the statistical significances have been estimated independently for each parameter; thus different parameters (StAR (iii) activin A treatment decreases basal and cAMP- and P450c17) should not be compared with each other. A induced StAR and P450c17 mRNA accumulation in representative Northern blot labeled with StAR, P450c17 and 28S cultured adrenocortical NCI-H295R cells; (iv) activin A ribosomal RNA probes is presented below the bar Figure. inhibits the secretion of cortisol, androstenedione, DHEA and DHEA-S in NCI-H295R cells; and (v) activin A induces apoptotic cell death in NCI-H295R cells. 8-BrcAMP stimulates and activin A inhibits steroid secretion This and previous studies (Vänttinen et al. 2002) Steroid secretion by cultured NCI-H295R cells increased showed that human adrenocortical cells express mRNAs in relation to the cell number and reached maximum for the activin/inhibin receptors and the R-, Co-, and

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Figure 4 Time- (3–48 h; 20 ng/ml) and dose- (0–100 ng/ml; 48 h) dependent effects of activin A on StAR and P450c17 (c17) mRNA expression in cultured NCI-H295R cells. 28S rRNA was used as a loading control. The bars represent StAR/28S and P450c17/28S mRNA expression (meansS.E.M.) in five separate experiments when control (0 ng/ml) is adjusted to 100%. *P,0·05 comparing control vs treatment. A representative Northern blot hybridized with StAR, P450c17 and 28S ribosomal RNA probes is presented below each bar figure. Lane C, control; lane A, activin 20 ng/ml; in pairs at 3, 6, 12, 24 and 48 h of incubation.

I-Smads. The coexpression of these components in the connection between steroidogenesis and activins has been adrenal cortex confirms the presence of a functional reported in primary cultures of adrenal cells where spon- activin/inhibin signaling system in the adult adrenal gland. taneously increasing activin A gene expression is associ- Despite the expression of activin receptors and R-Smads ated with decreasing steroid production and inhibin throughout the cortex, the expression of Co-Smads seems -gene expression (Voutilainen et al. 1991). In contrast, to limit activin signaling mainly to the ZR and the one study suggested that activin A stimulates ACTH- innermost ZF. The significance of differential expression induced cortisol secretion in human fetal, but not in adult of activin receptors and Smads in the adrenocortical zones adrenal cells (Spencer et al. 1992). These discrepancies is not known but it may represent a regulatory mechanism could be explained by the different developmental stage of for activin actions. Interestingly, the innermost ZF and the cells used in these studies, which has been reported to ZR have previously been shown to express inhibin - and influence the response to activin treatment (Miró & -subunit peptides (Munro et al. 1999, Arola et al. 2000). Hillier 1992, Di Simone et al. 1996). Relatively high In connection with G expression, this suggests that activin concentration was needed to cause effects on adrenal inhibins may interfere with activin signaling in the NCI-H295R cells. This may be explained by the high adrenal gland. In addition, the I-Smads may also regulate endogenous (an activin-binding protein) pro- activin signaling in the adrenal cortex. duction in these cell cultures (T Vänttinen, J Liu, T The expression of activin signaling system in the ZR Kuulasmaa, P Rasmus and R Voutilainen, unpublished and ZF suggests a role for adrenal activins in glucocorticoid observations). and androgen production. In our study activin treatment Activins seem to regulate steroidogenesis at the tran- reduced both cortisol and androgen production in NCI- scriptional level. In human ovarian theca-like tumor cells H295R cells. Similarly, a previous report (Nishi et al. activin decreased forskolin-induced P450c17 (Sawetawan 1992) shows that activin A inhibits ACTH-induced cor- et al. 1996) and StAR expression (Dooley et al. 2000). In tisol and DHEA secretion in bovine adrenal cells. A similar our study activins decreased P450c17 and StAR gene www.endocrinology.org Journal of Endocrinology (2003) 178, 479–489

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expression also in adrenocortical cells. The effects of activins are very similar to those of TGF1, another member of the TGF superfamily and a well-known inhibitor of adrenal steroidogenesis (reviewed in Chambaz et al. 1996). This is not surprising as TGFs share a common Smad pathway with activins. TGF1 has been reported to decrease both human adrenal P450c17 (Lebrethon et al. 1994) and StAR (Brand et al. 1998) mRNA expression. Thus, it seems that the human adrenal Smad pathway is anti-steroidogenic and can be activated by several para/autocrine peptides, including activins and TGFs. Stimulation of androgen production in ovarian theca cells in response to inhibins (reviewed in Findlay 1993) and high inhibin -subunit mRNA and peptide expres- sion in adrenal virilizing tumors (Nishi et al. 1995, Arola et al. 2000, Vänttinen et al. 2002), suggest that inhibins might stimulate adrenal androgen production. On the other hand, inhibins have not been found to have any direct effects in adrenal cells. Inhibins could regulate Figure 5 The effect of cycloheximide (CH; 20 µg/ml), activin A adrenal function via two indirect mechanisms. First, the (Act; 20 ng/ml), and their combination on StAR and P450c17 (c17) mRNA expression in cultured NCI-H295R cells. 28S rRNA expression of the inhibin receptor G in adrenal cells was used as a loading control. The bars represent StAR/28S and suggests that inhibins could antagonize the effects of P450c17/28S mRNA expression (meansS.E.M.) in three 48 h activins. Secondly, each dimeric inhibin molecule pro- experiments when control (0 µg/ml or ng/ml) is adjusted to 100%. duced limits the availability of -subunits for activin Different letters above the bars indicate statistically significant synthesis. The parallel effects of ACTH on steroidogenesis differences (P,0·05) between the treatments. Please note that the statistical significances have been estimated independently for and inhibin production on the one hand, and increasing each parameter; thus different parameters (StAR and P450c17) activin production and decreasing steroidogenesis in the should not be compared with each other. A representative absence of ACTH on the other hand support these Northern blot hybridized with StAR, P450c17 and 28S ribosomal hypotheses (Voutilainen et al. 1991, Vänttinen et al. 2002). RNA probes is presented below the bar figure.

Figure 6 The effect of 8-BrcAMP (0·1 mM; cAMP), activin A (40 ng/ml; Act), inhibin A (40 ng/ml; Inh), and their combinations on cortisol, androstenedione, DHEA and DHEA-S secretion in cultured NCI-H295R cells. The bars represent relative steroid secretion (meansS.E.M.) in four separate 48 h experiments with the control (C) adjusted to 100%. Different letters above the bars indicate statistically significant differences (P,0·05) between the treatments. Please note that the statistical significances have been estimated independently for each parameter; thus different parameters (e.g. cortisol and DHEA) should not be compared with each other.

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Figure 7 Time- (6–48 h; 40 ng/ml) and dose- (0–100 ng/ml; 48 h) dependent effects of activin A on cortisol, androstenedione, DHEA and DHEA-S secretion in cultured NCI-H295R cells. The bars represent relative steroid secretion (meansS.E.M.) in three (time response) or five (dose response) separate experiments with the control (0 ng/ml) adjusted to 100%. *P,0·05 comparing control vs treatment.

Activin A induced apoptotic changes in cultured NCI- overexpressing cells could contribute to increased H295R cells. This effect has also been shown in fetal steroidogenesis and cell mass in these tumors. adrenal cells and activins are therefore believed to contrib- In summary, we have shown that human adrenocortical ute to the postnatal remodeling of the adrenal cortex cells express activin/inhibin receptors and intracellular (Spencer et al. 1999). Apoptosis is also known to occur in signaling molecules suggesting activin/inhibin signaling the adrenal cortex during the adult period. It has been potential especially in the ZR and the innermost ZF of the assumed that adrenocortical cells proliferate in the outer- human adrenal cortex. Activin decreases StAR and most layers of the cortex and then migrate towards the P450c17 gene expression leading to decreased glucocorti- inner cortex and die through an apoptotic mechanism in coid and androgen secretion, and causes apoptotic cell the ZR (reviewed in Wolkersdörfer & Bornstein 1998). death. Thus, the activin–inhibin system may regulate The mechanisms controlling apoptosis in the adrenal steroid production and apoptotic cell death in the inner cortex are complicated, but it is possible that activins play layers of the adrenal cortex and may also be involved in the a role in this process as their signaling system is expressed development of hormonally active tumors in these zones. in the same area as the most active apoptosis (Sasano et al. 1995). Interestingly, increased inhibin -subunit expres- sion has been detected in cortisol-producing and virilizing Acknowledgements tumors probably originating from the inner cortical cells (Pelkey et al. 1998, Arola et al. 2000). Therefore, it is The laboratory technicians Merja Haukka and Minna possible that decreased activin production or disturbed Heiskanen are thanked for their skillful assistance. Teemu activin signal transduction in these inhibin -subunit Kuulasmaa is thanked for providing the oligo design www.endocrinology.org Journal of Endocrinology (2003) 178, 479–489

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