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479 Expression of activin/inhibin signaling components in the human adrenal gland and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis 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 glycoprotein hydroxylase/17,20-lyase mRNA accumulation as evalu- hormones modulating pituitary FSH secretion and gonadal ated by the Northern blot technique, and decreased steroidogenesis. Activins and inhibins are also produced in cortisol, androstenedione, dehydroepiandrosterone and the adrenal cortex where their physiological role is poorly dehydroepiandrosterone sulfate secretion as determined known. Hormonally active human adrenocortical tumors by specific enzyme 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 transferase 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 receptor type II/IIB and IB, activin signal activins and inhibins in these layers. Activin inhibits transduction proteins Smad2/3, and inhibin receptor beta- steroidogenic enzyme gene expression and steroid secre- glycan 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 androgen by immunohistochemistry. 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 protein 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 Gonads 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- hormone 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 peptide expres- widely expressed and they have been shown to regulate a sion, and increased secretion of immunoreactive inhibins, variety of cell functions, including steroid hormone 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 Downloaded from Bioscientifica.com at 09/28/2021 06:30:55PM via free access 480 TVA}NTTINEN and others · Activin regulation of adrenal cell function 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 enzymes, 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 staining 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 antigens in the sections were detected with corresponding Itoh et al. 2000, Pangas & Woodruff 2000). In brief, both affinity-purified goat polyclonal antibodies (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 antibody 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 activin and inhibin bind to rat adrenals and that activin and biotin-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 signal transduction exclude the effect of possible endogenous biotin, biotin peptide localizations in the adrenal cortex were studied by blocking (avidin-biotin blocking kit; Vector Laboratories) immunohistochemistry. Human adrenocortical carcinoma was performed in two samples for each antigen detection cell line NCI-H295R, expressing all adrenal steroidogenic before the addition of the primary antibodies. enzymes,