WNT-4 mRNA Expression in Human Adrenocortical Tumors and Cultured Adrenal Cells
Authors
T. Kuulasmaa1, J. Jääskeläinen1, S. Suppola1, T. Pietiläinen2, P. Heikkila3, S. Aaltomaa4, V .- M. Kosma2, 5, R. Voutilainen1
Affiliations
Affiliation addresses are listed at the end of the article
Key words
WNT-4
· adrenal glands
B-catenin adrenal cortex
· neoplasms (human)
Abstract
&
The members of the Wnt glycoprotein family are important in embryogenesis and adult tissue homeostasis, and deletion of Wnt-4 gene in mice leads to improper development of many organs including the adrenals. The objective of this study was to investigate the expression of WNT- 4 gene in human adrenals and adrenocortical tumors. The WNT-4 mRNA expression (analyzed by quantitative real-time RT-PCR) was signifi- cantly higher in Conn’s adenomas (p<0.01) and lower in Cushing’s adenomas, virilizing carcino- mas and fetal adrenals (p<0.05) compared with
normal adult adrenals. WNT-4 mRNA expression was clearly upregulated by ACTH and 8-bromo- CAMP (8-BrcAMP) in primary cultures of normal adult adrenocortical cells, but downregulated by 8-BrcAMP and 12- O -tetradecanoylphorbol-13- acetate (TPA) in human NCI-H295R adrenocor- tical carcinoma cells. Angiotensin II tended to increase WNT-4 mRNA expression at 24 hours and decreased it at 48 hours time point in both cell culture types. The abundant WNT-4 mRNA expression in Conn’s adenomas and its hormonal regulation in adrenocortical cells suggest a role for WNT-4 in human adrenocortical function.
Introduction
received 06.08.2007 accepted 14.02.2008
Bibliography DOI 10.1055/s-2008-1078716 Published online: June 13, 2008 Horm Metab Res 2008; 40: 668-673 @ Georg Thieme Verlag KG Stuttgart . New York ISSN 0018-5043
Correspondence
R. Voutilainen Department of Pediatrics Kuopio University Hospital P.O. Box 1777 70211 Kuopio Finland Tel .: +358/17/17 23 91
Fax: +358/17/17 24 10 Raimo.Voutilainen@uku.fi
& Wnts are secreted, lipid modified glycoproteins with auto- and paracrine effects. They bind to seven-transmembrane receptors known as Friz- zleds, and the ligand binding is modulated by several secreted antagonists [1-3]. Intracellular Wnt signaling uses at least three different path- ways: 1) the canonical Wnt/B-catenin pathway, which upon activation leads to stabilization of cytosolic ß-catenin and subsequent regulation of the target genes, 2) the planar cell polarity path- way, which activates c-Jun N-terminal kinase (JNK) directing cytoskeletal organization, and 3) the Wnt/Ca2+ pathway in which phospholipase C (PLC) and protein kinase C (PKC) are activated and intracellular calcium is released causing cel- lular response [4]. In humans, 19 WNTs have been identified [5]. Correct Wnt signaling is essential for appropriate embryogenesis and org- anogenesis, and aberrant Wnt signaling has been detected in several clinical conditions ranging from various cancers to sex reversal and schizo- phrenia [1,6].
Wnt-4 has been shown to be a critical component in the development of the reproductive system.
It is needed for the formation of the Müllerian ducts in both males and females at the early stages of gonadal development [7,8]. At later stages of the ovarian development, Wnt-4 upreg- ulates Dax-1, which antagonizes male determin- ing factor Sry and inhibits ovarian steroidogenesis including testosterone biosynthesis. Thus, insuf- ficient activation of Dax-1 and abnormal biosyn- thesis of testosterone in the ovaries lead to masculinization of Wnt-4 deficient female mice [7,9,10]. Aberrant Wnt-4 expression disrupts Sertoli cell differentation, testosterone produc- tion, and testicular vasculature in male mice [11, 12]. In agreement with these findings, Jordan and co-workers reported an XY female sex- reversal patient carrying a cytogenetic duplica- tion of the WNT-4 gene locus and elevated WNT-4 expression [13]. In contrast, a patient carrying a heterozygous loss-of-function mutation in WNT- 4 gene resembled the Wnt-4 deficient female mouse phenotype [14].
Relatively little is known about the role of Wnt-4 in the adrenals. WNT-4 mRNA is found in the human adrenal cortex, the expression being stronger in zona fasciculata/zona glomerulosa than in zona reticularis [15]. There are three
WNT-4 mRNA transcripts of 1.5, 2.4, and 4.3kb in the human adrenal gland, the smallest transcript being dominant [16]. In the Wnt-4 knockout mice, adrenals are normal in shape and size at birth, but the development of zona glomerulosa is abnormal leading to reduced aldosterone synthase (Cyp11B2) expression and hence reduced aldosterone production. In the female knock- out mice, the late embryonic adrenal expression of 17x- hydroxylase/C17, 20-lyase (Cyp17) resembles that in males [17]. The patient carrying heterozygous loss-of-function mutation in the WNT-4 gene had strikingly similar features with Wnt-4 knockout female mice thus indicating that WNT-4 is important also in the regulation of human steroidogenesis [14].
In the present study, our aim was to investigate the mRNA expression of WNT-4 in normal adrenals as well as in several adrenocortical tumors to find out whether WNT-4 expression associates with the steroid secretory pattern of these tumors. As WNT-4 could be a potential regulator of adrenal steroidogenesis, the regulation of WNT-4 mRNA expression by angiotensin II (AngII), adrenocorticotrophic hormone (ACTH), 8-bromo-cAMP (8-BrcAMP, a protein kinase A activator), and 12-O-tetra- decanoylphorbol-13-acetate (TPA, a protein kinase C modulator) was investigated in primary adrenocortical cell cultures (derived from normal human adrenals) and in the human adrenocortical cell line NCI-H895R.
Materials and Methods
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Tissue material
Adult adrenals were obtained from patients undergoing surgery for adrenal or renal tumors. The malignancy of the tumors was estimated according to the Weiss criteria as described previ- ously [18]. In short, adrenal tumor samples were classified into five groups according to the clinical features and predominant hormone production of a given patient as well as the histological features of the samples. The classification is as follows: normal adrenals (n=6), Conn’s (aldosterone producing) adenomas (n=11), Cushing’s (cortisol producing) adenomas (n=9), Cush- ing’s carcinomas (n=5), virilizing (androgen producing) adeno- mas (n=4), and virilizing carcinomas (n=4). Human fetal adrenals (n=7, gestational age 13-20 weeks) were obtained from legal abortions performed for social or medical reasons. The study was approved by the Research Ethics Committees of the Kuopio and Helsinki University Hospitals, and the patients gave an informed written consent.
Cell cultures
In order to prepare primary cell cultures from normal adrenals (originating from patients undergoing surgery for renal tumors), excess adipose tissue was removed from adrenal tissue sam- ples before they were decapsulated and finely minced with scis- sors. Tissue pieces were dispersed enzymatically with 0.4% collagenase/dispase (Roche Diagnostics GmbH, Penzberg, Germany) and 0.005 % DNaseI (Sigma, St. Louis, MO, USA) in Hanks’ balanced salt solution (Gibco Invitrogen corporation, Paisley, Scotland, UK) for two hours at 37 ℃, put through 60 mesh cell dissociation sieve, washed and plated on 22-mm plas- tic cell culture dishes (Nunc, Roskilde, Denmark). The medium used was DMEM-F12 containing 10% fetal calf serum, penicillin (100 IU/ml), streptomycin sulfate (100 µg/ml), and glutamine (0.5 mM). All the medium components were purchased from Gibco. The cells were maintained at 37 ℃ in 95% air/5% CO2 in
humidified atmosphere, and the culture media were changed every three days. The experiments were performed with or without Ang II, 8-BrcAMP, and ACTH (all from Sigma) seven days after plating when these cells are the most responsive to ACTH [19,20]. The NCI-H295R human adrenocortical carcinoma cell line was obtained from American Type Culture Collection (Manassas, VA, USA). It was plated, maintained, and the experi- mental procedures were performed with or without 8-BrcAMP, 12- O -tetradecanoylphorbol-13-acetate (TPA, Sigma), and Ang II as described previously [20].
RNA extraction, DNase treatment, reverse transcription, and quantitative real-time PCR
Total RNA was extracted from cultured cells using TriZol® Rea- gent (Life Technologies, Rockville, MD, USA) according to the manufacturer’s protocol and from in vivo tissues by ultracen- trifugation through a cesium chloride cushion. DNase treatment and reverse transcription were performed as described previ- ously [21] except that total RNA concentration was 50ng/ul in reverse transcription reactions.
Quantitative real-time PCR was carried out in the Applied Biosys- tems 7500 Real Time PCR System using ABsolute QPCR ROX Mix (ABgene, Epsom, Surrey, UK) and TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) for WNT-4 (assay ID Hs00229142_m1), ß-catenin (assay ID Hs00170025_m1), glyceraldehyde-3-phosphate dehydrogenase (GAPD; assay ID Hs99999905_m1) and transferrin receptor (TFRC; assay ID Hs99999911_m1). GAPD and TFRC were chosen for endogenous controls for cell culture experiments and adrenal tissue samples, respectively, as they showed least variation after several different treatments and in different tissue sample groups in TaqMan® Human Endogenous Control Plate (Applied Biosystems). In order to use standard curve method for the quantitative real-time PCR analysis, standard series of five dilutions containing 48, 12, 6, 1.5 and 0.5ng template cDNA were prepared from pooled sample cDNAs. Sample dilutions comprised of 6ng template cDNA. All standards and samples were run in the total volume of 20 ul in triplicate using universal PCR conditions recommended by Applied Biosystems.
Statistical analyses
Single cell culture experiments consisted of several manipula- tions each in duplicate or triplicate and the experiments were repeated at least three times. Relative expression levels are shown as arithmetic means±SEM in relation to the control adjusted to 1 (or 100%) for cell culture experiments, and to the mean of the normal adult adrenal samples adjusted to 1 (or 100%) for in vivo samples. Statistical significances between con- trol and treatments in cell culture experiments or normal adult adrenal tissue and other adrenal tissue types in vivo were esti- mated by the nonparametric Kruskal-Wallis test followed by two independent group comparisons using the Mann-Whitney test. A p-value of less than 0.05 was considered significant.
Results
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WNT-4 and ß-catenin mRNA expressions in adult and fetal adrenals, and adrenocortical tumors
WNT-4 and ß-catenin mRNAs were detected in all adrenal sam- ples by quantitative real-time RT-PCR ( Fig. 1A, B). However, in some samples, including three of the four virilizing carcinomas,
Relative WNT4 mRNA expression
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Relative beta-catenin mRNA expression w
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the expression of WNT-4 mRNA was only slightly above the detection limit (data for individual samples not shown). In the fetal adrenals, the expression of WNT-4 was 27±8% of the level of the normal adult adrenals (p<0.05). The expression was lower also in the virilizing carcinomas (21 ±20%) and Cushing’s adeno- mas (33±9%) compared with the normal adult adrenals (p<0.05 for both). In contrast, the expression in the Conn’s adenomas was 360±100% compared with the normal adult adrenals (p<0.01). The expression of WNT-4 in the virilizing adenomas and Cushing’s carcinomas did not differ from that in the normal adrenals (· Fig. 1A).
The expression of B-catenin mRNA was higher in the Conn’s adenomas (220±27%) compared with the normal adult adrenals (p<0.01). In the other adrenocortical tumor types, ß-catenin mRNA expression did not differ significantly from that in the normal adult adrenals ( Fig. 1B).
8
☐ primary culture
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Relative WNT4 mRNA expression
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Regulation of WNT-4 expression by 8-BrcAMP, ACTH, Ang II, and TPA in adrenal cell cultures
In primary adrenocortical cell cultures, 8-BrcAMP (0.1 mM) and ACTH (10 nM) increased WNT-4 mRNA expression remarkably (up to 480±100% and 540±150% of the control, respectively; p<0.05 for both) after 24 and 48 hours. The effects of Ang II (10 nM) were minor: a trend to increase at 24 hours followed by a slight decrease at 48 hours (down to 93±3% of the control, p<0.05) ( Fig. 2). In NCI-H295R cells, 1 mM 8-BrcAMP decreased WNT-4 mRNA expression (down to 31+4% of the control, p<0.05) (· Fig. 2), and the effect of 8-BrcAMP was dose dependent and detectable already after a 3-hour treatment ( Fig. 3). The effect of Ang II on WNT-4 mRNA expression in the cell line followed the same pattern as in primary cultures: a slight increase at 24 hours (up to 116±4% of the control, p<0.05) followed by a decrease (down to 55±6% of the control, p<0.05) after 48 hours ( Fig. 2). TPA was tested only in the cell line, and it (100 ng/ml, 3-48 h) significantly decreased WNT-4 mRNA expression with maxi- mal inhibition (down to 17±1% of the control; p<0.05) after 6 hours of incubation ( Fig. 4A). The inhibitory effect of TPA (0.3-300ng/ml, 48h) on WNT mRA expression was dose- dependent reaching maximal inhibition (down to 17±2% of the control; p<0.05) at 100 ng/ml (· Fig. 4B).
Discussion
&
In the present study we show that WNT-4 mRNA is differentially expressed in fetal and adult adrenal glands and in different types of adrenocortical tumors. WNT-4 mRNA expression was upregu-
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Relative WNT4 mRNA expression >
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lated by 8-BrcAMP and ACTH in primary adrenocortical cell cul- tures, but downregulated by 8-BrcAMP in the adrenocortical cell line NCI-H295R. Ang II tended to increase WNT-4 mRNA expres- sion at 24-hour time point and decreased it at 48 hours in both cell culture types. TPA decreased WNT-4 mRNA expression in the NCI-H295R cell line. WNT-4 mRNA expression (Northern blot analysis) has previously been reported in human adult adrenals [15, 16]. Within the adrenal cortex, the expression was higher in the zona glomerulosa/fasciculata than in the zona reticularis [15]. Nuclear ß-catenin immunoreactivity has also been detected in adrenals, localizing mainly in the outer cortex [22]. There is evidence that Wnt-4 may signal through either canonical Wnt/B- catenin or noncanonical pathway depending on the cell type and the set of available Frizzled receptors [11,23,24]. The present study together with the ß-catenin immunoreactivity findings [22] suggests that WNT-4 could signal via the WNT/B-catenin pathway in the human adrenals.
During the human embryogenesis, WNT-4 gene has been shown to be expressed at least in the fetal liver and kidney [16]. Here we show that WNT-4 mRNA is present also in the fetal adrenals
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implicating a possible role for WNT-4 in the human adrenal development. This is consistent with the Wnt-4 knockout mouse model in which Wnt-4 deficiency results in disrupted develop- ment of the zona glomerulosa and subsequent reduction of aldosterone production [17]. In our study, WNT-4 mRNA expres- sion in fetal adrenals was significantly lower than in adult adrenals. This difference may at least partly arise from the greater relative proportion of the zona glomerulosa in the adult than in the fetal adrenals.
In vitro studies of a loss-of-function mutation in the WNT-4 gene in the ovarian adenocarcinoma cell line OVCAR3 suggest that in normal conditions WNT-4 inhibits the expression and activity of the steroidogenic enzymes 3ß-hydroxysteroid dehydrogenase II (3B-HSDII) and 17x-hydroxylase (CYP17) thus regulating steroid production [14]. In the present study, we found that WNT-4 expression was higher in Conn’s adenomas than in normal adult adrenals. This further implicates that WNT-4 is involved not only in the development of zona glomerulosa, as indicated in the Wnt- 4 deficient mouse model [17], but also in the appropriate regula- tion of aldosterone synthesis in human adrenals. Even though
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B-catenin mediated signal transduction is mainly regulated by cytoplasmic stabilization, our finding that ß-catenin expression is elevated in Conn’s adenomas could be a further sign of activation of WNT/B-catenin signal transduction in these tumors.
In contrast to the aldosterone producing tumors, WNT-4 expres- sion in the virilizing and Cushing’s tumors was lower (virilizing carcinomas and Cushing’s adenomas) or at the same level (viri- lizing adenomas and Cushing’s carcinomas) as in normal adrenals. Thus WNT-4 expression does not seem to correlate with the malignancy or steroid secretory pattern in virilizing and cortisol producing adrenal tumors. As we did not analyze proliferation markers in our tumor samples, we cannot evaluate the possible association of WNT-4 expression with the cell pro- liferation activity in different adrenocortical tumors. Further studies of larger tumor groups and immunohistochemistry with specific WNT-4 antibodies may help in verifying our mRNA expression data at the protein level.
Tissier and colleagues showed recently that activating ß-catenin mutations are frequent in both benign and malignant adreno- cortical tumors [25]. Their tumor series did not contain Conn’s adenomas. In our study ß-catenin expression correlated with WNT-4 expression in Conn’s adenomas but not in the other tumor groups. Unfortunately we did not have a chance to study the possible activating ß-catenin mutations in our samples.
Relatively little is known about the factors regulating WNT-4 expression in the human adrenal glands. In this study we showed that both 8-BrcAMP (a protein kinase A activator) and ACTH upregulated WNT-4 mRNA in primary cultures of human adreno- cortical cells, but the effects of Ang II on WNT-4 expression were minor. Most of the cells in our primary cultures represent zona fasciculate-reticularis cells, which should be taken into account when interpreting these results. In any case, our cell culture reg- ulation data suggest a role for WNT-4 in adrenal steroidogenesis. This view is supported by the recent report of Chen and Hornsby in which adenovirus-mediated WNT-4 expression in cultured human adrenocortical cells increased steroidogenesis and expression of steroidogenic genes including aldosterone syn- thase [26]. In contrast to primary cultures, 8-BrcAMP decreased WNT-4 mRNA expression in the NCI-H295R cell line, while the effect of Ang II on WNT-4 mRNA expression followed the same pattern in both cell culture types. The partly controversial results in normal adrenal cells in primary cultures and NCI-H295R cells may result from the activating ß-catenin mutation found in the cell line by Tissier and colleagues [25]. This may indicate that the cell line is not a suitable model for studying the role of WNT-4 in normal human adrenals. Our results from NCI-H295R experi- ments may therefore reflect the behavior of WNT-4 expression in certain adrenal tumors rather than in normal adrenals.
Taken together, we demonstrated the expression of WNT-4 mRNA in the human adrenal gland and several adrenocortical tumors and revealed basic regulation of WNT-4 mRNA in pri- mary cultures of human adrenal cells and in the adrenocortical carcinoma cell line NCI-H295R. The findings suggest that WNT-4 may be an important factor in the regulation of human adrenal function, especially in aldosterone synthesis.
Acknowledgments
&
We thank Ms Minna Heiskanen, Ms Aija Parkkinen, and Ms Merja Haukka for their skillful technical assistance. This study was financially supported by the Jalmari and Rauha Ahokas
Foundation, Pediatric Research Foundation, Finnish Medical Foundation, Sigrid Juselius Foundation, Academy of Finland, and Kuopio University Hospital.
Affiliations
1 Department of Pediatrics, Kuopio University and University Hospital, Kuopio, Finland
2 Department of Pathology, Kuopio University Hospital, Kuopio, Finland
3 Department of Pathology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland
4 Department of Urology, Kuopio University Hospital, Kuopio, Finland
5 Department of Pathology and Forensic Medicine, University of Kuopio, Kuopio, Finland
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