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Extracellular Matrix and Hormones Modulate DAX-1 Localization in the Human Fetal Adrenal Gland

Service of Endocrinology, Department of Medicine, Faculty of Medicine, University of Sherbrooke (M.-C.B., M.O., M.C., A.L., N.G.-P.), Sherbrooke, Quebec, Canada J1H 5N4; Screening Laboratory Hannover (M.P.), D30430 Hannover, Germany; and Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6097 (E.L.), 06560 Valbonne, France

Address all correspondence and requests for reprints to: Dr. Nicole Gallo-Payet, Service of Endocrinology, Faculty of Medicine, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Québec, Canada J1H 5N4. E-mail: nicole.gallo-payet{at}usherbrooke.ca.

	Abstract

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Context: The orphan nuclear receptor DAX-1 is essential for human adrenal cortex development and functions as a transcriptional repressor of multiple genes implicated in steroidogenic pathways.

Objective: The aim of this study was to investigate the localization of the DAX-1 protein in human fetal adrenal glands and to assess whether this protein can be modulated by the extracellular matrix and hormones.

Results: DAX-1 is localized mainly in the nucleus in the outer definitive zone and in the cytoplasm in the fetal zone, whereas the number of DAX-1 positive cells decreases from the external to the internal portion of the gland. When cultured on a collagen or a fibronectin matrix, DAX-1 is localized in the nucleus of the definitive cells and exhibits a nucleocytoplasmic distribution in the fetal cells. ACTH stimulation induces nuclear localization of DAX-1 in fetal cells cultured on collagen without modifying nucleocytoplasmic localization on fibronectin. In contrast, angiotensin II induces the protein to be localized only in the cytoplasm in fetal cells cultured on either collagen or fibronectin.

Conclusions: The localization of DAX-1 is compatible with the known functional properties of DAX-1 regarding the steroidogenic activity of adrenal cells. Moreover, this study suggests that modulation of DAX-1 localization in the fetal adrenal gland by hormones and components of the extracellular matrix may represent a mechanism for controlling the expression of steroidogenic enzymes in the definitive and fetal zones.

	Introduction

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THE HUMAN FETAL adrenal gland from the second trimester of gestation is composed of the definitive zone and the fetal zone; the latter represents 85% of the entire gland (for review, see Ref. 1). The fetal zone primarily expresses the 450C17 enzyme, inducing the production of dehydroepiandrostenedione (DHEA)/DHEA sulfate (DHEAS) (2). On approaching term, the transitional zone becomes immunoreactive for 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and begins to produce cortisol (1, 3).

Among the known hormonal stimuli of the adult adrenal gland are ACTH and angiotensin II (Ang II). ACTH is known to regulate DHEAS production by the fetal zone (4), whereas we have previously shown that Ang II, acting through the AT₂ receptor (the major Ang II receptor present during the second trimester of gestation) (5) also elicits DHEA production (6) and is involved in the programmed cell death seen in the central portion of the gland (7). We have also shown that extracellular matrices are able to interact with hormones to orchestrate cell behavior (proliferation and cell death) and secretion (6).

Steroidogenesis is regulated by two important transcription factors steroidogenic factor-1 (SF-1) and DAX-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenita (AHC) critical region on the X-chromosome gene 1]. DAX-1 is an unusual member of the nuclear hormone receptor superfamily in which mutations cause the X-linked form of AHC (8, 9), and the adrenal gland is characterized by the absence of the permanent zone and by the presence of large vacuolated cells, similar to fetal adrenal cells (9, 10, 11). SF-1 is an important transcriptional activator of a large number of genes involved in steroid hormone production (12), whereas DAX-1 functions as a negative regulator of SF-1-induced transactivation (for review, see Ref. 13).

The first aim of this study was to investigate the cellular distribution of the DAX-1 protein throughout the adrenal gland starting from the second trimester of gestation, when steroidogenesis is absent in the definitive zone and abundant in the fetal zone. The second aim was to investigate whether the composition of the extracellular matrix and stimulation with ACTH and Ang II can influence the cellular localization of DAX-1 in cells from the definitive and fetal zones.

	Materials and Methods

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Chemicals

Chemicals used in the present study were obtained from Tissue-Tek (Miles, Elkhart, IN); tools for immunochemistry were purchased from Vector Laboratories, Inc. (Burlingame, CA); matrix-coated dishes were obtained from BD-VWR Canlab (Ville Mont-Royal, Canada); ACTH-(1–24) peptide (Cortrosyn) was purchased from Organon (Toronto, Canada); Ang II was purchased from Bachem (Marina Delphen, CA). The anti-DAX-1 2F4 antibody was raised against a peptide corresponding to amino acids 135–166 of the human DAX-1 protein (14, 15); 3ß-HSD and P450C17 antibodies were provided by Drs. Van Luu-The and Alain Bélanger (Centre de Recherche Hospitalier de Québec, Ste-Foy, Canada); SF-1 antibody was purchased from Upstate Biotechnology, Inc (Lake Placid, NY); secondary antibodies coupled to Alexa 488, Alexa 594, and 4',6-diamido-2-phenylindole hydrochloride (DAPI) were obtained from Molecular Probes (Eugene, OR); horseradish peroxidase-conjugated antirabbit antibody was purchased from Amersham Biosciences (Oakville, Canada); the enhanced chemiluminescence system was obtained from Roche (Montréal, Canada). All other chemicals were of A grade purity.

Retrieval and preparation of glands

Fetal adrenal glands were obtained from fetuses, 14–21 wk of age, at the time of therapeutic abortion. Fetal ages were estimated by foot length and time after last menstruation (16). The project was approved by the human subject review committee of our institution. After retrieval, glands were cleansed of fat and immediately processed for cellular preparation, frozen in liquid nitrogen for Western blotting analyses, or included in cryoprotectant (OCT, Tissue Tek).

Immunohistochemical studies

Adrenal glands were fixed in 3.7% paraformaldehyde, embedded in paraffin, cut into 3-µm sections, and processed for indirect immunoperoxidase immunohistochemistry. After toluene and hydration processing, sections were incubated in 0.3% H₂O₂ and 1 mM KCN to quench endogenous peroxidase activity. The sections were incubated with the primary anti-DAX-1 2F4 antibody (1:1000) for 30 min at room temperature. Where indicated, 2F4 peptide antigen was added in a 20-fold molar excess to the antibody immediately before incubation. The sections were incubated with antimouse biotinylated secondary antibody for 1 h at room temperature, washed, and incubated with Vectastain Elite ABC reagent, followed by detection using the 3,3'-diaminobenzidine reaction. Counterstaining was performed using hematoxylin, and slides were mounted in VectaMount nonaqueous mounting medium. Immunolabeling was observed using an Eclipse 300 microscope (Nikon, Mississauga, Canada) equipped with a CoolSnap FX color digital camera (Roper Scientific, Tucson, AZ). Images were taken using a x20 objective.

Cell culture

Whole adrenal tissues were used for cell preparation as described previously (5, 6). Cells were plated at a density of 1 x 10⁵ cells in 35-mm diameter tissue culture dishes coated with collagen IV or fibronectin. Cells were grown for 2 d in a humidified atmosphere of 5% CO₂ at 37 C, in the absence or presence of 10 nM ACTH or 100 nM Ang II after an initial resting period of 24 h. More than 300 cells from five different cell cultures performed with adrenal glands from 16 (n = 2), 18 (n = 2), and 19 (n = 1) wk gestation were analyzed.

Immunofluorescence studies in whole glands and cell culture

Tissue sections (3–5 µm) and cells grown in culture were fixed in 3.7% formaldehyde. Tissue sections were incubated with the primary anti-DAX-1 2F4 antibody (1:100) overnight at 4 C, whereas cells were incubated with anti-DAX-1 2F4 antibody (1:1000) for 60 min at room temperature. Where indicated, 2F4 peptide antigen was added in a 20-fold molar excess to the antibody immediately before incubation. Tissue sections were incubated with the secondary antimouse Alexa 594-coupled antibody (1:500; red) for 1 h at room temperature and cells were incubated with the secondary antimouse Alexa 488-coupled antibody (1:500; green). Cells were also incubated with an anti-P450C17 (1:1000) or an anti-SF-1 antibody (1:1000), followed by the secondary antirabbit Alexa 594-coupled antibody (1:500; red). Tissue and cells were stained with DAPI (1:1000) to visualize nuclei. Slides were mounted with Vectashield mounting medium and examined under a Nikon Eclipse 2000 inverted fluorescence microscope equipped for epiillumination. Tissue images were taken using a x20 objective, whereas cell images were taken using a x100 objective. Images were processed by Photoshop 4.0 (Adobe Systems, San Jose, CA). For quantification in tissue sections, DAX-1-positive cells and the total number of cells were counted in the number of fields indicated in parentheses. In all cases, no specific staining was observed when primary antibodies were replaced by nonimmune mouse serum (data not shown). Note that all illustrations (pictures and micrographs) were taken with the same camera settings for contrast and brightness.

Western blotting

Frozen tissues were used for determination of DAX-1 and steroidogenic enzymes (3ß-HSD and P450C17). Cell homogenates were prepared and separated as previously described (17). Membranes were blocked and incubated with the primary antibodies at dilutions of 1:1000 (DAX-1), 1:500 (3ß-HSD), and 1:2000 (P450C17) overnight at 4 C. Membranes were incubated for 1 h with horseradish peroxidase-conjugated antimouse or antirabbit antibody (Amersham Biosciences). Detection was performed using an enhanced chemiluminescence detection system from Roche on Kodak XK-1 films (Eastman Kodak Co., Rochester, NY).

Data analysis

Images were processed using Adobe Photoshop 4.0. Ten glands from various ages were examined. Unless indicated, data are presented as the mean ± SE of the number of cells indicated in the text. Statistical analyses of the data were performed by one-way ANOVA. Homogeneity of variance was assessed by Bartlett’s test, and P values were obtained from Dunnett’s tables.

	Results

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Localization of DAX-1 in human fetal adrenal gland from second trimester of gestation

The distribution and cellular localization of DAX-1 were examined in adrenal glands from 15-, 16-, 19-, and 21-wk-old fetuses. Similar results were obtained for all samples. As illustrated in a gland from a 22-wk-old fetus, the 2F4 antibody produced specific labeling (Fig. 1, A, Ab, and Ac), which was completely abrogated after 2F4 peptide adsorption (Fig. 1B). Most, if not all, cells from the definitive zone were labeled with varying intensity and with labeling present mainly in the nucleus, although some staining was observed in the cytoplasm (Fig. 1A). In the fetal zone, which also displayed a nucleocytoplasmic labeling pattern, the number of DAX-1-negative cells increased from the outer region, which lies adjacent to the definitive zone (Fig. 1Ac), toward the central portion of the gland (Fig. 1Ab).

View larger version (128K): [in this window] [in a new window]   FIG. 1. Immunohistochemical detection of DAX-1 in a human fetal adrenal gland. Adrenal glands were processed as described in Materials and Methods. A, DAX-1 labeling in the external definitive zone (DZ) and the fetal zone (FZ). B, Nonspecific binding, as revealed by peptide adsorption of 2F4 antibody. Ab and Ac are magnifications of the selected portion of the fetal and definitive zones shown in A; Bb and Bc are magnifications of the selected portion of the fetal and definitive zones shown in B. The lower panel illustrates a schematic representation of the adrenal gland section indicating the location of the pictures. Images are from a 22-wk-old fetal gland. Images were taken with a x20 objective. Scale bars, 26 and 13 µm (magnification, x385 and x770), respectively, for the figure and the inset.

View larger version (55K): [in this window] [in a new window]   FIG. 2. Immunofluorescence detection of DAX-1 in a human fetal adrenal gland. Frozen sections of a 20-wk-old human fetus were processed as described in Materials and Methods. A, a–c, DAX-1 labeling in the external definitive zone (DZ) and the fetal zone (FZ). B, a–c, DAPI staining. C, a–c, Merged images. Ab, Bb, and Cb are magnifications of the selected portions of the definitive zone shown in Aa, Ba, and Ca; Ac, Bc, and Cc are magnifications of the selected portion of the fetal zone shown in Aa, Ba, and Ca. D, Specific labeling of a similar region. E, Nonspecific binding, as revealed by peptide adsorption of 2F4 antibody, taken in a portion of the fetal zone. D and E, Labeling without pseudocolor. DAX-1 localization in nuclei of definitive zone (arrowheads) and in cytoplasm of fetal cells (arrows). DAX-1 localization at the cell periphery is evident (open arrows). Images were taken with a x20 objective. Scale bars, 70 µm for Aa, Ba, and Ca; 10 µm for Ab, Bb, and Cb (magnification, x143 and x1000, respectively).

View larger version (53K): [in this window] [in a new window]   FIG. 3. Western blot analyses of DAX-1, 3ß-HSD, and P450C17 in human fetal adrenal (HFA), aged 16, 17, 19, and 20 wk, and from adult adrenal glands. Analyses were performed on homogenates of whole adrenal glands, as described in Materials and Methods. Cell lysates containing 30 µg proteins were subjected to Western blot analysis using specific antibodies against DAX-1 (1:1000), 3ß-HSD (1:500), and P450C17 (1:2000) proteins. Numbers on the left indicate the molecular masses of proteins in kilodaltons.

As shown in Fig. 4A, fetal zone cells in culture (arrow) were distinguishable from the small definitive zone cells (arrowhead) by their size, polygonal morphology, presence of bright lipid droplets, and, more specifically, their specific expression of P450C17 (Fig. 4Ab). In definitive cells cultured on collagen IV (Fig. 4B, arrowheads), 73.5 ± 3.9% of cells (344 of a total of 472 counted cells in 12 different fields) exhibited nuclear DAX-1, as shown by the azure blue color in the superimposed images of DAX-1 and DAPI (Fig. 4B, arrowheads). However, in fetal cells, which represented 34.6 ± 8.2% of the cell population in culture, DAX-1 was expressed in both nucleus and cytoplasm (Fig. 4B, arrows). On collagen IV-cultured cells, stimulation with ACTH increased the number of DAX-1-positive cells to 92.8 ± 8.1% for both definitive and fetal cells (Fig. 4C, overlay between DAX-1 and P450C17). Conversely, although stimulation with Ang II did not modify nuclear localization of DAX-1 in definitive zone cells (Fig. 4D, arrowheads), DAX-1 exhibited either nucleocytoplasmic or cytoplasmic localization in the fetal cells (Fig. 4D, arrows 1 and 2).

View larger version (47K): [in this window] [in a new window]   FIG. 4. Localization of DAX-1 in cells cultured on collagen IV and stimulated with ACTH or Ang II. Human fetal adrenal cells were plated on collagen-coated dishes (1 x 105 cells) as described in Materials and Methods. Cells were incubated for 2 d in the absence (A and B) or presence of (C) 10 nM ACTH or 100 nM Ang II (D). Cells were processed for immunofluorescence labeling using the anti-DAX-1 2F4 antibody (green; a) and anti-P450C17 antibody (red; b). Cells were incubated with DAPI to visualize nuclei (c) with phase contrast morphology of cells shown in d. The azure blue color in the overlay images indicates exact overlap. Images are representative illustrations of more than 300 cells originating from at least four different cell cultures and specimens. Scale bar, 20 µm (magnification, x500).

View larger version (32K): [in this window] [in a new window]   FIG. 5. Localization of DAX-1 in cells cultured on fibronectin and stimulated with ACTH or Ang II. Human fetal adrenal cells were plated on fibronectin-coated dishes (1 x 105 cells) as described in Materials and Methods. Cells were incubated for 2 d in the absence (A) or presence (B) of 10 nM ACTH or 100 nM Ang II (C). Cells were processed for immunofluorescence labeling using the anti-DAX-1 2F4 antibody (green; a) and with an anti-P450C17 antibody (red; b). Cells were incubated with DAPI to visualize nuclei (c); phase contrast morphology of the cells is shown in d. The azure blue color in the overlay images indicates exact overlap of DAX-1 and DAPI, and the yellow color shows the overlap of DAX-1 and P450C17 in the cytoplasm. Images are representative illustrations of more than 300 cells originating from at least four different cell cultures and specimens. Scale bar, 20 µm (magnification, x500).

View larger version (23K): [in this window] [in a new window]   FIG. 6. Comparative localization of DAX-1 and SF-1 in human fetal adrenal cells cultured on fibronectin. Definitive cells (A) and fetal cells (B) were cultured for 2 d on fibronectin-coated dishes (1 x 105 cells), then fixed and processed for immunofluorescence labeling as described in Materials and Methods. DAX-1 was stained using the anti-DAX-1 2F4 antibody (green; Aa and Ba), and SF-1 was stained using the anti-SF-1 antibody (red; Ab and Bb). Overlay images are shown in Ac and Bc, with the yellow color indicating exact overlap. Ad and Bd, Phase contrast morphology of corresponding cells. Images are representative illustrations of more than 100 cells originating from at least four different cell cultures and specimens. Scale bar, 20 µm (magnification, x500).

	Discussion

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Differential subcellular localization of DAX-1 is likely to have important physiological consequences. The nucleocytoplasmic localization of the DAX-1 protein has been recently shown by studies performed in the National Cancer Institute H295R adrenal cell line (19). DAX-1 is a nucleocytoplasmic shuttling protein associated with ribonucleoprotein structures in the nucleus and polyribosomes in the cytoplasm (for review, see Ref. 13). Using cells transfected with various DAX-1 constructs, we recently found an inverse relationship between the extent of nuclear localization and the transcriptional repression activity of individual DAX-1 AHC missense mutants (20).

In the adrenal gland, we have previously shown that the composition of the extracellular matrix plays a critical role in modulating cell proliferation, programmed cell death, and steroidogenesis (6, 7, 21, 22, 23). In this study we provide evidence that collagen IV and fibronectin modulate DAX-1 localization in a cell type-specific manner. DAX-1 is localized in the nucleus of definitive cells cultured on collagen IV or fibronectin and exhibits nucleocytoplasmic localization in fetal cells. In addition, ACTH stimulation clearly favors nuclear labeling in both cell types regardless of the matrix used for cultures. DAX-1 is known to function as a powerful negative regulator of steroidogenesis both in vitro and in vivo (15, 24, 25, 26, 27, 28). In adrenocortical tumors, DAX-1 expression and the capacity of tumors to produce steroid hormones are also inversely related (29, 30). The nuclear localization of DAX-1 in the definitive cells is thus compatible with the negative modulation of steroidogenesis in these cells during the second trimester of gestation. Our results using cell culture also provide evidence that the percentage of DAX-1 immunoreactive cells is higher in definitive cells compared with fetal cells, whereas in situ, the fetal zone represents the major component of this gland. This observation in culture reflects the high proliferative capacity of definitive cells compared with fetal cells, which do not proliferate, as observed in the whole gland (1, 6, 31). Our results thus corroborate data obtained in the adult or from in vitro assays, showing that in the definitive zone, nuclear DAX-1 may act to down-regulate the expression of 3ß-HSD.

In contrast, localization of DAX-1 in the cytoplasm of fetal cells may allow for their high expression of P450C17 and their secretion of DHEA/DHEAS. In addition, Ang II induces cytoplasmic localization of DAX-1 in fetal zone cells (the only adrenal cell type expressing AT₂ receptor) (5) when these cells are cultured on collagen IV or fibronectin. The cytoplasmic localization in situ and the observations in cell lines (19) suggest that DAX-1 may play additional regulatory functions in posttranscriptional processes. A role in cell migration or organogenesis may be proposed based on the observations that in the X-linked form of AHC, the adrenal gland is characterized by the absence of the permanent zone and the presence of large vacuolated cells, similar to fetal adrenal cells (9, 32).

Specific nuclear or cytoplasmic localization further strengthens the hypothesis of an important role for DAX-1 in the regulation of 3ß-HSD, which needs to be down-regulated during the second trimester of pregnancy. This contrasts with the up-regulation of P450C17 expression, which is necessary to promote the intense production of DHEA/DHEAS, stimulated by the combined presence of ACTH and AT₂ receptors of Ang II in the fetal zone (6).

In summary, our results strongly suggest that both extracellular matrix and hormones such as ACTH and Ang II may modulate the localization of DAX-1 in the human fetal adrenal gland. This may represent an important mechanism for these factors to operate their well-known actions on adrenal cell behavior and hormonal secretion.

	Acknowledgments

 
We thank Lucie Chouinard and Claude Roberge for their invaluable experimental assistance and stimulating discussions. We express our gratitude for the generous gift of antisera from our colleagues Drs. Van Luu-The and Alain Bélanger (Centre de Recherche Hospitalier de Québec, Ste-Foy, Canada).

	Footnotes

 
This work was supported by grants from the Fondation pour la Recherche sur les Maladies Infantiles, the Canadian Institutes for Health Research (to N.G.-P.), the Fondation pour la Recherche Médicale (to E.L.), and Institut National de la Santé et de la Recherche Médicale-Fonds de la Recherche en Santé du Québec (Grant 7252). N.G.-P. is the recipient of a Canada Research Chair in Endocrinology of the Adrenal Gland, and E.L. is the recipient of a Centre National de la Recherche Scientifique Actions Thèmatiques et Initiatives sur programme grant.

First Published Online June 14, 2005

¹ M.-C.B. and M.O. contributed equally to this work and should both be considered first authors.

Abbreviations: AHC, Adrenal hypoplasia congenita; Ang II, angiotensin II; DAPI, 4',6-diamido-2-phenylindole hydrochloride; DHEA, dehydroepiandrostenedione; DHEAS, dehydroepiandrostenedione sulfate; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; SF-1, steroidogenic factor-1.

Received March 25, 2005.

Accepted June 6, 2005.

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