Conditional Inactivation of Glucocorticoid Receptor Gene in Dopamine ...

GLUCOCORTICOIDS-CRH-ACTH-ADRENAL

Conditional Inactivation of Glucocorticoid Receptor Gene in Dopamine-␤-Hydroxylase Cells Impairs Chromaffin Cell Survival Rosanna Parlato, Christiane Otto, Jan Tuckermann, Stefanie Stotz, Sylvia Kaden, Hermann-Josef Gro¨ne, Klaus Unsicker, and Gu¨nther Schu¨tz Departments of Molecular Biology of the Cell I (R.P., C.O., S.S., G.S.) and Cellular and Molecular Pathology (S.K., H.-J.G.), German Cancer Research Center, 69120 Heidelberg, Germany; Leibniz Institute for Age Research (J.T.), Fritz Lipmann Institute, 07445 Jena, Germany; and Department of Neuroanatomy and Interdisciplinary Center for Neurosciences (K.U.), University of Heidelberg, 69120 Heidelberg, Germany

Glucocorticoid hormones (GCs) have been thought to determine the fate of chromaffin cells from sympathoadrenal progenitor cells. The analysis of mice carrying a germ line deletion of the glucocorticoid receptor (GR) gene has challenged these previous results because the embryonic development of adrenal chromaffin cells is largely unaltered. In the present study, we have analyzed the role of GC-dependent signaling in the postnatal development of adrenal chromaffin cells by conditional inactivation of the GR gene in cells expressing dopamine-␤-hydroxylase, an enzyme required for the synthesis of noradrenaline and adrenaline. These mutant mice are viable, allowing to study whether in the absence of GC signaling further development of the adrenal medulla is affected. Our analysis shows that the loss of GR leads not only to the loss of phenylethanolamineN-methyl-transferase expression and, therefore, to inhibition of adrenaline synthesis, but also to a dramatic reduction in the number of adrenal chromaffin cells. We provide evidence that increased apoptotic cell death is the main consequence of GR loss. These findings define the essential role of GCs for survival of chromaffin cells and underscore the specific requirement of GCs for adrenergic chromaffin cell differentiation and maintenance. (Endocrinology 150: 1775–1781, 2009)

hromaffin cells of the adrenal medulla are neuroendocrine cells producing noradrenaline (NA) and adrenaline (A), which originate from the sympathoadrenal cell lineage, a major derivative of the neural crest cells (1). Different environmental signals influence the differentiation of sympathoadrenal precursors to sympathetic neurons or chromaffin cells. Among these factors, neurotrophins and glucocorticoid hormones (GCs) are well characterized (2–5). GCs act via the glucocorticoid receptor (GR), a ligand-dependent transcription factor regulating the expression of specific target genes (6). Classical in vitro experiments indicated that GC-mediated signaling is responsible for differentiation of chromaffin cells by inhibiting the differentiation of sympathoadrenal precursors toward a default neuronal phenotype. This function was thought to be performed along with the GC-dependent prosurvival role on chromaffin progenitor cells in culture (3). GCs would also stimulate the synthesis of enzymes, such as phenylethanolamine-N-methyl-transferase

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(PNMT), the enzyme responsible for establishing the adrenergic phenotype (7), and inhibit proliferation of progenitors in vitro when stimulated with nerve growth factor (8). According to these in vitro studies, it was hypothesized that the loss of GCdependent signaling would affect the development of chromaffin cells. However, these expectations were not validated in vivo by the phenotype of mutant mice carrying a germ line ablation of the GR gene (9). Unexpectedly, chromaffin cell determination occurs in these mice, and no significant differences were found in the number of chromaffin cells between controls and mutants, excluding an effect of GCs on chromaffin cell survival. In GR null mice, no alteration in proliferation of chromaffin cells was found. Only the expression of a restricted number of molecular markers, such as PNMT, was dependent on the GR activity (9). Because GR null mutants are perinatally lethal, due to lung failure, the role of GCs in the postnatal adrenal gland remains unknown.

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2008-1107 Received July 28, 2008. Accepted November 18, 2008. First Published Online November 26, 2008

Abbreviations: A, Adrenaline; DBH, dopamine-␤-hydroxylase; GC, glucocorticoid hormone; GR, glucocorticoid receptor; GRDBHCre, GRfl/fl; DBHCre; IHC, immunohistochemistry; MR, mineralocorticoid receptor; NA, noradrenaline; PNMT, phenylethanolamine-Nmethyl-transferase; P7, postnatal d 7; TH, tyrosine hydroxylase; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling.

Endocrinology, April 2009, 150(4):1775–1781

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Deletion of GR in Chromaffin Cells

To investigate whether the postnatal development of chromaffin cells is dependent on GC signaling, we have generated mouse mutants lacking GR in chromaffin cells using the Cre/loxP recombination system (10). We took advantage of a transgenic line expressing the Cre recombinase under control of the regulatory elements of the dopamine-␤-hydroxylase (DBH) gene. This line reproduces the expression pattern of the DBH gene, an enzyme required for the synthesis of catecholamines, and specifically expressed in noradrenergic and adrenergic cells (11, 12). The generation of the mutant mice lacking the GR in chromaffin cells enabled us to study the consequences of GR loss also postnatally because in contrast to the germ line mutants, their viability is not altered. The conditional GR mutants show a significant loss of chromaffin cells, providing the first evidence that GCs are required for survival of chromaffin cells.


brown color cytoplasm and clear nucleus were counted as positive in sections containing the adrenal medulla for both control and mutant mice. The total number of positive cells per adrenal was counted at postnatal d 7 (P7) and P20. At other stages TH⫹ cells in 12 serial sections per specimen were counted, and the results are presented as the percentage (%) of positive cells in comparison to the respective control. For the analysis of proliferation, cells TH⫹ and Ki67⫹ were counted in 12 serial sections per specimen. The results are expressed as percentage (%) of TH⫹ cells that are Ki67⫹. For analysis of apoptosis, TH positive cells also stained with activated caspase-3 antibody were counted in 12 serial sections per specimen. The results are expressed as fold change in comparison to the respective controls. Values shown are mean ⫾ SEM for at least three mice per each genotype. Statistical significance was analyzed using a homoscedastic Student’s t test. Values were considered significantly different with P ⬍ 0.05.

Electron microscopy For electron microscopy, adrenals were fixed and treated as previously described (14).

Materials and Methods Generation of GRfl/fl; DBHCre (GRDBHCre) mutant mice Mice carrying the GR conditional alleles (GRflox/flox) (13) were crossed to the transgenic line DBHCre (11) to generate GR⫹/flox; DBHCre mice. Mutant mice GRflox/flox; DBHCre, indicated as GRDBHCre, were generated by crossing GR⫹/flox; DBHCre mice with GRflox/flox. The analysis of the genotype was performed as previously described. In all the experiments, we used GRflox/flox littermate mice as control.

Histological and immunohistochemical analysis Embryos or adrenal glands were fixed in 4% paraformaldehyde (pH 7.2), overnight processed for paraffin sections, sectioned at 7 ␮m on a microtome. For immunohistochemistry (IHC) the following primary antibodies were used: anti-GR (1:2000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-tyrosine hydroxylase (TH) (1:500; CHEMICON International, Inc., Temecula, CA); anti-DBH polyclonal (1:500; Alpha Diagnostics International Inc., San Antonio, TX); anti-PNMT (1:100; CHEMICON International); anti-Ki67 (1:500; Abcam, Inc., Cambridge, MA); antiactivated caspase-3 antibody (1:800; Cell Signaling Technologies, Beverly, MA); anti-NeuN (1:2000; CHEMICON International); and anti-mineralocorticoid receptor (MR) (1:50) (gift of Celso Gomez-Sanchez; University of Mississippi Medical Center, Jackson, MS). Antigen unmasking was used for IHC on paraffin sections. The sections were incubated in citrate buffer (pH 6.0), and boiled in a microwave oven. As blocking solution, 5% pig serum in PBS was used. The primary antibodies diluted in blocking solution were incubated overnight at 4 C. Biotin-conjugated secondary antibody was diluted 1:400 in PBS, and detection was performed using the avidin-biotin system (Vector Laboratories, Burlingame, CA) with the VECTOR peroxidase kit. The staining was developed with 3⬘3-diaminobenzidine HCl and H2O2 (Sigma-Aldrich Corp., St. Louis, MO). Analysis of apoptosis was performed by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5⬘-triphosphate nick-end labeling (TUNEL) assay using DeadEnd Fluorometric TUNEL System (Promega Corp., Madison, WI) according to the manufacturer’s instructions. To identify chromaffin cells, the sections were further immunostained with anti-TH antibody and fluorescent secondary antibody Alexa594 (Invitrogen Corp., Carlsbad, CA). Sections were then rinsed and mounted with Fluorescent mounting medium (Vector Laboratories).

Cell counts and statistical analysis After immunolabeling, cell counts were performed at ⫻40 magnification in bright-field. Clearly identified TH⫹ cells characterized by

FIG. 1. Loss of GR and PNMT expression in adrenal chromaffin cells. Paraffin sections from embryonic d 18.5 control (A) and GRDBHCre mutant (B) mice stained with a GR-specific antibody reveal specific loss of GR immunoreactivity in the adrenal medulla of the mutant, whereas GR expression in the cortex is unaffected (arrowhead). The adrenal medulla is indicated by a dashed line in A and B. Expression of PNMT is lost in GRDBHCre mutants (C and D). The arrowheads in the inset highlight few immunoreactive cells at this stage. Scale bars, 100 ␮m (A and B); 250 ␮m (C and D).



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Results and Discussion Targeted disruption of GR in adrenal chromaffin cells results in severe loss of chromaffin cells To study the specific role of glucocorticoid signaling in adrenal chromaffin cells, we generated a conditional mutant of the GR gene using the Cre-loxP system (10). We crossed a mouse strain carrying the conditional (GRflox) allele of GR (13) and a transgenic mouse line expressing the Cre recombinase under the control of the DBH gene (11). The DBHCre mice express the Cre recombinase in noradrenergic and adrenergic neurons of the central and peripheral nervous system recapitulating the expression pattern of DBH (11). We verified the loss of the GR protein in adrenal chromaffin cells of GRDBHCre mutants by IHC at embryonic d 18.5. In adrenals of control mice, GR expression is found in the adrenal cortex and in chromaffin cells of the medulla (Fig. 1A). In the GRDBHCre mice, GR immunoreactivity is lost in chromaffin cells but is unaltered in the cortex (Fig. 1B). The loss of GR leads to the loss of the A-synthesizing enzyme PNMT (Fig. 1, C and D, inset), as also reported in germ line mutants (9). The GRDBHCre mutants are viable and fertile, and they do not appear different from control littermates. Therefore, we analyzed the adrenal glands in 1-, 3-, and 12-month-old mice (Fig. 2). Size and weight of the adrenal glands are alike in control and mutants (data not shown). In comparison to a representative 12-month-

FIG. 3. TH, DBH, and PNMT immunoreactivities in adrenal glands of adult GRDBHCre mutants. IHC using antibodies against TH, DBH, or PNMT antibodies performed on serial sections of adrenal glands isolated from control and GRDBHCre mutant mice (3 months old). Dashed line in F defines the adrenal medulla in the mutant. A, C, and E, Control. B, D, and F, GRDBHCre mutant. Scale bar, 125 ␮m.

FIG. 2. Degeneration of adrenal medulla in adult GRDBHCre mutant mice. Hematoxylin and eosin staining shows a severe degeneration of the adrenal medulla in 1-, 3- and 12-month-old GRDBHCre mutants (B–D) in comparison to a representative 12-month-old control (A). A dashed line indicates the adrenal medulla. Bracket in C indicates increased vascularization. Arrowheads in D indicate infiltrates. Scale bar, 125 ␮m.

old control (Fig. 2A), a dramatic degeneration of the adrenal medulla is clearly visible in young GRDBHCre mutants by hematoxylin and eosin (Fig. 2B). Although size and morphology of the chromaffin cells are indistinguishable between control and mutant, the number of chromaffin cells is markedly decreased. No changes were observed in the cortical cell size and morphology. At later stages the degenerative process is connected to increased vascularization in the space between the cortex and the medullary cells (Fig. 2C), and macrophage infiltration is clearly visible in older mice (Fig. 2D, arrowheads, and sup-

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Several studies indicated that not only the expression of PNMT but also of the other catecholamine enzymes, TH and DBH (15–17), are in part dependent on glucocorticoids (GCs), based on examination of hypophysectomized rats with undetectable levels of plasma corticosterone and cultures of PC12 cells treated with the synthetic glucocorticoid dexamethasone. To assess whether the structural changes observed in the adrenal medulla of GRDBHCre mutants are related to impaired expression of catecholaminergic markers, we performed IHC with specific antibodies for TH, DBH, and PNMT in controls and GRDBHCre mutants (3 month old) (Fig. 3). This analysis reveals the presence of a thin layer of chromaffin cells still expressing TH (Fig. 3, A and B) and DBH (Fig. 3, C and D), but not PNMT (Fig. 3, E and F) in GRDBHCre mutants. We conclude that at this stage, the basal levels of TH and DBH are independent of GCs. On the other hand, it has to be indicated that GCs are required for PNMT expression, but several different factors, such as low permissive hypoxic conditions and depolarizing agents, also influence its expression (18, 19). To study whether the expression of the MRs is influenced in the adrenal medulla of GRDBHCre mutants by the loss of GR, we have performed IHC with a MR specific antibody. This analysis reveals low levels of immunoreactivity in both control and mutant chromaffin cells, and a partial effect on the nuclear distribution of MR in GRDBHCre mutants (supplemental Fig. 2). GR can regulate gene expression either by binding to DNA as a homodimer or by interaction with other DNA-binding transcription factors as a monomer. Mice carrying a point mutation in the dimerization loop of the DNA binding domain of the GR (GRdim) have a defect of GR DNA binding but retain the protein interaction of the GR (20). We used this mouse model to determine the contribution of GR dimerization to the survival and presence of chromaffin cells. Interestingly, the morphology of the FIG. 4. Early effects on chromaffin cell number in GRDBHCre mutant (M) mice. IHC performed on serial sections from adrenal glands of controls (C) and GRDBHCre mutant mice with TH antibody at P7 (A and B) adrenal glands in GRdim mutants differs and P20 (C and D). E, Quantification of numbers of TH positive cells in the adrenal glands of control (black from the respective controls because of the bar) and GRDBHCre (white bar). Shown are the means ⫾ SEM for at least three to four mice of each expansion of the blood vessels between the genotype. F, TH positive cells, expressed as percentage (%) of respective controls counted at P0, P3, P7, and P20. Values were considered significantly different with P ⬍ 0.05 (*) compared with the respective most inner zone of the cortex, the zona recontrols. Scale bar, 250 ␮m. ticularis, and the adjacent medullary tissue, but not in the chromaffin cells. Indeed, these mice still expressing GR do not show alterations in TH and plemental Fig. 1, which is published as supplemental data on PNMT expression (20) (supplemental Fig. 3, A and B). These The Endocrine Society’s Journals Online web site at results suggest that the impaired survival of chromaffin cells and http://endo.endojournals.org).



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expression of PNMT observed in the GRDBHCre mice is due to a DNA-independent transcriptional function of GR. GCs are required for survival of adrenal chromaffin cells To elucidate the onset of the severe morphological changes observed in the adrenal medulla of GRDBHCre mice, we performed a detailed analysis of the early postnatal stages. The overall morphology of the adrenal medulla appears similar in controls and GRDBHCre mutants analyzed at P7 by TH immunoreactivity (Fig. 4, A and B). Differently from controls (Fig. 4C), at P20 TH immunoreactive cells form in the mutant a thin layer underlying the cortex (Fig. 4D), as observed in the adult mutant mice (Fig. 3). Unexpectedly, the quantification of the total number of TH positive cells reveals that already at P7 there is a significant loss of TH immunoreactive cells in comparison to controls. A similar decrease is also found at P20 (Fig. 4E). The quantification at earlier stages (P0 and P3) detects a significant decrease in the number of TH positive cells at birth and in the following postnatal days (Fig. 4F). Thus, we investigated whether one possible cause for the decreased number of chromaffin cells can be ascribed to reduced proliferation. However, this is unlikely, because of the proposed role of GCs (8), one would expect increased cell proliferation in the absence of GR. Moreover, during the stress hyporesponsive period occurring in mice after birth until d 12, GCs have low influence (21). By counting TH positive cells immunolabeled with an antibody recognizing the Ki67 antigen as marker of proliferating cells, we found no changes at P0 between controls and mutants, and a slight increase in the number of proliferating cells in the GRDBHCre mutants at P3 (Fig. 5A). This is consistent with the antiproliferative effect of GCs observed in other organs, such as the developing lung (22). Therefore, we exclude that reduced proliferation of chromaffin cells in GRDBHCre mutants is responsible for the lower number of chromaffin cells. To investigate whether the reduction of chromaffin cells resulted from increased cell death, we performed terminal TUNEL assay, and analyzed the number of apoptotic cells labeled with activated caspase-3 antibody at P1 and P3 (Fig. 5, B–F). Thus, we found an increased number of TUNEL-positive cells in the GRDBHCre mutants at both stages (Fig. 5, D and E) and also of caspase-3 positive cells (Fig. 5F). Based on these results, we conclude that GCs are required for survival of chromaffin cells, and one mechanism activated by the loss of GR is apoptotic cell death. NA producing cells are only partially affected by loss of GR To establish whether the loss of GR leads to dedifferentiation and acquisition of a neuronal phenotype, we performed immunohistochemical analysis with NeuN antibody, a marker for neuronal nuclei at P7 and P20 in control and GRDBHCre mutant mice (Fig. 6, A and B). The percentage of chromaffin cells that are NeuN⫹ cells is similar in control and GRDBHCre mutants at P7 (33.1 ⫾ 3.2 and 45.1 ⫾ 16.1%, respectively). At the same stage, virtually all chromaffin cells still present in the adrenal medulla

FIG. 5. Proliferation and survival of chromaffin cells in GRDBHCre mutant mice. A, Percentage of proliferating TH⫹ cells in the adrenal glands of control (black bar) and GRDBHCre (white bar) at P0 and P3 after IHC with Ki67 and TH specific antibodies on serial sections. B–E, TUNEL staining (green) performed on paraffin sections from adrenal glands and immunostained with TH antibody (red) to identify cell death in control and GRDBHCre mutants at P1 and P3. F, Quantification of apoptotic cells analyzed by IHC with an antibody recognizing activated caspase-3 in combination with TH at P1 and P3. The measurements are expressed as fold change in comparison to the respective controls. Shown are the means ⫾ SEM for at least three to four mice of each genotype. Values were considered significantly different with P ⬍ 0.05 (*) compared with the respective controls. Scale bar, 60 ␮m.

of the GRDBHCre mutants are NeuN⫹, as indicated by the number of TH⫹ cells in comparison to control (44.1 ⫾ 14.4%). This observation suggested that a subset of chromaffin cells is less severely affected by the loss of GR. To elucidate the nature of the remaining cells, we have performed silver stain analysis on semithin sections at P20. This method allows to identify noradrenergic chromaffin cells because of the intense dark staining of the NA granules in comparison to adrenergic granules having a lower density, as shown in controls (Fig. 6C). Interestingly, in the mutants the chromaffin cells still present belong to the noradrenergic subtype (Fig. 6D). These results support the existence of specific programs for the induction of the adrenergic vs. noradrenergic phenotype in

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antibody against mineralocorticoid receptor was kindly provided by Celso Gomez-Sanchez. Address all correspondence and requests for reprints to: Gu¨nther Schu¨tz, Department. of Molecular Biology of the Cell I, German Cancer Research Center, 69120 Heidelberg, Germany. E-mail: g.schuetz@ dkfz.de. Present address for C.O.: Women’s Healthcare, Bayer Schering Pharma AG, 13353 Berlin, Germany. This work was supported by the “Deutsche Forschungsgemeinschaft” through Collaborative Research Centres SFB 488 and SFB 636, FOR Ot 165/2-2, GRK 791/1.02, and Sachbeihilfe Schu 51/7-2, by the “Fonds der Chemischen Industrie,” the European Union through Grant LSHM-CT-2005-018652 (CRESCENDO), the Bundesministerium fu¨r Bildung und Forschung through NGFN Grants FZK 01GS01117, 01GS0477, and KGCV1/01GS0416, German-Polish Cooperation project 01GZ0310, and Systems Biology Projects No. 0313074C (HepatoSys) and NW5 (CoReNe). Disclosure Summary: The authors have nothing to disclose.

FIG. 6. NeuN as a marker for noradrenergic chromaffin cells. A, IHC with NeuN antibody identifies a subset of immunoreactive cells in the adrenal medulla (dashed area) of control mice. B, Virtually all the remaining chromaffin cells in GRDBHCre mutants are NeuN positive in the mutants at P20. C and D, Silver stain on semithin sections identifies groups of noradrenergic cells (arrowheads) in control and in GRDBHCre mutants. Scale bars, 250 ␮m (A and B); 125 ␮m (C and D).

sympathoadrenal cells. According to recent studies performed on mouse pheochromocytome cell lines, a model of adrenergic differentiation, cAMP, and/or glial cell line-derived neurotrophic factor-dependent signaling is involved in suppression of PNMT expression (23). It would be interesting to elucidate further the contribution of these pathways in an animal mouse model. The analysis of the GR germ line mutants (9) provided the first evidence that GCs are not playing a major role in the early phases of adrenal gland organogenesis. These conclusions have been also supported by the analysis of the nuclear orphan receptor steroidogenic factor-1 null mice. These mutants lack the adrenal cortex but show correct determination and differentiation of chromaffin cells (24). The phenotype of both mutants proves that GCs produced by the adrenal cortex are not necessary for the determination of the chromaffin cell fate. Although GCs do not play a role in the chromaffin cell developmental decisions, our study performed with conditional mutants lacking GR in noradrenergic and adrenergic cells provides the first in vivo evidence that chromaffin cells require GC-dependent signaling for their survival and that the lack of GR results in increased cell death, likely due to apoptosis. In addition, our data indicate a differential effect of GR loss in the survival of chromaffin cells, underscoring that GC-dependent signaling is necessary for the maintenance of the adrenergic phenotype, whereas noradrenergic cells are only partially affected.

Acknowledgments We thank R. Hertel for excellent technical help, Dr. G. Erdmann for providing floxed glucocorticoid receptor mice, and Dr. S. Berger for advice about the mineralocorticoid receptor expression analysis. The

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