help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Beuschlein, F.
Right arrow Articles by Hammer, G. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Beuschlein, F.
Right arrow Articles by Hammer, G. D.
Endocrinology Vol. 143, No. 8 3122-3135
Copyright © 2002 by The Endocrine Society

ARTICLE

Steroidogenic Factor-1 Is Essential for Compensatory Adrenal Growth Following Unilateral Adrenalectomy

Felix Beuschlein, Chris Mutch, David L. Bavers, Yvonne M. Ulrich-Lai, William C. Engeland, Catherine Keegan and Gary D. Hammer

Division of Endocrinology and Metabolism (F.B., C.M., D.L.B., G.D.H.), Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109-0678; Departments of Surgery and Neuroscience (Y.M.U.-L., W.C.E.), University of Minneapolis, Minneapolis, Minnesota 55455; and Division of Genetics (C.K.), Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109-0318

Address all correspondence and requests for reprints to: Gary D. Hammer, M.D., Ph.D., Division of Endocrinology and Metabolism, Department of Internal Medicine, 5560A MSRB II, 1150 West Medical Center Dr, University of Michigan, Ann Arbor, Michigan 48109-0678. E-mail: . ghammer{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
While the orphan nuclear receptor steroidogenic factor-1 (SF-1) has been shown to function as an induction factor to define adrenocortical cell lineage, it remains unclear whether SF-1 plays an additional role as a growth promoting agent in the adult adrenal cortex. The proliferative potential of the adrenal cortex in adult SF-1+/- mice was examined using the model of compensatory adrenal growth following unilateral adrenalectomy (uADX). While the right adrenal gland of wild-type (wt) mice grew significantly after uADX, the adrenal of SF-1+/- mice exhibited a blunted, statistically nonsignificant weight increase. Accordingly, a profound increase in the proliferation marker proliferating cell nuclear antigen could be detected only in wt mice after uADX but not in the SF-1+/- mice. The proposed key regulator in adrenal compensatory growth, the recently cloned adrenal secretory serine protease was up-regulated in the remaining adrenal of wt mice, whereas this increase was blunted in SF-1+/- mice. While no differences in preadipocyte factor-1, the presumed marker of primitive adrenocortical cells, were detectable in the adrenals of wt and SF-1+/- mice, an increase in the ACTH receptor as well as agouti-related protein was observed only in wt animals but not in the SF-1+/- mice following uADX. Taken together, these results reflect a primary inability of adrenal cortical cells of SF-1+/- mice to undergo compensatory adrenal growth in response to uADX.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE REGULATION OF adrenal cell growth and differentiation is under complex and multifactoral control, including both hormonal and neural regulatory cascades (1). The experimental model of compensatory adrenal growth after unilateral adrenalectomy (uADX) in which the remaining adrenal gland undergoes hyperplasia and hypertrophy has been extensively used as an experimental paradigm for adrenal growth in the adult organism. In a series of animal experiments involving disruption of the ipsilateral ventral hypothalamus (2), spinal cord hemisections (3), and sympathectomy (4), it has been demonstrated that compensatory adrenal growth is mediated by a neural loop including afferent and efferent limbs between the adrenals and the ventromedial hypothalamus. In addition, the cloning and characterization of the adrenal secretory serine-protease (AsP), which is specifically up-regulated after uADX and is capable of cleaving the N-terminal proopiomelanocortin (POMC) peptide into a potent adrenal mitogen, has provided insight into important downstream mediators of compensatory adrenal growth and presumably adrenocortical growth in general (5).

The orphan nuclear receptor steroidogenic factor-1 (SF-1) has been shown to be essential for the development of the adrenal cortex. SF-1 was initially cloned as a transcription factor regulating the expression of the various steroid hydroxylase genes. However, SF-1 expression at embryonic d 9 in the urogenital ridge corresponding to the adreno-gonadal primordia predates the onset of hydroxylase expression by 2 d, suggesting that SF-1 is also a developmental factor involved in adrenal organogenesis (6). In accordance with this hypothesis, SF-1-/- knockout (7, 8) and heterozygous (SF-1+/-) mice (9, 10) are born with adrenal aplasia and hypoplasia, respectively. In addition, the recent description of two patients with heterozygous mutations in SF-1 presenting with adrenal insufficiency highlights the concept that gene dosage is essential for SF-1-dependent transcription in the adrenal cortex (11, 12). Specifically, the proximal N-terminal mutation in one of the patients generated a premature stop codon in the DNA binding domain, suggesting that rather than a dominant negative effect, true haplo-insufficiency underlies this defect (11). Whether these phenotypes are indicative of a primary role of SF-1 in cell lineage determination vs. proliferation of the adrenal cortex has remained elusive. Forced expression of SF-1 in embryonic stem cells is alone sufficient for the partial differentiation of these cells into steroidogenically competent cells, suggesting a role for SF-1 in cell lineage determination (13). This project aims to determine whether SF-1 is also required for the proliferation of the adult adrenal cortex. To answer this question, we subjected SF-1+/- mice to uADX to examine the proliferative potential of the remaining adrenal in the context of SF-1 haplo-insufficiency.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
All experiments involving animals were performed in accordance with institutionally approved and current animal care guidelines. Male SF-1+/- haplo-insufficient and wild-type (wt) mice (DBA/2J, The Jackson Laboratory, Bar Harbor, ME) were obtained by breeding SF-1+/- mice with wt animals. After weaning, the resulting male offspring were genotyped by genomic PCR of tail DNA as described previously (10). All animals were maintained under standard conditions of temperature (22 C) and lighting (12-h light, 12-h dark) and with food and water given ad libitum. Six-week-old male SF-1+/- or wt mice were subjected to left adrenalectomy (n = 10 per genotype) or sham surgery (n = 6 per genotype) on d 0 following standard procedures. Sham surgery included a similar surgical procedure including opening of the peritoneum but without touching the adrenal. Mice were decapitated in the morning of d 3, trunk blood for hormonal measurements was taken within 60 sec after initial mouse handling and the remaining adrenal(s) were collected. Two mice per genotype were lost after uADX and thus were excluded from data analysis for postadrenalectomy adrenal weights. Additional mice of each group were euthanized at 12 h and 24 h after the surgical procedure for AsP measurements (n = 5 per time-point and genotype). Following microdissection, adrenal weights were measured and the tissues snap frozen for protein/RNA extraction or immersed in paraformaldehyde or Zamboni’s fixative for histochemistry.

Immunoblotting
Individual adrenals from each group were homogenized in lysis buffer (50 mM HEPES, pH 7.6; 250 mM NaCl; 0.5 mM EDTA; 0.5% Igepal; and protease inhibitors cocktail; Roche, Indianapolis, IN). The homogenate was allowed to rotate at 4 C for 1 h and the protein contents of the high-speed supernatant samples were measured using the Bio-Rad Laboratories, Inc. (Hercules, CA) Dc protein assay kit.

Protein samples (6 µg) from organ tissue solubilized fractions were separated by 10% SDS-PAGE minigel and transferred to immunoblot polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.) for immunoblotting. After blocking of nonspecific sites, membranes were incubated overnight at 4 C in blocking buffer [TBS containing 5% (wt/vol) skim milk powder and 0.05% Tween-20] with primary antibodies to proliferating cell nuclear antigen (PCNA) (1:750, rabbit polyclonal from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), steroidogenic acute regulatory protein (StAR) (1:2500; Hales, D. B.), ACTH receptor (ACTH-R) (1:100, Santa Cruz Biotechnology, Inc.), agouti-related protein (AGRP) (1:250, Chemicon International, Temecula, CA), AsP (1:500; Lowry, P.), preadipocyte factor-1 (Pref-1, 1:2000; Teisner, B.), or ß-actin (1:5000, Sigma, St. Louis, MO). The washed blots were then incubated with suitable secondary antibodies conjugated to horseradish peroxidase (1:7000, Pierce Chemical Co., Rockford, IL). Antibody binding to the membrane was visualized using the ECL Plus (Amersham Pharmacia Biotech, Piscataway, NJ) chemiluminescent detection system. All immunoblots were performed at least three times on adrenal samples from at least two different animals. Bands were quantified using volume quantification (Quantity One, version 4.2.0, Bio-Rad Laboratories, Inc.) and resulting values were expressed as percental changes compared with baseline (wt animal before uADX as 100%).

Histology and immunohistochemistry
Adrenal sections.
Adrenal glands from both wt and SF-1+/- animals were rapidly dissected and placed in 4% paraformaldehyde overnight. Tissues for histochemistry (n = 4 per genotype) were dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) using standard protocols.

For PCNA immunohistochemistry, paraffin-embedded sections were rehydrated, boiled in 10 mM Na citrate (pH 6.0) for 5 min, blocked with 0.3% H2O2 in methanol for 10 min, and incubated overnight with a rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) in a dilution of 1:100 in blocking buffer containing 3% BSA (Roche), 5% goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and 0.5% Tween 20. Bound antibody was detected using the Santa Cruz Biotechnology, Inc. Immunocruz Kit according to the manufacturer’s protocol. Sections were counterstained with methyl green. Pref-1 immunohistochemistry was performed accordingly with a rabbit polyclonal antibody (1:1000, B. Teisner) without boiling.

For immunofluorescent localization of neural elements, adrenal glands (n = 4 per genotype and surgical procedure) were frozen-sectioned (50 µm) and placed into wells containing 0.1 M PBS. Sections were blocked with 5% normal donkey serum in PBS containing 0.3% Triton X-100 and then incubated with primary antibodies in wash buffer (1% normal donkey serum in PBS-0.3% Triton X-100) overnight. The primary antibodies used included rabbit antisera directed against calcitonin gene-related peptide (CGRP, 1:1000; DiaSorin, Inc., Stillwater, MN), vasoactive intestinal peptide (VIP, 1:1000, DiaSorin, Inc.), and neuropeptide Y (NPY, 1:2500, DiaSorin, Inc.) and goat antisera directed against vesicular acetylcholine transporter (VAChT, 1:1000, DiaSorin, Inc.). Labeling for each of the antibodies was eliminated when primary antibodies were omitted or preabsorbed with their cognate peptides (5 µg/ml and/or 40 µg/ml, 4 C, overnight). Sections were then rinsed and incubated with secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) overnight. The secondary antibodies used included donkey F(ab')2 fragments of antirabbit IgG conjugated to indocarbinocyanine (1:400) or cyanine (CY2, 1:100), and donkey F(ab')2 fragments of antigoat IgG conjugated to indocarbinocyanine (1:100) or CY2 (1:100). After rinsing, sections were then mounted in 1.35% melted noble agar (55–60 C; Difco Laboratories, Detroit, MI), dehydrated in ethanol, cleared in methyl salicylate, and coverslipped with p-xylene-bis(N-pyridinium bromide. Sections were viewed with a fluorescent microscope (Zeiss Axiovert 10, Carl Zeiss, Jena, Germany). Images were made using a monochrome charge coupled device camera (Cohu, San Diego, CA), captured with a Scion LG-3 frame grabber and processed using the public domain NIH image 1.6 program [W. Rasband (NIH) and available from the Internet by anonymous ftp at zippy.nimh.nih.gov].

Cell counts.
H&E-stained adrenal sections from wt and SF-1+/- mice were examined with a standard light microscope using x400 magnification. Cell nuclei within the zona fasciculata of three independent sections from three different animals per group were counted under standardized conditions. Cell counts were expressed as cell number/high power field (HPF).

Calculation of adrenal areas.
H&E-stained adrenal sections from four animals of wt mice and SF-1+/- mice before and after uADX were examined with a standard light microscope using x50 magnification. Areas were quantified using the Quantity One software (Version 4.2.0, Bio-Rad Laboratories, Inc.). To ensure for a reliable comparison between the specimen, three adjacent sections from the middle portion of each individual adrenal were examined. To control for the spherical shape of the mouse adrenal gland, the cortical area was normalized for the medullary area and expressed as the cortical/medullary area ratio.

Brain sections.
Brains were collected without fixation and immediately frozen in 2-methyl butane and stored at –80 C until used. After cryosectioning of 16-µm sections (n = 3 per group), cresyl violet staining of every fifth section was performed following standard protocols for morphological studies and to identify sections with central parts of the ventromedial hypothalamus. Adjacent sections were used for immunohistochemistry of SF-1. Sections were fixed in 4% paraformaldehyde for 10 min and blocked in blocking buffer containing 3% BSA (Roche), 5% goat serum, and 0.5% Tween 20 for 10 min at room temperature. SF-1 was detected using a rabbit polyclonal antibody (K. Morahashi) in a dilution of 1:1500 which was incubated overnight in a blocking buffer containing 3% BSA, 0.5% goat serum (Jackson ImmunoResearch Laboratories, Inc.) and 0.5% Tween-20. Bound antibody was visualized with a biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA) and streptavidin-conjugated CY2 (Jackson ImmunoResearch Laboratories, Inc.) and covered with an aqueous mounting media. Sections were observed using a Nikon Optiphot-2 fluorescence microscope and pictures were captured with Spot advance software (Diagnostic Instruments Inc., Sterling Heights, MI).

Northern blot analysis
For the evaluation of AsP mRNA expression, a mouse AsP probe was generated by RT-PCR using total RNA from the murine adrenocortical tumor cell line Y1 as a template and rat AsP specific primers (5'-ACA CTG TCA GAA GAG AGA ATC ATT GGA GGC-3' and 5'-TCC GAA CTT ACT ATT CTG ACC TCT CCT TGC-3'). The amplification product (corresponding to nucleotide position 155–618 of the rat AsP homolog) was gel separated and sequenced. Sequence comparison with the rat AsP sequence revealed a 98% identity on the DNA level. Total RNA from adrenal tissues (baseline, 12 h and 24 h after uADX from wt mice and SF-1+/- animals) was extracted using the QIAGEN RNA mini kit (QIAGEN, Valencia, CA). To obtain a sufficient amount of RNA, adrenals from three animals of the same genotype and treatment group were pooled. Fifteen micrograms of total RNA were separated on a 1% formaldehyde gel and transferred to a nylon membrane (Hybond XL, Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was prehybridized for 3 h at 42 C in the Ultra Hyb solution (Ambion, Inc., Houston, TX). This was followed by hybridization under the same conditions overnight with 1 x 106 cpm/ml of 32P-labeled AsP probe with a specific activity of 10 9cpm/µg. After hybridization, the membrane was washed twice in 2x sodium chloride/sodium citrate, 0.1% sodium dodecyl sulfate, at room temperature, followed by two washes under high stringency conditions (0.1x sodium chloride/sodium citrate, 0.1% sodium dodecyl sulfate at 42 C) before exposure to Bio-Max film (Eastman Kodak Co., Rochester, NY) with intensifying screens (Amersham Pharmacia Biotech). To monitor the loading of RNA samples from the different samples, the membrane was stripped and rehybridized with a 32P-labled-mouse ß-actin cDNA probe (Ambion, Inc.).

RT-PCR
Individual adrenals from each group (baseline and 72 h after uADX from wt and SF-1+/- animals) were used for RNA extraction using the QIAGEN RNA mini kit following instructions of the manufacturer. Multiplex relative RT-PCR was used as described earlier (10) to determine the expression of ACTH-R and AGRP transcripts with 18S RNA serving as a control in each amplification reaction. In brief, cDNA was created using a RT kit (Ambion, Inc.) with a standardized amount of total RNA. Aliquots of the cDNA samples were subjected to the subsequent PCR reactions. For detection of ACTH-R transcripts, specific primers in exons 1 (nucleotide position 23) and 4 (position 663, spanning small intron 2 and 1.5 kb intron 3) were used to amplify a 640-bp product (Table 1Go). For detection of AGRP expression, primers in exons 2 (position 5) and 4 (position 345, spanning 159 bp intron 2 and 182 bp intron 3) were used to amplify a 340-bp product (Table 1Go). Pilot studies were performed to optimize PCR conditions that resulted in the exponential amplification of the ACTH-R and AGRP mRNAs (data not shown). Maximal differences were obtained between cycles 28 and 32 for ACTH-R and 27 and 30 for AGRP. The final conditions for amplification of ACTH-R and AGRP are shown in Table 1Go. Two different sets of primers were used for coamplification of 18S ribosomal RNA as internal standards, which resulted in a 315-bp product and a 488-bp product, respectively (Ambion, Inc.). Amplification products were separated on 1.5% agarose gel and stained with ethidium bromide. Resulting bands of the PCR product of the ACTH-R, AGRP, and 18s controls from four independent experiments were quantified using volume quantification (Quantity One, version 4.2.0, Bio-Rad Laboratories, Inc.).


View this table:
[in this window]
[in a new window]
  		Table 1. Primers and PCR conditions used in RT-PCR experiments

 
Plasma hormone measurements
Plasma corticosterone and ACTH levels were determined by RIA using an 125I RIA kit according to the manufacturer’s protocols (ICN Biomedicals, Costa Mesa, CA). Samples were run in duplicate and concentrations were expressed either as ng/ml (corticosterone) or pg/ml (ACTH).

Statistical analysis
All results are expressed as mean ± SEM. Statistical comparisons were analyzed by ANOVA and Fisher’s protective least significant difference test. Statistical significance is defined as P < 0.05 and is indicated as a lowercase letter (a) in the tables and asterisk in figures; n.s. designates nonsignificant changes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SF-1 haplo-insufficiency attenuates the increase in adrenal weight after uADX due to blunted hypertrophy and hyperplasia of the adrenal cortex
Adrenals of SF-1+/- mice were significantly smaller than those of wt animals at baseline (left adrenal, 1.3 ± 0.1 mg vs. 1.7 ± 0.1 mg), as shown in earlier reports (9, 10), and after both uADX (right adrenal, 1.6 ± 0.1 mg vs. 2.6 ± 0.2 mg) and sham surgery (left adrenal, 1.3 ± 0.1 mg vs. 2.1 ± 0.1 mg and right adrenal, 1.4 ± 0.1 mg vs. 2.0 ± 0.1 mg). As expected the weights of the remaining adrenal in wt mice increased significantly from 1.7 ± 0.1 mg to 2.6 ± 0.2 mg 3 d after left adrenalectomy. In contrast, SF-1+/- mice showed a blunted and statistically insignificant increase in adrenal weight (right adrenal, 1.3 ± 0.1 mg vs. 1.6 ± 0.1 mg). Because in addition to the specific effect of uADX on adrenal growth, the difference in left-to-right adrenal weight may simply reflect changes mediated by the stress-induced activation of the hypothalamo-pituitary-adrenal axis, following the surgical procedure, a group of mice was subjected to sham surgery alone. Sham surgery itself did not change adrenal weight significantly when compared with pre adrenalectomy in either genotype (left adrenal, 2.1 ± 0.1 mg vs. 1.7 ± 0.1 mg for wt animals; 1.3 ± 0.1 mg vs. 1.3 ± 0.1 mg for SF-1+/- mice). However, the right adrenal weight in wt animals after ADX was significantly higher than after sham surgery (right adrenal, 2.6 ± 0.2 mg vs. 2.0 ± 0.1 mg) arguing for a specific effect of uADX on the weight increase of the remaining adrenal (Fig. 1Go). Furthermore, adrenal weights in the sham surgery group demonstrated no significant left to right differences in animals from both genotypes.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Adrenal weights of wt and SF-1+/- mice before (left adrenal; n = 10) and 3 d after adrenalectomy (right adrenal, ADX; n=8) or after sham surgery (n = 6). Whereas the weight of the right adrenal in wt mice increases significantly after left sided adrenalectomy, SF-1+/- mice show a blunted and insignificant increase in adrenal weight. Animals of both genotypes that underwent sham surgery show no side difference in adrenal weight. Adrenals of SF-1+/- mice are significantly smaller than the wt counterparts under each experimental condition. For complete statistical analysis see text.

 
Histologically, an increase in adrenal size was evident in wt animals after adrenalectomy, whereas no significant change was observed in the SF-1+/- mice (Fig. 2Go). Adrenal hypertrophy following uADX was quantified both by counting cells per HPF within the zona fasciculata and by measurement of the adrenal cortical/medullary area ratio at baseline and after surgery. Consistent with earlier reports (9, 10), SF-1+/- mice had a significantly lower cell count/HPF compared with wt animals at baseline. As expected, wt mice showed a statistically significant decrease in cell number after uADX indicating an increase in cell size (102.9 ± 3.3 cells/HPF vs. 84.0 ± 3.2 cells/HPF; Fig. 3AGo). In contrast, cell counts in SF-1+/- mice did not change significantly (84.4 ± 3.7 cells/HPF vs. 77.2 ± 5.0 cells/HPF). In accordance with the cell counts, cortical/medullary area ratios indicated a significant hypertrophic response in wt animals (cortical/medullary area ratio 2.5 ± 0.2 to 3.1 ± 0.2), but no significant increase in SF-1+/- mice (2.0 ± 0.1 to 2.1 ± 0.1; Fig. 3BGo) after uADX. In addition, cortical/medullary area ratio was significant higher in wt animals compared with SF-1+/- mice both before (2.5 ± 0.2 vs. 2.0 ± 0.1) and after (3.1 ± 0.2 vs. 2.1 ± 0.1) adrenalectomy.



View larger version (73K):
[in this window]
[in a new window]
  		Figure 2. Low (x40) and high power (x200) magnification of H&E-stained adrenal sections from a wt animal (A, B, E, F) and SF-1+/- mouse (C, D, G, H) before (A, C, E, G) and after adrenalectomy (B, D, F, H). Note the increased adrenocortical width in the wt mouse after adrenalectomy (B, F) but no change in adrenal size and no significant change in the SF-1+/- mouse (D, H) compared with baseline.

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 3. Quantification of histological changes as observed on H&E-stained sections following uADX (ADX). Upper panel (A), Cell count of HPF within the zona fasciculata of adrenals of wt and SF-1+/- mice before and after adrenalectomy. Cell nuclei in three HPF of the adrenal areas of each of three individual animals in each group were counted. Significant decrease of cell counts per HPF in wt mice after ADX, indicative of cell hypertrophy, does not occur in SF-1+/- animals. Lower panel (B), Cortical/medullary area ratios from wt and SF-1+/- mice before and after adrenalectomy show a significant increase in wt animals after uADX, whereas no significant increase is detectable in SF-1+/- mice. Areas were calculated from three adjacent sections of four different animals in each group.

 
Adrenal expression of the proliferation marker PCNA, an auxiliary protein of DNA polymerase that is specifically expressed in cells during the S-phase (14) was investigated by immunoblot and immunohistochemistry to evaluate the extent of proliferation. Immunohistochemistry revealed significant nuclear staining for PCNA in the adrenal cortex of wt animals after adrenalectomy, whereas no specific staining could be detected in SF-1+/- mice (Fig. 4Go, lower panel). PCNA staining was restricted to a circumscribed zone beneath the adrenal capsule, which may represent the precursor cell zone of the rodent adrenal cortex (15). Consistent with the findings of the immunohistochemistry, immunoblotting revealed a significant increase in adrenal PCNA expression after uADX in wt mice suggesting an induction of a marked proliferative response in the remaining adrenal (Fig. 4Go, upper panel). In contrast, SF-1+/- mice exhibited no significant increase in adrenal PCNA levels after uADX. None of the animals of either genotype that underwent sham surgery exhibited any increase in adrenal PCNA expression (data not shown). This suggests that the increase in adrenal PCNA expression is a specific response to uADX as opposed to surgery related stress.



View larger version (118K):
[in this window]
[in a new window]
  		Figure 4. Upper panel, Immunoblot for proliferation marker PCNA in adrenal glands before (A, C) and after (B, D) uADX in wt (A, B) and SF-1+/- mice (C, D). Notable induction of PCNA expression occurs only in the remaining adrenal of wt animals but not in SF-1+/- mice after adrenalectomy. Actin serves as a control for equal loading of protein. Lower panel, PCNA immunohistochemistry of adrenal sections from a wt animal (A, B) and SF-1+/- mouse (C, D) before (A, C) and after (B, D) adrenalectomy. Note the increased nuclear staining of cells under the capsule in the wt mouse after adrenalectomy but no specific staining in the SF-1+/- mouse. Magnification, x400.

 
SF-1+/- mice show no gross differences in innervation of the adrenal gland and the overall structure and SF-1 content of the ventromedial hypothalamus (VMH)
Because compensatory adrenal growth is mediated by a neural circuit including nerve fibers to and from the VMH we analyzed the distribution of afferent and efferent nerve fibers within the adrenal cortex. Immunohistochemistry for CGRP-positive (primary afferent/sensory) fibers, NPY-positive (postganglionic sympathetic and/or type I ganglion cell) fibers, VIP-positive (type II ganglion cell) fibers and VAChT-positive (preganglionic sympathetic) fibers did not show marked differences between adrenals of wt and SF-1+/- mice (Fig. 5Go). While homozygous SF-1-/- knockout mice are known to exhibit markedly impaired VMH structure (16), no gross differences were evident in the morphological structure of the VMH in SF-1+/- mice compared with wt animals after cresyl violet staining (data not shown). Moreover, by means of immunohistochemistry, the distribution of SF-1 positive cells within the VMH did not greatly vary between wt and SF-1+/- mice after uADX or sham surgery (Fig. 6Go). In addition, no lateralizing differences in the expression pattern of SF-1 were observed in any of the groups following uADX. Taken together, these data suggest that the observed phenotype in SF-1+/- mice is not caused by major morphological defects in adrenal innervation or in the VMH.



View larger version (110K):
[in this window]
[in a new window]
  		Figure 5. Immunohistochemistry for CGRP-positive fibers in the cortex and medulla (A, B), VIP-positive fibers in the capsule and cortex (C, D), NPY-positive fibers in the capsule and cortex (E, F), and VAChT-positive fibers in the medulla (G, H) do not show marked differences between adrenals of wt (A, C, E, G) and SF-1+/- mice (B, D, F, H). Arrows in A–F point out examples of labeled nerve fibers, asterisks in E and F indicate a NPY-positive chromaffin cell in the medulla, and arrows in G and H mark the border between adrenal cortex and medulla in which positive labeling appears as light spots throughout the medulla. Magnification, x200.

 


View larger version (139K):
[in this window]
[in a new window]
  		Figure 6. No gross differences in the immunohistochemical staining pattern for SF-1 are evident in the ventromedial hypothalamus in a wt animal after sham surgery (A) or after adrenalectomy (B) compared with a SF-1+/- mouse after sham surgery (C) and after adrenalectomy (D). Magnification, x40.

 
The AsP is up-regulated in the remaining adrenal of wt mice but not in SF-1+/- mice after uADX
mRNA levels of the AsP have been shown to be up-regulated in rats in the remaining adrenal after uADX (5). In our mouse model, similar changes on the mRNA level were observed by Northern blot 12 h and 24 h after uADX in wt mice. However, no such increase was detected in SF-1+/- mice (Fig. 7Go, upper panel). Similarly, AsP protein levels were up-regulated only in wt animals 12 h after uADX, whereas no significant increase was observed in SF-1+/- mice (Fig. 7Go, lower panel, and Table 2Go). Because AsP regulation is believed to play a crucial role in the regulation of proliferation in compensatory adrenal growth, the blunted increase in AsP in SF-1+/- mice may account for the observed proliferative defect in these mice.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 7. Upper panel (A), Northern blot for AsP for wt mice before and after adrenalectomy as well as SF-1+/- mice before and after uADX, with adrenals collected 12 h and 24 h after the surgery, respectively. AsP mRNA levels are up-regulated in wt animals 12 h and 24 h after adrenalectomy but not in SF-1+/- mice. The murine adrenocortical cell line Y1 serves as a positive control for AsP expression. Lower panel (B), AsP Immunoblot for wt mice and SF-1+/- mice before and 12 h after uADX. AsP protein levels are up-regulated in wt animals but not in SF-1+/- mice after uADX.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Quantification of the immunoblots (three individual experiments each) and significances between the groups

 
Pref-1 levels by immunoblotting and immunohistochemistry are not different between SF-1+/- and wt mice
Pref-1, isolated from a regenerating rat adrenal gland and shown to be expressed in a discrete subcapsular cell layer has been proposed to serve as a marker for precursors of steroidogenic active adrenocortical cells (15). Pref-1-positive cells within the adrenal cortex in both wt and SF-1+/- mice were present only in the cell layer directly under the capsule. No gross differences in either the thickness of the Pref-1-positive cell layer nor the intensity of the staining were apparent between both groups of mice (Fig. 8Go, lower panel). Furthermore, no obvious changes in immunohistochemistry were observed following uADX in wt or SF-1+/- mice. Accordingly, no significant differences in the expression of Pref-1 were evident by immunoblotting in the examined adrenal samples of any group (Fig. 8Go, upper panel). Thus, the indistinguishable Pref-1 expression between wt and SF-1+/- mice argues against a quantitative defect in this putative precursor cell population as the cause of the proliferative defect in SF-1+/- mice.



View larger version (124K):
[in this window]
[in a new window]
  		Figure 8. Upper panel, Immunoblot for Pref-1. Similar protein levels were observed in wt animals before (A) and after adrenalectomy (B) as well as in SF-1+/- mice before (C) and after adrenalectomy (D). Lower panel, Pref-1 immunohistochemistry of adrenal sections from a wt animal (A, B) and SF-1+/- mouse (C, D) before (A, C) and after adrenalectomy (B, D) reveals a specific staining of a cell layer adjacent to the adrenal capsule (arrows) without gross changes in the thickness of the Pref-1-positive cell layer. Note the intensity of the staining is independent of the genotype or surgical procedure. Magnification, x400.

 
SF-1+/- mice show no difference in basal plasma ACTH and corticosterone levels but display a blunted increase of ACTH-R and AGRP expression after uADX compared with wt animals
As opposed to adrenal growth, the regulation of adrenal steroidogenesis in SF-1+/- mice following uADX was not different from wt mice. When compared with wt animals, no significant differences in plasma ACTH and corticosterone levels could be detected in SF-1+/- mice in the morning at 3 d after uADX or sham surgery (Table 3Go). Overall, these levels were comparable to levels that have been reported in nonstressed wt and SF-1+/- mice (9, 10). While no significant differences in basal plasma hormone levels were observed, a significant increase in the adrenal ACTH-R expression was evident in wt mice after adrenalectomy but not in the SF-1+/- mice. As reported earlier by Bland et al. (9), ACTH-R mRNA in SF-1+/- mice was significantly higher compared with wt mice at baseline. However, after uADX ACTH-R mRNA did increase significantly only in wt animals and not in SF-1+/- mice (Fig. 9AGo and Table 4Go). In accordance with earlier findings (9, 10), adrenal protein levels of StAR were higher in SF-1+/- mice compared with wt animals at baseline (Fig. 9BGo). This difference was still evident in the remaining adrenal 72 h following uADX (Fig. 9BGo). uADX in rats has been shown to result in an increase of AGRP in the contralateral adrenal (17). In accordance with this finding, the expression level of adrenal AGRP was significant elevated in wt mice following adrenalectomy, but this response was blunted in SF-1+/- mice (Fig. 9CGo).


View this table:
[in this window]
[in a new window]
  		Table 3. Plasma ACTH and corticosterone levels in wt and SF-1+/- animals 3 d after uADX or sham surgery

 


View larger version (52K):
[in this window]
[in a new window]
  		Figure 9. Upper panel (A), Increase of ACTH-R expression in wt but not SF-1+/- mice after uADX by means of semiquantitative RT-PCR and immunoblot. Middle panel (B), Increase of AGRP after uADX in wt mice. In contrast, SF-1+/- mice show a blunted increase in AGRP by semiquantitative RT-PCR and immunoblot. Lower panel (C), Higher protein level of StAR in SF-1+/- mice compared with wt animals before and after uADX.

 

View this table:
[in this window]
[in a new window]
  		Table 4. Quantification of RT-PCR experiments

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The compensatory adrenal growth response following uADX is tightly controlled by both humoral and neuronal factors that have been elucidated in increasing detail over the last decades. As shown in extensive ablation studies, compensatory adrenal growth is dependent upon a neural reflex arc with afferent nerve connections from one adrenal gland to the hypothalamus and an efferent limb back to the other adrenal (2, 3, 4). In addition, there is an increasing body of evidence indicating that postsecretory cleavage of the N terminus of POMC is also required for the proper initiation of compensatory growth (5). The recent description of a putative precursor cell zone between the zona glomerulosa and fasciculata in the rat adrenal together with the concept of cell migration involved in adrenocortical zonation predicts a role of these pluripotent cells in the proliferative response of the adrenal cortex to diverse growth stimuli (15). To characterize the growth defect(s) in SF-1 haploinsufficient mice, we explored the integrity of neuronal and hormonal inputs to the adrenal as well as specific cellular markers involved in adrenal proliferation and differentiation in the context of compensatory adrenal growth.

As expected, the remaining adrenal in wt mice increased significantly in weight following uADX, showed a widening of the adrenal cortex together with a significant decrease in cell count per HPF in the zona fasciculata and a marked increase in the proliferation marker PCNA indicating both a hypertrophic and hyperplastic response, respectively. Because the adrenal weight of wt mice following uADX is significantly higher than that after sham surgery, the weight increase after uADX is considered to be a specific consequence of the removal of one adrenal in contrast to an unspecific stress-related effect of the surgical procedure. SF-1+/- mice, in contrast, showed no significant increase in adrenal weight, no significant decrease in cell number per HPF in the zona fasciculata, no significant changes in cortical/medullary area ratios and no up-regulation in PCNA expression after uADX. These findings indicate that in addition to playing a role in adrenocortical lineage determination and/or differentiation, a full complement of SF-1 is required for the proliferative and hyperplastic response of the adult adrenal cortex.

The VMH is an essential component of the neuronal circuit involved in compensatory adrenal growth. Morphological markers such as nuclear enlargement (18) and functional criteria such as a higher uptake of tritiated-leucine in VMH neurons (19) indicated an activation of the contralateral VMH and reduced activity of the ipsilateral VMH after uADX. Lesions of the VMH ipsilateral, but not contralateral, to the side of the removed adrenal prevent the compensatory growth response most likely due to the disruption of a neural pathway (2). Interestingly, SF-1 is not only essential for the proper development of the adrenals but is also required for the formation of the ventromedial hypothalamus as evident by the lack of the VMH in the newborn SF-1-/- knockout mouse (16, 20). It is intriguing to speculate that SF-1 itself could be involved in the neural reflex at the level of the VMH. It is certainly possible that functional defects in the hypothalamus of the SF-1+/- mouse could account for the blunted compensatory adrenal growth observed in these mice. However, no gross structural differences in the VMH of SF-1+/- mice could be detected compared with wt animals. Furthermore, the SF-1 expression pattern by means of immunohistochemistry was not markedly altered in the VMH of SF-1+/- mice and revealed no noticeable sided differences in unilateral adrenalectomized animals of either group. These findings do not preclude the possibility that posttranslational modification of SF-1 [e.g. phosphorylation (21) or acetylation (22)] might be a regulated step in the activation of the VMH.

The adrenal gland is innervated by a variety of different nerves that have been implicated in modulating adrenal cortical function (23). The extrinsic innervation of the adrenal gland includes primary afferent (sensory) fibers positive for CGRP, catecholaminergic postganglionic sympathetic fibers positive for NPY, and cholinergic preganglionic sympathetic fibers expressing VAChT (reviewed in Ref. 24). The intrinsic innervation of the adrenal gland arises from two types of medullary ganglion cells: type I cells are noradrenergic and NPY-positive, whereas type II cells are VIP-positive (reviewed in Ref. 24). Ipsilateral anesthetic nerve blockade or contralateral spinal cord hemi-transsection performed before uADX prohibits compensatory growth of the other gland (3), suggesting that the stimulus initiating compensatory adrenal growth is dependent on neural input from the removed adrenal gland. Additionally, enucleation experiments on rat adrenals support the concept, that adrenal innervation modulates tissue regeneration and the rate of functional recovery (25). Therefore, it is possible that defects in adrenal innervation might theoretically contribute to the observed adrenal growth deficiency in SF-1+/- mice. However, immunolabeling for markers of adrenal innervation showed no clear morphological differences in SF-1+/- mice, arguing against a gross defect in the adrenal innervation in these mice.

In addition to neuronal regulation, adrenocortical growth is dependent upon peptide hormone stimulation. Whereas ACTH is the primary hormone responsible for the induction of adrenal steroidogenesis, there is compelling evidence indicating that ACTH is not the main POMC-derived peptide with mitogenic influence on the adrenal cortex (26, 27). It has been suggested that the mitogenic peptides reside in the N terminal 16-kDa fragment known as pro-{gamma}-MSH. Although this peptide itself does not have mitogenic potential, it has been shown, that the specific cleavage of pro-{gamma}-MSH after its secretion from the pituitary is required to release shorter fragments that exhibit potent mitogenic actions on adrenal cells (27, 28). To reflect their mitogenic properties on the adrenal cortex, together, these peptides have been termed Adrenoproliferin (29). In a recent paper by Bicknell and co-workers (5), a novel adrenal serine-protease (AsP), that is highly up-regulated in the remaining adrenal after uADX and is capable of cleaving pro-{gamma}-MSH into the mitogenic POMC fragment 1–52 was cloned in rats. It is intriguing to speculate that the observed up-regulation of AsP after uADX is driven by the neuronal efferents from the VMH, which would potentially bridge the gap between hormonal (e.g. POMC-derived peptides) and neuronal (e.g. adrenal-hypothalamic circuit) regulation (30).

In vitro studies on the murine Y1 cells showed that treatment with a serine protease inhibitor and—more specifically—transfection with antisense AsP RNA decrease the growth rate of the cells in the presence of pro-{gamma}-MSH (5). In addition, application of synthetic 1–28 N-POMC to Y1 cells increases cell growth significantly in a dose-dependent manner (29). These in vitro studies, however, cannot provide direct evidence that up-regulation of AsP expression is an important and rate limiting step for adrenal growth after uADX in the mouse. As presented herein, the regulation of AsP in the mouse is similar to the regulation in the rat. The murine AsP is highly homologous to its rat counterpart (31), it is highly expressed in the zona fasciculata derived murine tumor cell line Y1 (5), and it is detectable by immunoblot in the mouse adrenal cortex. In accordance with the findings in the rat, AsP mRNA levels were found to be up-regulated in the remaining adrenal of wt animals 12 h and 24 h after uADX. Similarly, AsP protein levels were significantly increased 12 h after the surgery. In contrast, SF-1+/- mice did not respond with a significant increase in AsP levels after uADX indicating that a full complement of SF-1 is required for the regulation of AsP. In the light of in vitro and in vivo experimental data indicating a potent mitogenic effect of locally generated adrenoproliferin on the adrenal cortex, the blunted increase in AsP in SF-1+/- mice provides a potential mechanism for the observed proliferative defect in these mice. Ongoing in vivo studies using the treatment of SF-1+/- mice with synthetic adrenoproliferin will provide additional insights whether SF-1 is also a downstream effector of adrenoproliferin action.

Staining for PCNA is an established and widely used technique for the detection and quantification of cell proliferation and has been applied to the adrenal by several groups during fetal adrenal growth in humans (32) and rodents (33). The marked increase in PCNA expression in wt mice after adrenalectomy is consistent with earlier experiments in which an increase in the mitotic index and tritiated thymidine uptake as well as DNA content in the remaining adrenal cortex was demonstrated (34, 35). These data strongly suggest that the compensatory growth response includes a marked adrenal hyperplasia, i.e. increase in the number of cells undergoing mitosis. In contrast, SF-1+/- mice lacked any increase in PCNA expression in response to uADX. PCNA-positive cells in wt animals were restricted to the outermost cell layers, which may represent precursor cells capable of responding to growth stimuli with an increase in mitotic activity. According to the cell migration model, each zone in the adrenal cortex is derived from a common pool of progenitor cells located in the periphery of the cortex, which migrates centripetally and populates the inner cortical zones upon differentiation (32, 36). Thus, the observed lack of specific PCNA staining in SF-1+/- mice after uADX can be explained by either a decreased number of precursor cells or the inability of an existing precursor cell population to respond to the growth stimulus.

In an attempt to characterize factors involved in adrenal proliferation and differentiation, Halder et al. (15), using a subtraction cloning strategy on enucleated rat adrenals, identified Pref-1 as a specific marker within the adrenal cortex, that is expressed in adrenocortical cells undergoing regeneration. Pref-1, also known as zona glomerulosa specific factor, encodes a putative transmembrane protein containing six tandem epidermal growth factor-like repeats in the extracellular domain. Pref-1 and other members of the epidermal growth factor-like protein family are involved in cell-fate determination during organogenesis and cell differentiation by keeping cells in an undifferentiated state (37). In rats, Pref-1-positive cells in the adrenal cortex have been shown to be restricted to the zona glomerulosa and a layer of cells in the outer zona intermedia that do not express either aldosterone synthase or 11ß-hydroxylase (15). As shown herein, the Pref-1 staining pattern in mouse adrenals differs to some extent from that in rats in that it is restricted to a cell layer under the adrenal capsule. Because a soluble form of Pref-1, designated as fetal antigen 1, has been identified (38), it is possible that the action of Pref-1 might be mediated in a paracrine fashion to adjacent cell layers. No apparent differences in Pref-1 immunoblotting and immunohistochemical staining pattern between wt and SF-1+/- mice were detectable. These findings—together with the lack of PCNA induction—suggest that the growth defect in the adrenal cortex of SF-1+/- mice is not due to a decreased population of putative adrenocortical precursor cells but is a result of the inability of these cells to respond with proliferation to uADX.

By definition, uADX removes half of the tissue capable of the secretion of adrenal steroids. To maintain hormonal homeostasis, rapid compensatory mechanisms must occur in addition to the proliferative response discussed above. It could be speculated that an increase in ACTH binding sites and thus an increase in ACTH responsiveness of the remaining adrenal gland might be important to maintain a sufficient corticosterone output. The ACTH-R (melanocortin 2 receptor) is a central part of the hypothalamo-pituitary-adrenal axis in that it mediates the adrenal steroid output in response to pituitary ACTH (39). As shown in in vitro studies the ACTH-R is up-regulated by its own ligand ACTH, which not only increases the transcriptional rate of ACTH-R message but also prolongs the ACTH-R mRNA half-life (40, 41). As presented herein, after uADX the remaining adrenal of wt mice responds with a marked increase in both ACTH-R mRNA and protein content. This response is unlikely to be the result of an increase in plasma ACTH, since prior reports revealed that plasma ACTH after uADX increases only for the first 12 h and normalizes soon thereafter (42). Consistently, our data show no differences in nonstress plasma ACTH or corticosterone levels in wt animals that underwent uADX or sham surgery at 72 h after surgery.

The ACTH-R is a known SF-1 target gene (43). In accordance with this notion, the observed increase in ACTH-R expression in wt animals after adrenalectomy was absent in SF-1+/- mice suggesting that a critical dose of SF-1 is needed for the proper up-regulation of ACTH-R in the context of uADX. However, the higher ACTH-R mRNA levels in SF-1+/- mice at baseline compared with wt animals as shown herein and by others (9) clearly indicate the existence of additional, SF-1 independent factors that regulate ACTH-R expression in vivo. Because plasma hormone levels in SF-1+/- mice were not different from wt animals despite the lack of ACTH-R up-regulation, the maintenance of corticosterone output after uADX cannot solely be dependent on the up-regulation of ACTH-R expression. In accordance with earlier findings (9, 10), the expression of StAR, the rate-limiting enzyme in steroidogenesis, is up-regulated in SF-1+/- mice in comparison to wt mice at baseline indicating a probable compensatory SF-1-independent up-regulation of StAR transcription. As shown herein, differences in StAR protein levels are still apparent between wt and SF-1+/- mice in the remaining adrenal 72 h after uADX. Thus, it is likely that higher StAR protein levels in SF-1+/- mice are sufficient to maintain a normal corticosterone output despite the lack of an ACTH-R up-regulation.

In addition to its steroidogenic properties, ACTH has been reported to induce adrenocortical hypertrophy when injected in rats at pharmacologic doses (44). When given at the time of uADX, pharmacologic doses of ACTH induce adrenal hypertrophy but surprisingly inhibit adrenocortical hyperplasia (45). Predicated on the common origin of ACTH and adrenoproliferin from the POMC precursor peptide, the inhibition of adrenocortical hyperplasia by exogenous ACTH is most likely the result of an ACTH-induced compensatory increase in adrenal steroidogenesis and subsequent inhibition of endogenous POMC-derived peptides (including pro-{gamma}-MSH) resulting in a decreased adrenoproliferin-induced adrenal hyperplasia. The induction of adrenal hypertrophy, however, presumably reflects a direct effect of exogenous ACTH on the steroidogenic capacity of the adrenal cortex. In addition, a persistent, albeit partial, compensatory adrenal growth following hypophysectomy portends an additional pituitary-independent and/or POMC-independent component of this adrenal response that remains to be integrated into the current model of adrenoproliferin action (45). In the current study, the observed ACTH levels 72 h following uADX are well within the range of nonstressed baseline ACTH levels reported previously (9, 10). It is intriguing to speculate that the observed up-regulation of the ACTH-R in wt mice contributes to the ACTH-dependent adrenal hypertrophy in the remaining adrenal following uADX. The lack of ACTH-R up-regulation in SF-1+/- mice is consistent with the blunted adrenocortical hypertrophy observed in these mice.

AGRP has been cloned as a highly homologous peptide to the agouti protein (46), which is a paracrine signaling molecule that antagonizes melanocortin 1 receptor (MC1-R) and results in the mouse Agouti coat phenotype (47). Furthermore, both AGRP and agouti are potent antagonists of the MC4-R and the MC3-R, which are expressed mainly in the hypothalamus including the VMH and have been implicated in the regulation of feeding behavior and metabolism (48). AGRP expression is confined to the arcuate nucleus of the hypothalamus and the adrenal and is furthermore found at low levels in the testis, lung and kidney (17, 46, 49). Whereas the site of AGRP expression in the adrenal was initially believed to be restricted to the medulla (46), studies using in situ hybridization have been shown that AGRP expression in the rat is localized in the adrenal cortex (17). Interestingly, the adrenal expression of AGRP has been reported to be up-regulated after fasting and after uADX indicating an as yet undefined role of AGRP in the adrenal (17).

In our mouse system, we were able to reproduce the increase of AGRP expression in the remaining adrenal after uADX. Whereas we saw a robust increase of AGRP expression in wt mice, this increase was blunted in SF-1+/- mice after adrenalectomy. Because AGRP is only a weak antagonist to the ACTH-R and another peripheral binding site for peripheral AGRP has not been defined yet, the site of action of adrenal-derived AGRP is not well understood. It is intriguing to speculate, however, that AGRP could be involved in a negative feedback loop to the hypothalamus to terminate compensatory growth. In this regard, the attenuated increase of AGRP expression in the SF-1+/- mice could be interpreted as a diminished feedback in response to the blunted adrenal growth in those animals. However, it has been shown that peripheral injections of purified AGRP in mice as opposed to intracranial administration do not change body weight or food intake, suggesting that peripheral secreted AGRP might not have an antagonistic effect on central MC3-R or MC4-R (50). Although its active C-terminal fragment, AgRP(83–132), can cross the blood-brain barrier from the blood to the brain, the nonsaturable rate of entry is very slow (51). Thus it is not clear whether peripheral AGRP has any physiological effect on central AGRP binding sites. Current studies using mouse models with targeted deletion of the AGRP and MC3-R genes are aimed at further characterizing the potential role(s) of these molecules in the regulation of compensatory adrenal growth.

In conclusion, we have shown that SF-1, in addition to its known function as a regulator of adrenal steroidogenesis and its involvement in adrenal development, is also required for the growth maintenance of the adult mouse adrenal gland. We have presented evidence suggesting that the growth defects observed in SF-1 haplo-insufficient mice after uADX are not caused by a defect in adrenal cell lineage determination but by a specific defect in the ability of the adrenal cortex of SF-1+/- mice to respond to growth stimuli with compensatory proliferation and hypertrophy. The blunted up-regulation of the AsP and the ACTH-R in the remaining adrenal gland of SF-1+/- mice indicates an essential role of SF-1 in these regulation cascades and provide possible mechanisms for the observed proliferative and hypertrophic defect in SF-1+/- mice.


   Acknowledgments
 
We are indebted to Dr. K. Morohashi (NIBB, Japan), Dr. P. Lowry (Reading, UK), Dr. B. Teisner (Odense University Hospital, Denmark) and Dr. D. B. Hales (University of Illinois at Chicago, Chicago, IL) for the generous gifts of the anti-Ad4BP antibody, the anti-AsP antibody, the anti-Pref-1 and the anti-StAR antibody, respectively.


   Footnotes
 
This work was supported by NIH Grants DK-02393 and DK-58124 (to G.D.H.), NIH Grant R01-GM59732 (to W.C.E.), Emmy Noether Grant BE2177/3-1 awarded by the Deutsche Forschungsgemeinschaft (DFG) (to F.B.), and a Howard Hughes Medical Institute Predoctoral Fellowship (to Y.M.U.).

Abbreviations: ACTH-R, ACTH receptor; AGRP, Agouti-related protein; AsP, adrenal secretory serine-protease; CGRP, calcitonin gene-related peptide; CY2, cyanine; H&E, hematoxylin and eosin; HPF, high power field; MC1-R, MC3-R, or MC4-R, melanocortin 1, 3, or 4 receptor; NPY, neuropeptide Y; PCNA, proliferating cell nuclear antigen; Pref-1, preadipocyte factor 1; POMC, proopiomelanocortin; SF-1, steroidogenic factor 1; uADX, unilateral adrenalectomy; StAR, steroidogenic acute regulatory protein; VAChT, vesicular acetylcholine transporter; VMH, ventromedial hypothalamus; wt, wild-type.

Received January 9, 2002.

Accepted for publication April 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Dallman MF 1984 Control of adrenocortical growth in vivo. Endocr Res 10:213–242[Medline]
  2. Holzwarth MA, Dallman MF 1979 The effect of hypothalamic hemi-islands on compensatory adrenal growth. Brain Res 162:33–43[CrossRef][Medline]
  3. Engeland WC, Dallman MF 1976 Neural mediation of compensatory adrenal growth. Endocrinology 99:1659–1662[Abstract]
  4. Kleitman N, Holzwarth MA 1985 Compensatory adrenal cortical growth is inhibited by sympathectomy. Am J Physiol 248:E261–E263
  5. Bicknell AB, Lomthaisong K, Woods RJ, Hutchinson EG, Bennett HP, Gladwell RT, Lowry PJ 2001 Characterization of a serine protease that cleaves pro-{gamma}-melanotropin at the adrenal to stimulate growth. Cell 105:903–912[CrossRef][Medline]
  6. Ikeda Y, Shen WH, Ingraham HA, Parker KL 1994 Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 8:654–662[Abstract]
  7. Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[CrossRef][Medline]
  8. Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA, Tourtellotte LM, Simburger K, Milbrandt J 1995 Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci USA 92:10939–109343[Abstract/Free Full Text]
  9. Bland ML, Jamieson CA, Akana SF, Bornstein SR, Eisenhofer G, Dallman MF, Ingraham HA 2000 Haploinsufficiency of steroidogenic factor-1 in mice disrupts adrenal development leading to an impaired stress response. Proc Natl Acad Sci USA 97:14488–14493[Abstract/Free Full Text]
  10. Babu PS, Bavers DL, Beuschlein F, Shah S, Jeffs B, Jameson JL, Hammer GD 2002 Interaction between Dax-1 and SF-1 in vivo: Increased adrenal responsivness to ACTH in the absence of Dax-1. Endocrinology 143:665–673[Abstract/Free Full Text]
  11. Achermann JC, Ito M, Hindmarsh PC, Jameson JL 1999 A mutation in the gene encoding steroidogenic factor-1 causes XY sex reversal and adrenal failure in humans. Nat Genet 22:125–126[CrossRef][Medline]
  12. Biason-Lauber A, Schoenle EJ 2000 Apparently normal ovarian differentiation in a prepubertal girl with transcriptionally inactive steroidogenic factor 1 (NR5A1/SF-1) and adrenocortical insufficiency. Am J Hum Genet 67:1563–1568[CrossRef][Medline]
  13. Crawford PA, Sadovsky Y, Milbrandt J 1997 Nuclear receptor steroidogenic factor 1 directs embryonic stem cells toward the steroidogenic lineage. Mol Cell Biol 17:3997–4006[Abstract]
  14. Kelman Z 1997 PCNA: structure, functions and interactions. Oncogene 14:629–640[CrossRef][Medline]
  15. Halder SK, Takemori H, Hatano O, Nonaka Y, Wada A, Okamoto M 1998 Cloning of a membrane-spanning protein with epidermal growth factor-like repeat motifs from adrenal glomerulosa cells. Endocrinology 139:3316–3328[Abstract/Free Full Text]
  16. Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL 1995 The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 9:478–486[Abstract]
  17. Bicknell AB, Lomthaisong K, Gladwell RT, Lowry PJ 2000 Agouti related protein in the rat adrenal cortex: implications for novel autocrine mechanisms modulating the actions of pro-opiomelanocortin peptides. J Neuroendocrinol 12:977–982[CrossRef][Medline]
  18. Halasz B, Szentagothai J 1959 Histologischer beweis einer nervösen signalübermittlung von der nebennierenrinde zum hypothalamus. Histochemie 50:297–306
  19. Gerendai I, Kiss J, Molnar J, Halasz B 1974 Further data on the existence of a neural pathway from the adrenal gland to the hypothalamus. Cell Tissue Res 153:559–564[Medline]
  20. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, Morohashi KI, Li E 1995 Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dyn 204:22–29[Medline]
  21. Hammer GD, Krylova I, Zhang Y, Darimont BD, Simpson K, Weigel NL, Ingraham HA 1999 Phosphorylation of the nuclear receptor SF-1 modulates cofactor recruitment: integration of hormone signaling in reproduction and stress. Mol Cell 3:521–526[CrossRef][Medline]
  22. Jacob AL, Lund J, Martinez P, Hedin L 2001 Acetylation of steroidogenic factor 1 protein regulates its transcriptional activity and recruits the coactivator gcn5. J Biol Chem 276:37659–37664[Abstract/Free Full Text]
  23. Engeland WC 1998 Functional innervation of the adrenal cortex by the splanchnic nerve. Horm Metab Res 30:311–314[Medline]
  24. Ulrich-Lai YM, Engeland WC 2000 Rat adrenal transplants are reinnervated: an invalid model of denervated adrenal cortical tissue. J Neuroendocrinol 12:881–893[CrossRef][Medline]
  25. Ulrich-Lai YM, Engeland WC 2000 Hyperinnervation during adrenal regeneration influences the rate of functional recovery. Neuroendocrinology 71:107–123[CrossRef][Medline]
  26. Estivariz FE, Carino M, Lowry PJ, Jackson S 1988 Further evidence that N-terminal pro-opiomelanocortin peptides are involved in adrenal mitogenesis. J Endocrinol 116:201–206[Abstract/Free Full Text]
  27. Estivariz FE, Iturriza F, McLean C, Hope J, Lowry PJ 1982 Stimulation of adrenal mitogenesis by N-terminal proopiocortin peptides. Nature 297:419–422[CrossRef][Medline]
  28. Ross JT, Bennett HP, James S, McMillen IC 2000 Infusion of N-proopiomelanocortin-(1–77) increases adrenal weight and messenger ribonucleic acid levels of cytochrome P450 17{alpha}-hydroxylase in the sheep fetus during late gestation. Endocrinology 141:2153–2158[Abstract/Free Full Text]
  29. Fassnacht M, Hahner S, Zink M, Adermann K, Beuschlein F, Allolio B, N-terminal pro-opiomelanocortin (N-POMC) acts as an adrenal mitogen. Program of the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000, p 211 (Abstract P864)
  30. Lowry PJ, Silas L, McLean C, Linton EA, Estivariz FE 1983 Pro-{gamma}-melanocyte-stimulating hormone cleavage in adrenal gland undergoing compensatory growth. Nature 306:70–73[CrossRef][Medline]
  31. Hansen I, Fassnacht M, Hammer F, Schammann M, Allolio B 2002 Is the human airway trypsin-like protease (HAT) a key regulator of adrenal growth? Exp Clin Endocrinol Diabetes 110(Suppl 1):p236
  32. Spencer SJ, Mesiano S, Lee JY, Jaffe RB 1999 Proliferation and apoptosis in the human adrenal cortex during the fetal and perinatal periods: implications for growth and remodeling. J Clin Endocrinol Metab 84:1110–1115[Abstract/Free Full Text]
  33. Mitani F, Mukai K, Miyamoto H, Suematsu M, Ishimura Y 1999 Development of functional zonation in the rat adrenal cortex. Endocrinology 140:3342–3353[Abstract/Free Full Text]
  34. Reiter RJ, Pizzarello DJ 1966 Radioautographic study of cellular replacement in the adrenal cortex of male rats. Texas Rep Biol Med 24:189–194
  35. Dallman MF, Engeland WC, Shinsako J 1976 Compensatory adrenal growth: a neurally mediated reflex. Am J Physiol 231:408–414[Abstract/Free Full Text]
  36. Belloni AS, Mazzocchi G, Meneghelli V, Nussdorfer GG 1978 Cytogenesis in the rat adrenal cortex: evidence for an ACTH-induced centripetal cell migration from the zona glomerulosa. Arch Anat Hist Norm Exp 61:195–206
  37. Smas CM, Sul HS 1993 Pref-1, a protein containing EGF-like repeats, inhibits adipocyte differentiation. Cell 73:725–734[CrossRef][Medline]
  38. Jensen CH, Krogh TN, Hojrup P, Clausen PP, Skjodt K, Larsson LI, Enghild JJ, Teisner B 1994 Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2. Eur J Biochem 225:83–92[Medline]
  39. Mountjoy KG, Robbins LS, Mortrud MT, Cone RD 1992 The cloning of a family of genes that encode the melanocortin receptors. Science 257:1248–1251[Abstract/Free Full Text]
  40. Lebrethon MC, Naville D, Begeot M, Saez JM 1994 Regulation of corticotropin receptor number and messenger RNA in cultured human adrenocortical cells by corticotropin and angiotensin II. J Clin Invest 93:1828–1833
  41. Penhoat A, Jaillard C, Begeot M, Durand P, Saez JM 1996 Cycloheximide enhances ACTH-receptor mRNA through transcriptional and post-transcriptional mechanisms in bovine adrenocortical cells. Mol Cell Endocrinol 121:57–63[CrossRef][Medline]
  42. Dallman MF, Engeland WC, McBride MH 1977 The neural regulation of compensatory adrenal growth. Ann NY Acad Sci 297:373–392
  43. Cammas FM, Pullinger GD, Barker S, Clark AJ 1997 The mouse adrenocorticotropin receptor gene: cloning and characterization of its promoter and evidence for a role for the orphan nuclear receptor steroidogenic factor 1. Mol Endocrinol 11:867–876[Abstract/Free Full Text]
  44. Farese RV, Reddy WJ 1963 Observations on the interrelations between adrenal protein, RNA and DNA during prolonged ACTH administration. Biochim Biophys Acta 76:145–148[CrossRef]
  45. Dallman MF, Engeland WC, Holzwarth MA, Scholz PM 1980 Adrenocorticotropin inhibits compensatory adrenal growth after unilateral adrenalectomy. Endocrinology 107:1397–404[Abstract]
  46. Shutter JR, Graham M, Kinsey AC, Scully S, Luthy R, Stark KL 1997 Hypothalamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice. Genes Dev 11:593–602[Abstract/Free Full Text]
  47. Robbins LS, Nadeau JH, Johnson KR, Kelly MA, Roselli-Rehfuss L, Baack E, Mountjoy KG, Cone RD 1993 Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72:827–834[CrossRef][Medline]
  48. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, Lee F 1997 Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88:131–141[CrossRef][Medline]
  49. Broberger C, Johansen J, Johansson C, Schalling M, Hokfelt T 1998 The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice. Proc Natl Acad Sci USA 95:15043–15048[Abstract/Free Full Text]
  50. Levin N, Davis JR, Komorowski R, Zeni L, Rosenfeld RD, Stark KL, Agouti-related protein (AGRP) profoundly increases food intake and body weight when administered centrally. Program of the 81st Annual Meeting of The Endocrine Society, San Diego, CA, 1999, p 66 (Abstract OR 3-5)
  51. Kastin AJ, Akerstrom V, Hackler L 2000 Agouti-related protein(83–132) aggregates and crosses the blood-brain barrier slowly. Metabolism 49:1444–1448[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Development