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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
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Abstract |
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Introduction |
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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.
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Materials and Methods |
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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 1
). 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 1). 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 1. 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.).
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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.
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). Furthermore, adrenal weights in the sham surgery group demonstrated no significant left to right differences in animals from both genotypes.
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). 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. 3A). 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. 3B) 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.
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, 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. 4, 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.
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). 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. 6). 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.
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, 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. 7, lower panel, and Table 2). 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.
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, 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. 8, 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.
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). 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. 9A and Table 4). 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. 9B). This difference was still evident in the remaining adrenal 72 h following uADX (Fig. 9B). 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. 9C).
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Discussion |
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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-
-MSH. Although this peptide itself does not have mitogenic potential, it has been shown, that the specific cleavage of pro--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--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--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--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.
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Acknowledgments |
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Footnotes |
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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.
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