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Endocrinology Vol. 148, No. 6 2644-2652
Copyright © 2007 by The Endocrine Society

Disabled-2 Is Expressed in Adrenal Zona Glomerulosa and Is Involved in Aldosterone Secretion

Damian G. Romero, Licy L. Yanes, Angela F. de Rodriguez, Maria W. Plonczynski, Bronwyn L. Welsh, Jane F. Reckelhoff, Elise P. Gomez-Sanchez and Celso E. Gomez-Sanchez

Division of Endocrinology (D.G.R., L.L.Y., A.F.d.R., M.W.P., B.L.W., E.P.G.-S., C.E.G.-S.), G. V. (Sonny) Montgomery Veterans Affairs Medical Center, and Departments of Medicine (D.G.R., L.L.Y., A.F.d.R., B.L.W., E.P.G.-S., C.E.G.-S.), Physiology and Biophysics (J.F.R.), and Pharmacology and Toxicology (E.P.G.-S.), University of Mississippi Medical Center, Jackson, Mississippi 39216

Address all correspondence and requests for reprints to: Damian G. Romero, Ph.D., Division of Endocrinology, Department of Medicine, The University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216. E-mail: dromero{at}medicine.umsmed.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The differentiation of the adrenal cortex into functionally specific zones is probably due to differential temporal gene expression during fetal growth, development, and adulthood. In our search for adrenal zona glomerulosa-specific genes, we found that Disabled-2 (Dab2) is expressed in the zona glomerulosa of the rat adrenal gland using a combination of laser capture microdissection, mRNA amplification, cDNA microarray hybridization, and real-time RT-PCR. Dab2 is an alternative spliced mitogen-regulated phosphoprotein with features of an adaptor protein and functions in signal transduction, endocytosis, and tissue morphogenesis during embryonic development. We performed further studies to analyze adrenal Dab2 localization, regulation, and role in aldosterone secretion. We found that Dab2 is expressed in the zona glomerulosa and zona intermedia of the rat adrenal cortex. Low-salt diet treatment increased Dab2-long isoform expression at the mRNA and protein level in the rat adrenal gland, whereas high-salt diet treatment did not cause any significant modification. Angiotensin II infusion caused a transient increase in both Dab2 isoform mRNAs in the rat adrenal gland. Dab2 overexpression in H295R human adrenocortical cells caused an increase in aldosterone synthase expression and up-regulated aldosterone secretion under angiotensin II-stimulated conditions. In conclusion, Dab2 is an adrenal gland zona glomerulosa- and intermedia-expressed gene that is regulated by aldosterone secretagogues such as low-salt diet or angiotensin II and is involved in aldosterone synthase expression and aldosterone secretion. Dab2 may therefore be a modulator of aldosterone secretion and be involved in mineralocorticoid secretion abnormalities.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAMMALIAN ADRENAL cortex is composed of three distinct zones: the zona glomerulosa, zona fasciculata, and zona reticularis (1, 2, 3). The cells in the zona glomerulosa, underlying the adrenal capsule, secrete aldosterone. The zona fasciculata cells, localized between the zona glomerulosa and the zona reticularis, secrete glucocorticoids. The zona reticularis cells surround the adrenal medulla and secrete adrenal androgens and small amounts of glucocorticoids. Each adrenal cortex zone has a differential regulation pattern as well as the hormones secreted due to such regulation. The differentiation of the adrenal cortex zones is probably due to differential temporal gene expression during fetal growth, development, and adulthood (4, 5, 6, 7, 8, 9).

In our search for adrenal zona glomerulosa-specific genes that may be involved in steroidogenesis and proliferation regulation, we performed a differential gene expression pattern analysis between rat adrenal zona glomerulosa and zona fasciculata-reticularis by a combination of laser capture microdissection, mRNA amplification, cDNA microarray hybridization, and real-time RT-PCR. We found that Dab2 (also known as Disabled-2 homologue, DOC2, C9, and p96/p67) was exclusively expressed in rat adrenal zona glomerulosa.

Dab2 is a mitogen-regulated phosphoprotein with features of an adaptor protein and functions in signal transduction, endocytosis, and tissue morphogenesis during embryonic development (10). Dab2 was initially described as a phosphoprotein in mitogenic signal transduction in murine macrophages (11). Dab2 was suggested to be involved in human ovarian cancer after being identified as a down-regulated gene in ovarian carcinomas compared with nontumor ovarian surface epithelial cells (12). Several studies confirmed its loss of expression in tumors (13, 14, 15) and growth-suppressive activity when exogenously overexpressed (14, 15, 16, 17). Dab2 loss of expression has been also reported in other epithelial tumor types (18, 19, 20). Dab2 is an adaptor protein that has been reported to interact with several proteins including clathrin (21, 22), activator protein-2 (21, 22), myosin VI (23, 24), and low-density lipoprotein family receptors (21, 25, 26, 27). Dab2 regulates mitogenic signal transduction by less defined signaling pathways (17, 28, 29, 30). The Dab2 gene is alternatively spliced to produce two major protein products originally identified as p96 and p67 in the mouse corresponding to polypeptides with apparent molecular masses of 96 and 67 kDa, respectively (11). Similar splice variants have been described in the human and rat (20, 31). Dab2 is essential in the visceral endoderm for embryonic development and the absence of its expression is embryonic lethal (32, 33).

To further understand the role and regulation of Dab2 in the adrenal gland, we performed studies of the localization of Dab2 in the rat adrenal gland, its regulation by salt diet and angiotensin II (Ang II) and its role in aldosterone secretion by adrenal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All animal protocols were approved by the Institutional Animal Care and Use Committee of the G. V. Montgomery Veterans Affairs Medical Center and the Central Office of the Department of Veterans Affairs as the Animal Component of Research Protocol. All animal protocols were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.

Male Sprague Dawley rats (3 months old) were obtained from Harlan Sprague Dawley (Indianapolis, IN) and maintained on standard rat chow (Teklad, Harlan, Indianapolis, IN) and tap water in an environment with 12-h light, 12-h dark cycles.

Salt diet manipulation.
Rats (n = 6 per group) were fed ad libitum a standard normal-salt diet (0.3% NaCl; Harlan, Madison, WI), low-salt diet (0.03% NaCl), or high-salt diet (standard chow plus 0.9% saline to drink). Salt diet manipulations were performed for 2, 9, or 15 d.

Ang II infusion.
Rats (n = 6 per group) were anesthetized by isoflurane inhalation and a catheter placed in the femoral vein. The catheter was exteriorized at the back of the neck and rats were allowed to recover for 2 d. Conscious unrestrained rats were connected to an infusion pump and infused for 0.5, 2, or 6 h with Ang II (American Peptide Co., Sunnyvale, CA) in saline at a dose of 100 ng/kg·min at rate of 500 µl/h. Control rats received saline infusion under similar conditions.

At the end of the experimental protocols, rats were anesthetized with isoflurane, adrenal glands were removed, excised of fat, flash frozen in liquid nitrogen, and stored at –80 C for real-time RT-PCR or Western blot analysis or processed as described below for immunohistochemistry.

Antibodies
Dab2 mouse monoclonal antibody raised against amino acids 31–45 of mouse Dab2, 100% identical with the rat sequence (BD Biosciences, San Jose, CA; catalog no. 610464) was used for immunohistochemistry. Dab2 rabbit polyclonal antibody raised against amino acids 661–770 of human Dab2, 72% identical and 80% similar to the rat sequence (Santa Cruz Biotechnology, Santa Cruz, CA; catalog no. H-110) was used for immunofluorescence and Western blots. Aldosterone synthase monoclonal mouse antibody was generated by immunizing mice with the peptide (KVRQNARGSLTMDVQQSL-MAP) as previously described (34). Delta-like 1 homolog (Dlk1) polyclonal antibody was generated by immunizing New Zealand White rabbits with the full-length rat Dlk1 recombinant protein.

Western-blot
Adrenal glands were homogenized in T-PER (Pierce Biotechnology, Rockford, IL) supplemented with 5 mM EDTA, 1 mM EGTA, protease inhibitor cocktail (Roche, Indianapolis, IN), and phosphatase inhibitor cocktail set I and II (EMD Biosciences, San Diego, CA) at 1 ml/adrenal. Homogenates were centrifuged at 12,000 x g for 15 min at 4 C and supernatants saved at –80 C. For Western blot-positive controls, CHO-K1 cells maintained in DMEM-high glucose supplemented with 10% fetal bovine serum were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s suggested protocol with plasmids expressing rat Dab2-p82 and Dab2-p59. Total protein was quantified with the bicinchoninic acid method (Pierce Biotechnology) using BSA as standard. Protein aliquots were separated by PAGE in 7.5% polyacrylamide gels, transferred to polyvinylidene difluoride membranes using a semidry technique, and blocked with 1% Carnation dry skim milk in 50 mM Tris-HCl buffer (pH 7.5) with 0.05% Tween 20. The membranes were incubated with the rabbit anti Dab2 polyclonal antibody overnight. The blots were then incubated with an affinity purified peroxidase-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h and developed using West Pico chemiluminescence substrate (Pierce Biotechnology). The films were scanned and quantified with a Image Station 440 CF (Kodak, Rochester, NY) using Kodak 1D image analysis software. The membranes were stripped and reprobed with anti-ß-tubulin antibody (monoclonal antibody developed by Michael Klymkowsky and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA). Results are expressed as arbitrary units (AU) and normalized against ß-tubulin.

Immunohistochemistry
Adrenal glands were collected from anesthetized rats perfused with heparinized saline followed by Streck Tissue Fixative (Streck Tissue Fixative, Omaha, NE). Adrenal glands were further fixed in Streck Tissue Fixative overnight and embedded in paraffin. Four-micrometer sections were cut, deparaffinized; treated with 0.1% phenylhydrazine for 10 min to inhibit peroxidases; and then blocked with 0.05 M Tris (pH 7.6), 2% Carnation dry skim milk, 5% normal goat serum, and 0.5% sodium dodecyl sulfate for 1 h. The slides were then incubated overnight with the primary antibodies in above buffer containing 0.05% Tween 20. For immunohistochemistry, sections were washed and then incubated for 1 h with an affinity purified donkey antimouse IgG biotin-labeled antibody (Jackson ImmunoResearch Laboratories), washed, and incubated with ZyMax horseradish peroxidase-conjugated streptavidin (Zymed, Invitrogen, Carlsbad, CA) in Superblocker (Pierce Biotechnology) for 30 min. The slides were developed using diaminobenzidine, counterstained with hematoxylin, and mounted with Permount (Fisher Scientific, Pittsburgh, PA) (35). Negative controls included incubation with no primary antibody. For immunofluorescence, sections were washed and then incubated for 1 h with Alexa Fluor-conjugated secondary antibodies (Molecular Probes, Carlsbad, CA). Sections were washed and mounted in 90% glycerol in PBS. Fluorescence images were acquired using Metavue software (version 6.2r6; Molecular Devices Corp., Downingtown, PA), and deconvoluted with Autoquant software version X1.3.0 (Media Cybernetics Inc., Silver Spring, MD).

RNA extraction and RT-PCR
Dab2 isoform mRNAs were quantified as previously described (36, 37). Briefly, total RNA was extracted with Tri-Reagent (MRC, Cincinnati, OH), resuspended in diethyl pyrocarbonate-H2O, DNase treated with Turbo DNA-free kit (Ambion, Austin, TX), and quantified by spectrophotometry. Five micrograms of RNA were reverse transcribed (RT) with 0.5 µg of T12VN primer and Superscript III (Invitrogen) in a final volume of 20 µl. The reaction was carried out for 60 min at 50 C and terminated by incubation at 75 C for 15 min. Dab2 isoform-specific primers and luciferase primers were designed with Primer3 software (38) (Table 1Go). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were previously described (39). Real-time PCR contained 1 µl of RT product, 0.1 µM each of primer, 0.2 mM deoxynucleotide triphosphates, SYBR green I (1:20,000 final concentration; Molecular Probes, Eugene, OR), and 1 µl of Titanium Taq DNA polymerase (CLONTECH, Palo Alto, CA). Amplifications were performed in a real-time thermal cycler (iCycler; Bio-Rad Laboratories, Hercules, CA). Cycling conditions were 1 min at 95 C, followed by 50 cycles of 15 sec at 95 C, 15 sec at 60 C, and 60 sec at 72 C. Fluorescence data were collected during the elongation step. After PCR amplification, the specificity of the PCR was confirmed by melting temperature determination of the PCR product and electrophoretic analysis in 4% NuSieve 3:1 agarose gels (Cambrex, Rockland, ME). PCR product quantification was performed by the relative quantification method (40), standardized against GAPDH, and expressed as arbitrary units. Efficiency for each primer pair was assessed by using serial dilutions of pooled RT product.


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  		TABLE 1. PCR primers

 
Plasmids
Rat Dab2 isoform expressing plasmids in the pCI-neo backbone, p82 (equivalent to p96 in mouse) and p59 (equivalent to p67 in mouse) were previously described (20). Reporter plasmid carrying the human aldosterone synthase promoter (41) was generated by transferring the promoter regions from pGL3-Basic to pGL4.10[luc2] (Promega, Madison, WI), as we have previously described (42). pCMVi-Luc+ was previously described (43).

Reporter assays
H295R human adrenocortical cells (44) were cultured in H295R complete media containing DMEM-F12 (1:1) supplemented with 2% Ultroser G (Biosepra, Villeneuve-la-Garenne, France), ITS-Plus (Discovery Labware, Bedford, MA), and antibiotic/antimycotic mixture (Invitrogen) as we previously described (36). H295R cells were plated in 24-well plates with media without antibiotic/antimycotics and grown until 90–95% confluent. H295R cells were transfected by a combination of cationic lipids (Lipofectamine 2000; Invitrogen) and magnetofection (CombiMag; Oz Biosciences, Marseille, France) as we previously described (45). Briefly, cells were transfected with 3 µg plasmid DNA/well (2 µg reporter plasmid plus 1 µg expression plasmid), 2 µl/well Lipofectamine 2000 (Invitrogen), and 4.5 µl/well CombiMag following the manufacturer’s suggested protocols. Cells were cultured overnight, media replaced with 1 ml/well fresh media with or without Ang II (10 nM), and cultured for an additional 24 h. Cells were lysed with Glo Lysis buffer (Promega) and luciferase activity quantified with Bright-Glo Luciferase assay kit (Promega). Reporter assays were performed in quadruplicates using three different plasmid DNA maxipreps in each experiment to avoid plasmid DNA preparation-related effects.

Aldosterone secretion
H295R cells were transfected using Nucleofector technology (Amaxa Biosystems, Gaithersburg, MD) as previously reported (45). Briefly, 3 million log-phase cells were resuspended in 100 µl Nucleofector Solution R, mixed with 3 µg plasmid DNA, and electroporated using the proprietary program P-20. Cells were allowed to recover for 15 min in RPMI 1640 media at 37 C and then plated in 24-well plates with 1 ml H295R complete media per well. Cells were cultured for 16 h. The media were then removed and the cells incubated with prewarmed media with or without 10 nM Ang II for 24 h. At the end of the incubation period, cell culture supernatants were saved for aldosterone determination by ELISA as previously reported (46). Cells were lysed with M-PER lysis buffer (Pierce) and protein concentration measured using the Coomassie Plus kit (Pierce).

Statistical analysis
All results were expressed as mean ± SEM. Multiple experimental groups under one treatment were analyzed by one-way ANOVA followed by Dunnett’s contrasts against the control group. Multiple experimental groups under two treatments were analyzed by two-way ANOVA followed by Bonferroni contrasts. Statistical calculations were performed with GraphPad Prism package (version 4.03; GraphPad Software, Inc., San Diego, CA). All experiments were repeated at least three times. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dab2 is expressed in rat adrenal zona glomerulosa
Rat adrenal gland expressed both the short and long isoforms of Dab2 protein as determined by Western blot analysis (Fig. 1Go). Both Dab2 isoforms migrated with apparent molecular weights similar to the proteins generated by transient transfection of the plasmids encoding the rat isoforms of Dab2.


Figure 1
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  		FIG. 1. Dab2 protein expression in rat adrenal gland. Total adrenal homogenate (100 µg), adrenal capsule homogenate (50 µg), and CHO-transfected cells (5 µg) were separated by PAGE, transferred to polyvinyl difluoride membranes, and probed with anti-Dab2 antibody.

 
To study the expression pattern of Dab2 in rat adrenal, we performed immunohistochemistry in normal adult adrenal glands. Figure 2AGo shows that Dab2 protein is expressed in the zona glomerulosa of the rat adrenal gland with no expression in the zona fasciculata-reticularis or medulla. Under higher magnification, it is clearly noticeable that Dab2 staining is strong in the outer layers of the adrenal cortex with no staining in the zona fasciculata-reticularis or capsule (Fig. 2Go, B and C). Negative control slides with no primary antibody show no staining (Fig. 2Go, D–F).


Figure 2
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  		FIG. 2. Dab2 immunolocalization in rat adrenal gland. A–F, Rat adrenal glands were immunostained with anti-Dab2 antibody (A–C) or control antibody (D–F). G–O, Rat adrenal glands from animals treated with high- (G–I), normal- (J–L), or low- (M–O) salt diet for 15 d were double immunostained with anti-Dab2 (G, J, and M, shown in red) and anti-Dlk1 (H, K, and N, shown in green) antibodies. Nuclei were stained with 4',6'-diamino-2-phenylindole (DAPI) and are shown in blue in merged pictures. Scale bars, 500 µm (A), 100 µm (B), 20 µm (C), 40 µm (O).

 
To determine more precise localization of the cells expressing Dab2, sections of adult male rat adrenal glands were double stained with antibodies against Dab2 and Dlk1 (also known as ZOG, Pref-1, FA1, pG2, SCP-1) (Fig. 2Go). Dab2 colocalized with Dlk1 expression in rat adrenal glands from control animals. Dlk1 has been reported to be expressed in both the zona glomerulosa and the outer zona intermedia (47). To further study whether salt diet treatment, a well-known stimulator of zona glomerulosa proliferation, modifies Dab2 colocalization with Dlk1, we performed double-staining studies with adrenal glands from animals treated with low- or high-salt diet for 15 d (Fig. 2Go, G–O). As expected, low-salt diet treatment caused proliferation of the adrenal zona glomerulosa, but neither low- nor high-salt diet treatment modified the colocalization pattern of Dab2 with Dlk1 as observed in the control animals.

Dab2 is regulated by low-salt diet
One of the main physiological modulators of adrenal gland zona glomerulosa physiology is salt intake. To study whether salt intake regulates Dab2 expression in adrenal gland, rats were placed on low- or high-salt diet for 2, 9, or 15 d. First we studied whether salt diet treatment modified the Dab2 expression pattern by immunolocalization in rat adrenal glands. As a control we studied the expression pattern of aldosterone synthase in the same adrenal glands. Low-salt diet treatment caused a continuous increase in zona glomerulosa width as evidenced by an increase in the number of cell layers expressing aldosterone synthase (Fig. 3Go, lower panel). In contrast, high-salt diet treatment caused a decrease in the number of cells expressing aldosterone synthase although some positive cells were still detectable after 15 d of treatment. Neither low- nor high-salt diet treatment modified the expression pattern of Dab2 in rat adrenal glands (Fig. 3Go, upper panel). There was no significant staining for Dab2 in either the zona fasciculata-reticularis or the medulla under any diet treatment condition. Low-salt diet treatment caused a noticeable increase in the number of cells stained for Dab2. In contrast, high-salt diet did not cause a significant change in Dab2 expression. We performed studies to quantify Dab2 mRNA and protein expression by real-time RT-PCR and Western blots, respectively, in whole adrenal glands from animals treated with different salt diets for 2, 9, and 15 d. Both Dab2 mRNA isoforms were quantified by real-time RT-PCR using isoform-specific primers. Low-salt diet caused a continuous increase in Dab2-long isoform mRNA reaching values of more than 3.5-fold induction after 15 d of treatment (3.51 ± 0.47 vs. 1.01 ± 0.08 AU, P < 0.01) (Fig. 4AGo). In contrast, high-salt diet did not modify Dab2-long isoform mRNA levels up to 15 d of treatment. Neither low- nor high-salt treatment up to 15 d modified Dab2-short isoform mRNA levels (Fig. 4BGo).


Figure 3
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  		FIG. 3. Dab2 and aldosterone synthase immunolocalization in rat adrenal glands under different salt diets. Rats were maintained in normal-, low-, or high-salt diet for 2, 9, or 15 d. Adrenal slices were stained with anti-Dab2 or antialdosterone synthase antibodies. Slides are representative of multiple slides from three animals per group. Scale bar, 100 µm.

 

Figure 4
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  		FIG. 4. Adrenal Dab2 mRNA levels under low/high-salt diet treatment. Rats were maintained in low/high-salt diet for 0, 2, 9, or 15 d and Dab2-long (A) and Dab2-short (B) mRNA isoforms quantified by real-time RT-PCR. GAPDH mRNA was used as housekeeping gene. Results are expressed as Dab2/GAPDH in arbitrary units. *, P < 0.05 vs. normal salt.

 
To confirm that increases in Dab2 mRNA levels produced by the low-sodium diet also increased Dab2 protein levels in adrenal glands, we quantified both Dab2 isoforms by Western blot analysis (Fig. 5Go). Low-salt diet caused a continuous increase in Dab2-long isoform protein expression that reach values of more than 2-fold induction after 15 d of treatment (2.18 ± 0.15 vs. 1.00 ± 0.34 AU, P < 0.05). In contrast, Dab2-short protein levels remained constant up through 15 d of low-salt treatment.


Figure 5
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  		FIG. 5. Adrenal Dab2 protein levels under low/high-salt diet treatment. Rats were maintained in low/high-salt diet for 0, 2, 9, or 15 d and Dab2 protein isoforms quantified by Western blot. Tubulin was used as loading control. Results are expressed as Dab2/tubulin in arbitrary units. *, P < 0.05, low salt vs. control.

 
Dab2 is regulated by Ang II infusion in vivo
To study whether Ang II, a well-known aldosterone secretagogue and modulator of adrenal zona glomerulosa physiology, regulates Dab2 expression in adrenal gland, rats were infused with Ang II. Conscious chronically cannulated rats were infused with Ang II for 0.5, 2, or 6 h, and Dab2 mRNA isoforms were quantified by real-time RT-PCR in adrenal glands. Ang II infusion caused a transient increase after 2 h in both Dab2 isoforms with mRNAs levels returning to basal levels after 6 h of Ang II infusion (Fig. 6Go). After 2 h of Ang II infusion, there was a significant increase, almost doubling of both the Dab2-long isoform (1.96 ± 0.21 vs. 0.87 ± 0.15 AU, P < 0.05) and the Dab2-short isoform (1.76 ± 0.17 vs. 1.01 ± 0.20 AU, P < 0.05) mRNA levels.


Figure 6
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  		FIG. 6. Adrenal Dab2 mRNA levels in Ang II-infused rats. Rats were cannulated through the femoral vein, allowed to recover for 2 d, and infused for 0.5, 2, or 6 h with or without Ang II (100 ng/kg·min, 500 µl/h). Control rats for each time point were infused with saline. Adrenals were removed and Dab2 isoforms quantified by real-time RT-PCR. GAPDH mRNA was used as housekeeping gene. Results are expressed as Dab2/GAPDH in arbitrary units. *, P < 0.05, Ang II vs. control.

 
Dab2 regulates aldosterone synthase expression in H295R cells
To determine whether Dab2 up-regulation modulates adrenal aldosterone synthesis, we performed reporter gene expression studies of aldosterone synthase in H295R human adrenocortical cells. The H295R human adrenocortical cell (44) is a widely used in vitro model for the study of adrenal cell physiology and metabolism because it is the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells (48). Aldosterone synthase is the last and unique enzyme in the aldosterone biosynthetic pathway exclusively expressed in adrenal gland zona glomerulosa. Two plasmids that constitutively expressed both Dab2 isoforms were used for Dab2 overexpression. Reporter assay studies were performed under basal and suboptimal Ang II stimulatory conditions. Both Dab2 isoforms significantly increased aldosterone synthase expression under Ang II-stimulated conditions (Fig. 7Go). Dab2-long and Dab2-short isoforms caused a 2.6-fold (194.9 ± 31.7 vs. 366.2 ± 6.1 AU, P < 0.05) and 2.9-fold (194.9 ± 31.7 vs. 393.3 ± 44.9 AU, P < 0.05) increase, respectively, of aldosterone synthase expression. In contrast, neither Dab2 isoform regulated aldosterone synthase expression under basal conditions.


Figure 7
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  		FIG. 7. Effect of Dab2 on aldosterone synthase reporter gene expression. H295R cells were cotransfected with aldosterone synthase reporter plasmid and Dab2-long, Dab2-short, or control plasmid (pCI-neo). Cells were allowed to recover overnight and then treated with or without Ang II (10 nM) for 24 h. Data are expressed as percentage of control-basal. *, P < 0.05 vs. control-basal; #, P < 0.05 vs. control-Ang II.

 
Dab2 regulates aldosterone secretion in H295R cells
To further study whether Dab2 effect on aldosterone synthase expression is translated into an increase in aldosterone secretion, we transfected H295R cells with plasmids overexpressing both Dab2 isoforms and determined aldosterone secretion under basal and suboptimal Ang II stimulatory conditions. Whereas only Dab2 short isoform caused a 2.1-fold increase in aldosterone secretion under basal conditions, both long and short Dab2 isoforms increased aldosterone secretion under suboptimal Ang II stimulatory conditions, 1.3- and 2.0-fold, respectively (Fig. 8AGo). Dab2 isoform-specific overexpressing plasmids caused an increase at the mRNA and protein level of their respective isoforms (Fig. 8Go, B and C). Cotransfection with a plasmid constitutively expressing luciferase indicates similar transfection efficiencies when quantified as plasmid-driven luciferase over endogenous GAPDH mRNAs ratios (Fig. 8DGo).


Figure 8
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  		FIG. 8. Effect of Dab2 on aldosterone secretion by H295R cells. A, H295R cells were transfected with Dab2-long, Dab-short, or control plasmid (pCI-neo), cultured overnight, and then treated with or without Ang II (10 nM) for 24 h. Aldosterone was measured in cell culture supernatants by ELISA. B, Dab2 mRNA isoform-specific overexpression quantified by real-time RT-PCR. C, Dab2 protein overexpression quantified by Western blot. D, Transfection efficiency quantified as luciferase/GAPDH mRNA. *, P < 0.05 vs. control-basal; #, P < 0.05 vs. control-Ang II. NS, Not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of the present study are: 1) Dab2 is exclusively expressed in the zonas glomerulosa and intermedia of the rat adrenal gland, 2) Dab2 long and short isoforms are expressed in the rat adrenal gland, 3) low-salt diet treatment increases Dab2-long isoform expression at the mRNA and protein level in the rat adrenal gland, 4) Ang II infusion causes a transient increase in both Dab2 mRNA isoforms in the rat adrenal gland, 5) Dab2 increases aldosterone synthase expression under Ang II-stimulated conditions in cotransfection reporter assays in H295R cells, and 6) overexpression of Dab2 isoforms in H295R cells increases aldosterone secretion under Ang II-stimulated conditions.

Our combined results by Western blot and immunohistochemistry confirmed that Dab2 is highly expressed in the outer cortical zone, more precisely the zona glomerulosa and the zona intermedia. In our study, Dab2 long and short isoforms migrated with an apparent molecular mass of approximately 100 and approximately 75 kDa, respectively. These bands correspond to the originally described rat Dab2 isoforms of apparent molecular mass of 82 and 59 kDa, respectively [C9-p82 and C9-p59 (20)]. Although there is a discrepancy in apparent molecular mass with that originally described for the rat, the apparent molecular mass for Dab2 isoforms that we observed are similar to the ones reported in mice [96 and 67 kDa (11)] and humans (31). We think this discrepancy is due to overestimated expected molecular mass because the rat, mouse, and human isoform sequences are very similar and both isoforms migrate with apparent molecular masses similar to the proteins generated by transient transfection of the plasmids encoding the rat isoforms of Dab2.

Double-staining immunofluorescence studies indicated that Dab2 colocalizes with Dlk1 in both the zona glomerulosa and zona intermedia. The zona intermedia of the adrenal cortex is localized between the zona glomerulosa and zona fasciculata. The zona intermedia was described more than 50 yr ago by its unique cytological characteristics (49), but it was not until a decade ago that it was characterized by the absence of expression of either of the last steroidogenic enzymes involved in mineralocorticoid or glucocorticoid biosynthesis, aldosterone synthase, or 11ß-hydroxylase, respectively (50).

Dlk1 (also known as ZOG, Pref-1, FA1, pG2, SCP-1) is a transmembrane and secreted protein. It is a member of the epidermal growth factor-like family with homologies to Notch/{delta}/Serrate, containing a signal peptide, followed by six epidermal growth factor-like repeats, a transmembrane domain, and a short intracellular tail (51). Dlk1 is expressed in a variety of murine and human fetal cells, but it is present only in a limited number of adult tissues (52, 53, 54, 55, 56). Dlk1 participates in several differentiation processes including adipogenesis (57, 58, 59), hematopoiesis (60, 61, 62), and adrenal gland development (52, 63) as well as wound healing (64); it is also involved in the control of embryonic growth (65, 66, 67). Detailed studies by Halder et al. (47) have shown that Dlk1 (reported as ZOG in the manuscript) was expressed in the zona glomerulosa and the zona intermedia of the rat adrenal cortex. They clearly show that there were cells expressing Dlk1 that did not express either aldosterone synthase or 11ß-hydroxylase. Our results indicated that Dab2 colocalizes with Dlk1 in the rat adrenal cortex in rats under normal-, low-, or high-salt diet intake.

In our study, both Dab2 isoforms were expressed in rat adrenal gland, but only Dab2-long isoform was up-regulated by a low-salt diet, suggesting that whereas the Dab2-long isoform plays a role in low-salt diet-induced aldosterone secretion, the Dab2-short isoform is probably involved in basal aldosterone secretion. On the other hand, both Dab2 isoform mRNAs are transiently up-regulated by Ang II infusion, suggesting that, although sharing some similarities, low-salt diet and Ang II differentially regulate Dab2 expression in the rat adrenal. It is important to point out that although one of the main physiological responses to low-salt diet is an up-regulation of the renin angiotensin system, a low-salt diet may cause a plethora of physiological responses to achieve a new equilibrium. Furthermore, low-salt diet treatment is a chronic condition, whereas Ang II infusion is a subacute treatment in which the animal does not reach homeostasis due to the exogenous hormone treatment.

Our transfection studies in H295R cells indicated that Dab2 up-regulated aldosterone synthase expression and aldosterone secretion under suboptimal Ang II stimulatory conditions. Although Dab2 up-regulates Ang II-mediated aldosterone synthase expression, it cannot be excluded that other steroidogenic steps are also modulated, thereby causing an increase in the secretion of the final product aldosterone. In fact, most steroidogenic regulatory inducers modulate several key regulatory processes or enzymes as cholesterol uptake, cholesterol-ester hydrolysis, or cholesterol transport to the inner mitochondrial membrane mediated by Star protein, 3ß-hydroxysteroid dehydrogenase activity, etc. (1, 2, 3, 68). The lack of effect of Dab2 on aldosterone synthase expression and aldosterone secretion (except Dab2-short isoform) under basal conditions probably indicates the requirement of other molecules to be generated or up-regulated under Ang II stimulation. Both Dab2 isoforms modulate aldosterone synthase expression and aldosterone secretion indicating that these effects are probably mediated by protein domains shared by both isoforms, the actin binding domain, the Dab homology domain, or the Ser/Pro-rich domain (11). Dab2-short isoform is generated by alternative splicing and lacks an internal fragment that is probably not involved in the above-mentioned aldosterone synthase induction effect. The mechanism(s) by which Dab2 up-regulates aldosterone synthase expression in adrenal cells is currently unknown. One possibility is that Dab2 may act as a transcription factor. Cho et al. (69) tested the ability to regulate the expression of a GAL4-responsive CAT reporter plasmid by fusion proteins between both Dab2 isoforms, Dab-p96 and Dab2-p67, to the GAL4 DNA binding domain. Both Dab2 isoforms were reported to up-regulate the reporter gene expression, indicating that Dab2 isoforms have an intrinsic transcriptional activation function. Another possibility by which Dab2 may modulate gene expression is that Dab2 has been shown to modulate several intracellular signaling pathways including the transforming growth factor-ß through phosphorylated mothers against decapentaplegic (Smad)-2 and -3 proteins (29), the Wnt/ß-catenin-mediated signaling pathway (70), and the son of sevenless (SOS)-ERK phosphorylation pathway (30).

In conclusion, we describe Dab2 as a gene expressed exclusively in the zonas glomerulosa and intermedia of the rat adrenal gland. Dab2 is up-regulated by both low-salt diet and Ang II infusion in the rat adrenal gland in vivo. Dab2 overexpression in H295R human adrenocortical cells up-regulated aldosterone synthase expression and aldosterone secretion. Dab2 is a key player in the physiology of rat adrenal cortex with an important role in the regulation of adrenal aldosterone secretion, although with the present data, we cannot exclude other roles in adrenal gland physiology. Dab2 may be one more component of the protein network that modulates aldosterone secretion. Although Dab2 caused a modest 2-fold increase in aldosterone secretion, previous studies have shown that plasma aldosterone concentration increases of only 2-fold are enough to cause profound physiological effects. Garwitz and Jones (71) performed elegant dose-response studies showing that aldosterone infusion at doses causing only a 2.3-fold increase in plasma aldosterone are enough to increase systolic blood pressure, and kidney and heart weight after 4 wk of treatment in salt-loaded uninephrectomized rats. Brilla and Weber (72) have shown that a 2-fold increase in plasma aldosterone levels increased left ventricle interstitial collagen volume fraction and perivascular collagen in intramyocardial coronary arteries after 8 wk of treatment in salt-loaded uninephrectomized rats. The clinical relevance of Dab2 in adrenal gland disorders is still unknown at present. However, our study suggests that Dab2 is a promising candidate gene whose dysregulation could lead to alterations of adrenal gland aldosterone secretion such as seen in hyperaldosteronism.


   Acknowledgments
 
The authors thank Dr. Donald. B. Sittman and Stephanie Warren from the Mississippi Functional Genomics Network-Genomics facility (Jackson, MS) for their assistance. The authors are greatly thankful to Dr. Shannon Matta and Kathleen Spencer (University of Tennessee Health Science Center, Memphis, TN) for the use of the PixCell II laser capture microdissection instrument and their assistance. The authors thank Dr. Jer-Tsong Hsieh (University of Texas Southwestern Medical Center, Dallas, TX) for the Dab2 plasmids, Dr. William E. Rainey (Medical College of Georgia, Augusta, GA) for the reporter plasmid and the H295R cells, and Dr. Stephen A. Johnston (Arizona State University, Tempe, AZ) for the control plasmid.


   Footnotes
 
This work was supported by Medical Research funds from the Department of Veterans Affairs, National Institutes of Health (NIH) Grants HL27255 and HL75321 from the National Heart, Lung, and Blood Institute and NIH Grant RR016476 from the Mississippi Functional Genomics Network-IDeA Networks of Biomedical Research Excellence (INBRE) Program of the National Center for Research Resources.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 15, 2007

Abbreviations: Ang II, Angiotensin II; AU, arbitrary unit; Dab2, Disabled-2; Dlk1, delta-like 1 homolog; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcribed.

Received November 13, 2006.

Accepted for publication February 1, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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D. G. Romero, M. W. Plonczynski, B. L. Welsh, C. E. Gomez-Sanchez, M. Y. Zhou, and E. P. Gomez-Sanchez
Gene expression profile in rat adrenal zona glomerulosa cells stimulated with aldosterone secretagogues
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