Physiol. Genomics AJP: Renal Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 30: 26-34, 2007. First published February 27, 2007; doi:10.1152/physiolgenomics.00187.2006
1094-8341/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Table
Right arrow All Versions of this Article:
30/1/26    most recent
00187.2006v1
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 Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Romero, D. G.
Right arrow Articles by Gomez-Sanchez, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Romero, D. G.
Right arrow Articles by Gomez-Sanchez, C. E.
Received 24 August 2006; accepted in final form 22 February 2007.
Physiological Genomics 30:26-34 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Adrenal transcription regulatory genes modulated by angiotensin II and their role in steroidogenesis

Damian G. Romero 1,2, Silvia Rilli 2, Maria W. Plonczynski 1, Licy L. Yanes 1,2, Ming Yi Zhou 3, Elise P. Gomez-Sanchez 1,2 and Celso E. Gomez-Sanchez 1,2

1 Division of Endocrinology, G.V. (Sonny) Montgomery Veterans Affairs Medical Center, and
2 Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi; and
3 DNA Core, University of Missouri-Columbia, Columbia, Missouri


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transcription regulatory genes are crucial modulators of cell physiology and metabolism whose intracellular levels are tightly controlled to respond to extracellular stimuli. We studied transcription regulatory genes modulated by angiotensin II, one of the most important regulators of adrenal cortical cell function, and their role in adrenal steroidogenesis in H295R human adrenocortical cells. Angiotensin II-modulated transcription regulatory genes were identified with high-density oligonucleotide microarrays and the results validated by real-time RT-PCR. Cotransfection reporter assays were performed in H295R cells to analyze the role of these transcription regulatory genes in the control of the expression of 11ß-hydroxylase and aldosterone synthase, the last and unique enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways, respectively. We selected a subset of the most regulated genes for reporter plasmid studies to determine the effect on these enzymes. BHLHB2, BTG2, and SALL1 decreased expression of both enzymes, whereas CITED2, EGR2, ELL2, FOS, FOSB, HDAC5, MAFF, MITF, NFIL3, NR4A1, NR4A2, NR4A3, PER1, and VDR increased expression for both enzymes. By the ratio of aldosterone synthase to 11ß-hydroxylase expression, NFIL3, NR4A1, NR4A2, and NR4A3 show the greatest selectivity toward upregulating expression of the mineralocorticoid biosynthetic pathway preferentially. In summary, this study reports for the first time a set of transcription regulatory genes that are modulated by angiotensin II and their role in adrenal gland steroidogenesis. Abnormal regulation of the mineralocorticoid or glucocorticoid biosynthesis pathways is involved in several pathophysiological conditions; hence the modulated transcription regulatory genes described may correlate with adrenal steroidogenesis pathologies.

adrenal cortex; transcription regulation; gene expression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ONE OF THE MOST BASIC LEVELS in the regulation of cellular physiology is at the level of control of gene transcription. Gene products with "transcription regulator activity" are defined by the Gene Ontology Database as any gene product that plays a role in regulating transcription, which may include binding a promoter or enhancer DNA sequence or interacting with a DNA binding transcription factor (1). Transcription regulatory proteins are key molecules because any alteration in their level or activity generally results in the modification of multiple cellular processes. Adrenal cortical cells secrete mineralocorticoids and glucocorticoids under the stimulatory effect of a great variety of molecules, some of the most important being angiotensin II (ANG II), adrenocorticotropic hormone (ACTH), and potassium. Transcription regulators whose levels are modified by any of these secretagogues are expected to be important for normal adrenal cell physiology and, consequently, crucial to the maintenance of mineralocorticoid- and glucocorticoid-regulated homeostasis. Abnormal regulation or function of transcription regulatory factors would be expected to lead to alterations in adrenal gland development, morphology, and function, resulting in a wide range of pathophysiological conditions associated with adrenal steroid excess or deficiency.

We have previously described several genes regulated by ANG II in H295R human adrenocortical cells using cDNA microarrays (44), including two transcription regulatory genes, the orphan nuclear receptors NR4A2 and NR4A3 from the NGFI-B gene family. NR4A2 has been reported to upregulate aldosterone synthase expression in H295R cells (5) and proposed to have clinical importance, since its expression levels are upregulated in aldosteronoma samples and correlate with aldosterone synthase expression levels (30).

Several high-throughput gene expression-profiling studies in the adrenal gland have been reported for human fetal and adult adrenal glands (42), adrenocortical tumors (20), normal subjects and patients with ACTH-dependent and -independent macronodular adrenal hyperplasia (9), steroidogenic enzymes in aldosterone-producing adenoma or cortisol-producing adenoma (3), human sporadic adrenocortical tumors (12), adrenocortical adenomas and carcinomas (52), primary pigmented nodular adrenocortical disease samples (24), and ACTH-induced gene expression in Y1 mouse adrenocortical cells (47). However, no study has focused specifically on transcription regulators that can be involved in both normal and abnormal adrenal physiology.

We used high-throughput screening with high-density oligonucleotide microarrays, a promising tool for the discovery of regulated genes, to identify genes for transcription regulators that are modulated by ANG II, potassium, and forskolin in H295R cells and validated the results by real-time RT-PCR. Human H295R adrenocortical cells were used as the experimental model, since it is the only available adrenal cell line that expresses all of the steroidogenic enzymes in the adrenal cortex and has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells (40, 43). We analyzed the role of ANG II-regulated transcription regulatory proteins in adrenal cell steroidogenesis, studying their role in modulating the expression of the two key enzymes in mineralocorticoid and glucocorticoid biosynthesis, aldosterone synthase and 11ß-hydroxylase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture.
H295R human adrenocortical cells (8) (a kind gift from W. E. Rainey, Medical College of Georgia, Augusta, GA) 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 an antibiotic-antimycotic mixture (Invitrogen, Carlsbad, CA), as previously described (44). Cells were incubated with ANG II (100 nM; PeproTech, Rocky Hill, NJ), potassium (16 mM), forskolin (10 µM; Sigma Chemical, St. Louis, MO), or vehicle for 3 h, media were removed, and cells were subjected to RNA extraction.

RNA extraction and microarray analysis.
Total RNA was extracted and DNase digested as previously described (44). RNA was biotin labeled, fragmented, and hybridized to the human Affymetrix Human Genome U133 array set following the manufacturer's suggested protocols (GeneChip Expression Analysis Manual) at the DNA core facility of the University of Missouri-Columbia. Microarrays were scanned, and raw data were extracted with GeneChip Operating Software version 1.2 (GCOS, Affymetrix). Data were normalized and modeled using the PM/MM model using dChip Software (version Sept. 23, 2005) (29). Transcription regulatory genes were extracted from up/down-regulated gene lists based on the Gene Ontology Molecular Function category (version 1.419) transcription regulator activity (GO:0030528) (1). Microarray hybridization data were submitted to the Gene Expression Omnibus database (GEO; National Center for Biotechnology Information, NCBI) under accession number GSE5553.

Real-time RT-PCR.
Real-time RT-PCR was performed as previously described (44). Primer pairs to analyze gene expression were designed with Primer3 Software (46) to generate 80- to 120-bp amplicons based on microarray target sequences (NETAFFX, Affymetrix) (Table 1). Aldosterone synthase and 11ß-hydroxylase Taqman primers and probes have been previously reported (16), except that 11ß-hydroxylase reverse primer was modified to 5'-CTCTTGGGTTAGTGTCTCCACCT-3'. Additional primer pairs used to quantify plasmid-driven overexpression in H295R cells are shown in Supplemental Table S1 (supplemental data are available at the online version of this article). Real-time data were obtained during the extension phase, and threshold cycle values were obtained at the log phase of each gene amplification. PCR product quantification was performed by the relative quantification method (39) and standardized against GAPDH. The efficiency for each primer pair was assessed by using serial dilutions of RT product. Results are expressed as arbitrary units normalized against GAPDH mRNA expression.


View this table:
[in this window]
[in a new window]

  		Table 1. Real-time PCR primers

 
Reporter assays.
H295R cells were grown in 24-well plates with H295R complete media without antibiotic/antimycotics until 90–95% confluent. H295R cells were transfected by a combination of cationic lipids (Lipofectamine 2000, Invitrogen) and magnetofection (CombiMag, OzBiosciences, France). Cells were transfected with 3 µg DNA/well (2 µg of reporter plasmid plus 1 µg of expression plasmid), 2 µl/well Lipofectamine 2000, and 4.5 µl/well CombiMag following the manufacturer's suggested protocols. Cells were cultured overnight, and media were replaced with 0.5 ml/well fresh media and cultured for an additional 24 h. Cells were lysed with Glo Lysis buffer (Promega, Madison, WI), and luciferase activity was quantified with the Bright-Glo Luciferase assay kit (Promega). Reporter assays were performed in quadruplicate using three different plasmid DNA maxipreps in each experiment to avoid plasmid DNA preparation-related effects at least three times.

Plasmids.
Reporter plasmids carrying human 11ß-hydroxylase and aldosterone synthase (49) (a kind gift from W. E. Rainey) were generated by transferring the promoter regions from pGL3-Basic to pGL4.10[luc2] (Promega), as we have previously described (45). Mammalian expression plasmids expressed human or mouse genes under the cytomegalovirus promoter. Table 2 lists the sequence accession numbers, gene species, and expression plasmid. All expression plasmids were obtained from Open Biosystems (Huntsville, AL) except NR4A3, which was purchased from Origene Technologies (Rockville, MD).


View this table:
[in this window]
[in a new window]

  		Table 2. Mammalian expression clones

 
Promoter and gene interaction analysis.
Genes and their promoters were analyzed for interactions at the literature and promoter level using the Bibliosphere PathwayEdition Software version 6.26 (Genomatix). Literature data mining was performed with all regulated transcription regulatory genes in addition to 11ß-hydroxylase and aldosterone synthase based on the more than 15 million NCBI PubMed collections of abstracts. Co-citation was set at the maximum stringency, showing genes connected by literature mining only if they are co-cited in the same sentence of the abstract with the order "gene... word function... gene". Co-cited genes were further analyzed for transcription factor binding sites in their promoters using the Genomatix MatInspector Software based on the Genomatix Promoter database containing information from more than 2,700 different transcription factors and more than 200,000 gene-transcription factor interactions.

Statistical analysis.
All results were expressed as means ± SE. Two groups were compared by t-test, and multiple groups were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. Differences were considered statistically significant at P < 0.05. Statistical calculations were performed with Graphpad Prism Package version 4.03 (Graphpad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ANG II-modulated transcription regulatory genes.
To determine which genes involved in transcription regulation are modulated by ANG II, H295R cells were incubated with ANG II (100 nM), potassium (16 mM), forskolin (10 µM), or vehicle for 3 h. RNA was extracted and subjected to microarray analysis. We set up an arbitrary cutoff of threefold change by ANG II and selected the subset of genes under the Gene Ontology Molecular Function category transcription regulator activity. Microarray-screened ANG II-modulated transcription regulatory genes were confirmed by real-time RT-PCR. Table 3 lists the mRNA fold changes observed both in the microarrays and by real-time RT-PCR under ANG II, potassium, and forskolin treatments. As a control for the cell treatments, mRNA levels of the last two key and unique enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways, 11ß-hydroxylase and aldosterone synthase, were quantified using a highly specific Taqman real-time RT-PCR assay. 11ß-Hydroxylase mRNA was upregulated by ANG II and forskolin, whereas aldosterone synthase mRNA was upregulated by ANG II and potassium (Table 3).


View this table:
[in this window]
[in a new window]

  		Table 3. Gene expression

 
ANG II-modulated transcription regulatory genes and steroidogenesis.
To study whether the transcription regulatory genes modulated by ANG II treatment are involved in adrenal steroidogenesis, we performed cotransfection reporter assays in H295R cells. Reporter plasmids were used to determine the effect of the transcription regulator genes on the last two key enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways. We used promoters of human 11ß-hydroxylase (CYP11B1 gene) and aldosterone synthase (CYP11B2 gene), which have been described previously (49). H295R cells are notoriously difficult to transfect. We greatly improved the transfection efficiency by using a combination of cationic lipids and magnetofection for plasmid delivery. All transfections were done using three different plasmid preparations to avoid plasmid DNA preparation-related effects. Figure 1 shows the effect of the ANG II-modulated transcription regulator genes on 11ß-hydroxylase (Fig. 1A) and aldosterone synthase reporter gene expression (Fig. 1B). Control transfections were assigned an arbitrary value of one. Eighty three percent (20/24) of the identified candidate genes modified 11ß-hydroxylase expression; 15 upregulated and 5 downregulated the last enzyme of glucocorticoid biosynthesis. Seventy percent (17/24) of the genes modified aldosterone synthase expression; 14 upregulated while 3 downregulated the last enzyme of mineralocorticoid biosynthesis. More specifically, the 24 genes can be divided into five trends: increased expression of both (n = 14; CITED2, EGR2, ELL2, FOS, FOSB, HDAC5, MAFF, MITF, NFIL3, NR4A1, NR4A2, NR4A3, PER1, VDR), decreased expression of both (n = 3; BHLHB2, BTG2, SALL1), no change for both (n = 4; RUNX1T1, SERTAD1, SIX2, ZNF295), or no change for aldosterone synthase but decreased expression of 11ß-hydroxylase (n = 2; BCL11B, HMGA1) or increased expression of 11ß-hydroxylase (n = 1; EGR1). Figure 1C shows the ratio of aldosterone synthase to 11ß-hydroxylase expression for each gene as a measure of the selectivity of each transcription regulatory gene toward modulating the mineralocorticoid or glucocorticoid biosynthetic pathway preferentially. By the ratio of aldosterone synthase to 11ß-hydroxylase expression, NFIL3, NR4A1, NR4A2, and NR4A3 show the greatest selectivity toward upregulating expression of the mineralocorticoid biosynthetic pathway preferentially. Figure 2 shows that plasmid transfection effectively increased the expression of each one of the genes under study. PCR reactions in the absence of RT showed no specific amplification product, indicating the absence of genomic or plasmid DNA contamination.


Figure 1
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 1. Effect of transcription regulatory genes on 11ß-hydroxylase and aldosterone synthase reporter expression. H295R cells were transfected with 11ß-hydroxylase (A) or aldosterone synthase (B) reporter plasmids and transcription regulatory genes expressing plasmid or control plasmid. Data are expressed as fold induction over control plasmid in arbitrary units (AU). Transfection experiments were performed in quadruplicate with 3 independent plasmid DNA preparations at least 3 times. *P < 0.05 vs. control. C: aldosterone synthase/11ß-hydroxylase expression log10 ratio for each gene.

 

Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 2. Plasmid-driven overexpression in transfected H295R cells. H295R cells were transfected with control plasmid or plasmids overexpressing the genes under study. RT-PCR products from reactions in the presence (+) or absence (–) of RT were detected by agarose gel electrophoresis. GAPDH mRNA expression was used as control, and a representative gel is shown.

 
Promoter and gene-gene interaction analysis.
Genes and their promoters were analyzed for interactions at the literature and promoter level using the Bibliosphere PathwayEdition Software (48). Figure 3 shows a gene-gene interaction network of reported regulated genes based on previously reported gene interactions in the literature. Analysis of the promoter sequences of the reporter genes indicated that 11ß-hydroxylase promoter has binding sites for FOS (V$AP1F), FOSB (V$AP1F), FOSL2 (V$AP1F), and NR4A2 (V$RORA), while aldosterone synthase promoter presents binding sites for EGR1 (V$EGRF), HMGA1 (V$SORY), NR4A1 (V$RORA), and NR4A2 (V$RORA), with transcription factor binding site matrixes between parenthesis.


Figure 3
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 3. 11ß-Hydroxylase and aldosterone synthase promoter and gene network analysis. Regulated genes are enclosed in a box divided in 3 segments representing expression levels under ANG II, potassium, and forskolin stimulation from left to right determined by real-time RT-PCR. If a gene that codes for a transcription factor is connected to a gene that is known to contain a binding site for this transcription factor in its promoter, the connecting line is colored green over one-half of its length near the gene containing the binding site. Arrows symbolize interaction between genes. Truncated lines symbolize inhibition. The length of the connection lines reflects the number of abstracts the genes are co-cited in; shorter lines mean more co-citations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we report for the first time a high-throughput screening of transcription regulatory genes modulated by ANG II, potassium, and forskolin in human H295R adrenocortical cells using high-density oligonucleotide microarrays and real-time RT-PCR validation. We also report the role of the aforementioned transcription regulatory genes in adrenal steroidogenesis regulation by analyzing their role in the expression of the last two key enzymes of the mineralocorticoid and glucocorticoid biosynthetic pathways.

There are several methodological and technical points that validate the experimental approach we performed. Cell culture conditions for H295R cells were chosen for optimal mineralocorticoid and glucocorticoid secretion. H295R cells are a widely used in vitro cell system in the study of adrenal cell physiology, since it is the only adrenal cell line whose regulation and steroid secretion mimic that of freshly isolated adrenal cells (40, 43). We studied gene expression regulation by several adrenal steroid secretion stimulators (ANG II, potassium, and forskolin) 3 h after stimulation, since we have already shown that this time point is very useful to detect early regulated genes in this cell line (44). ACTH is a segretagogue in vivo and in freshly isolated adrenal cortical cells that acts mainly through the adenylyl cyclase/cAMP/PKA pathway. H295R cells exhibit a minimal steroidogenic response to ACTH treatment (38, 41). Forskolin is an activator of adenylate cyclase that increases intracellular cAMP and elicits cAMP-dependent cellular responses mainly through protein kinase A activation. We used forskolin, which has been widely used in this cell line to study ACTH-mediated cell regulation, to bypass the ACTH receptor. All genes presented in this report were validated by real-time RT-PCR using experimentally determined primer pair efficiencies for each primer pair. Moreover, primer pairs were designed against the actual target sequences from which the microarray oligonucleotides were designed, rather than using database accession numbers, which eliminates the possibility of mistakes in gene annotation. To determine whether transcription regulation genes whose levels were modified by hormone treatment could be implicated in the regulation of steroid biosynthesis, we studied the effect of the candidate genes that were most altered by the secretagogues on the expression of the last and unique enzymes in the synthesis of cortisol and aldosterone, 11ß-hydroxylase and aldosterone synthase, respectively.

An important methodological consideration in this gene expression study was the decision to not use an internal control plasmid. It is common practice in reporter gene studies to cotransfect a second plasmid that expresses an easily quantifiable protein (for example, ß-galactosidase, Renilla luciferase, secreted alkaline phosphatase) to correct for transfection efficiency. It is assumed that the expression of the internal control plasmid is constant and independent of other contransfected plasmids or culture conditions. We tested several control plasmids using both strong (Cytomegalovirus, Rous Sarcoma Virus, Elongation Factor I) and moderate (herpes simplex virus thymidine kinase) promoters driving the expression of several control reporter genes (ß-galactosidase, secreted alkaline phosphatase, Renilla luciferase) as possible control genes. Unfortunately, most of the transcription regulation genes studied caused a dramatic change in the expression of the control genes, making them unsuitable to be used as a control. This is not surprising, as it has already been reported by several investigators in a variety of cells systems (6, 15, 17, 21, 25, 34, 37, 51). To overcome this methodological limitation, we performed the transfection studies using three different plasmid preparations for each gene. This experimental approach reflects a more accurate picture of the expression regulatory activity of the transcription regulatory genes under study.

Several of the genes found in the present study have been reported previously to be expressed in the adrenal gland or involved in adrenal cortex physiology, although, with some exceptions, they have not been studied in their role with adrenal steroidogenesis. For example, Fos family members (c-fos, FOSB) dimerize with Jun proteins to form the AP-1 transcription factor complex. ANG II and ACTH increase c-fos mRNA expression in bovine and ovine adrenal cells in vitro (53). ACTH treatment in vivo increases both c-fos and FOSB mRNA in rat adrenal zona glomerulosa and zona fasciculata (28). In H295R cells, it has been reported that ANG II increases expression of a reporter gene under c-fos promoter (54). Mukai et al. (35, 36) have reported that AP-1 is involved in 11ß-hydroxylase gene expression and that cotransfection of c-fos or FOSB increases the expression of an 11ß-hydroxylase reporter gene. CITED2 and SALL1 genes appear to be involved in adrenal gland development. CITED2 (also called MRG1, p35SRJ) is a transcriptional coactivator (7) implicated in adrenal development, since CITED2 knockout mice present complete adrenal agenesis (2). The transcription factor SALL1 is expressed in the adrenal gland (31), and mice expressing a truncated SALL1 also lack adrenal glands, mimicking Townes-Brocks syndrome (14, 26). We demonstrated that early growth response-2 (EGR2) was upregulated by ANG II but not significantly modified by potassium or forskolin, the surrogate for ACTH stimulation. The latter result agrees with previous reports that EGR2 mRNA in rat adrenal gland is not modified by stress in vivo (23). Expression of EGR1 and BHLHB2 is greater in adult, compared with fetal, adrenals (42). Elongation factor-2 (ELL2) was reported to be phosphorylated in rat adrenal glomerulosa cells through the calcium/calmodulin pathway (27).

The NGFI-B nuclear orphan receptor superfamily includes three members, NR4A1 (Nur77, NGFI-B), NR4A2 (Nurr1), and NR4A3 (Nor1) (19, 33), that are highly expressed in the adrenal cortex (5, 11, 18, 55). We and others have previously reported that NGFI-B family members are upregulated by ANG II in H295R cells (4, 5, 44) and regulate the expression of several steroidogenic enzymes, including aldosterone synthase, 11ß-hydroxylase, 3ß-hydroxysteroid dehydrogenase, and 21-hydroxylase in the adrenal gland (4, 5, 18, 32, 55, 56). NR4A1 and NR4A2 upregulate aldosterone synthase expression in reporter assays in H295R cells (5). Our results expand previous findings indicating that NR4A3 is also an activator of aldosterone synthase expression, but they differ from a previous study (5) by indicating that all three family members stimulate 11ß-hydroxylase expression in H295R cells. This is probably due to the use in the present study of improved reporter plasmids that lack spurious transcription factor binding sites in both the backbone and reporter gene, as well as to slightly different assay conditions.

The promoter fragments of aldosterone synthase and 11ß-hydroxylase present transcription factor binding sites for some of the transcription factors under study. We speculate that the other transcription factors that regulated the expression of either reporter gene are acting by interacting with other proteins or indirectly by modulating the expression of other transcription regulatory genes. Gene-gene interaction network based on literature data mining indicated that FOS is a gene described to interact with several of the genes that we found to be regulated by ANG II in human adrenal cells, probably suggesting a central position in the regulation of the ANG II signal transducing network. Further studies are necessary to elucidate whether similar gene regulatory networks take place in ANG II-stimulated adrenal cells.

The present study has several limitations. Because of the experimental design, we studied only the transcription regulator genes that are modified by ANG II at the mRNA level. Any posttranslational modifications such as protein half-life, protein phosphorylation, protein proteolysis, etc., have not been studied, since we focused on genes regulated at the mRNA level by ANG II. The time point chosen, 3 h of ANG II treatment, was selected based on previous studies showing this time point to be adequate to detect changes at the mRNA level in early regulated genes (44). Microarray studies are restricted to the gene probes and sequences spotted on them. Nevertheless, the microarrays used in the present report include almost 45,000 probe sets representing more than 39,000 transcripts derived from approximately 33,000 well-substantiated human genes that include more than 8,500 transcription regulators. Finally, reporter gene assays, although a useful tool used for decades, present some limitations, since it is difficult to know the exact size of the promoter, and in transient transfections the reporter plasmids are episomal. On the other hand, H295R cells are very difficult to transfect, and reporter gene expression assays are a practical way to analyze promoter activity. Mitigating these limitations is the finding by several laboratories that the aldosterone synthase and 11ß-hydroxylase reporter genes have been specifically proven to correlate well with endogenous promoter activity (10, 13, 22, 50).

In the present study, we report ANG II early regulated genes in H295R human adrenocortical cells. We focused on transcription regulatory genes because they are requisite parts of transcription networks involved in all aspects of cell function. The genes described in this report, some of them already implicated in adrenal physiology, are excellent potential candidates to be involved in adrenal pathophysiology. We have started analyzing their role in adrenal steroidogenesis by studying their capacity to modulate the expression of the last and only unique enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways. Since dysregulation of either glucocorticoid or mineralocorticoid production results in disease, elucidating the function of these genes in adrenal cells will lead to the understanding of the molecular basis of such pathological conditions.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Medical Research Funds from the Department of Veterans Affairs (to C. E. Gomez-Sanchez and E. P. Gomez-Sanchez), National Heart, Lung, and Blood Institute Grants HL-27255 (to C. E. Gomez-Sanchez) and HL-75321 (to E. P. Gomez-Sanchez), and University of Mississippi Medical Center Intramural Research Support Program (to D. G. Romero).


   FOOTNOTES
 
Address for reprint requests and other correspondence: D. G. Romero, Division of Endocrinology, Dept. of Medicine, The Univ. of Mississippi Medical Center, 2500 North State St., Jackson, MS 39216 (e-mail: dromero{at}medicine.umsmed.edu).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25–29, 2000.[CrossRef][Web of Science][Medline]
  2. Bamforth SD, Braganca J, Eloranta JJ, Murdoch JN, Marques FI, Kranc KR, Farza H, Henderson DJ, Hurst HC, Bhattacharya S. Cardiac malformations, adrenal agenesis, neural crest defects and exencephaly in mice lacking Cited2, a new Tfap2 co-activator. Nat Genet 29: 469–474, 2001.[CrossRef][Web of Science][Medline]
  3. Bassett MH, Mayhew B, Rehman K, White PC, Mantero F, Arnaldi G, Stewart PM, Bujalska I, Rainey WE. Expression profiles for steroidogenic enzymes in adrenocortical disease. J Clin Endocrinol Metab 90: 5446–5455, 2005.[Abstract/Free Full Text]
  4. Bassett MH, Suzuki T, Sasano H, De Vries CJ, Jimenez PT, Carr BR, Rainey WE. The orphan nuclear receptor NGFIB regulates transcription of 3beta-hydroxysteroid dehydrogenase. Implications for the control of adrenal functional zonation. J Biol Chem 279: 37622–37630, 2004.[Abstract/Free Full Text]
  5. Bassett MH, Suzuki T, Sasano H, White PC, Rainey WE. The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production. Mol Endocrinol 18: 279–290, 2004.[Abstract/Free Full Text]
  6. Bergeron D, Barbeau B, Leger C, Rassart E. Experimental bias in the evaluation of the cellular transient expression in DNA co-transfection experiments. Cell Mol Biol Res 41: 155–159, 1995.[Web of Science][Medline]
  7. Bhattacharya S, Michels CL, Leung MK, Arany ZP, Kung AL, Livingston DM. Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1. Genes Dev 13: 64–75, 1999.[Abstract/Free Full Text]
  8. Bird IM, Hanley NA, Word RA, Mathis JM, McCarthy JL, Mason JI, Rainey WE. Human NCI-H295 adrenocortical carcinoma cells: a model for angiotensin-II-responsive aldosterone secretion. Endocrinology 133: 1555–1561, 1993.[Abstract/Free Full Text]
  9. Bourdeau I, Antonini SR, Lacroix A, Kirschner LS, Matyakhina L, Lorang D, Libutti SK, Stratakis CA. Gene array analysis of macronodular adrenal hyperplasia confirms clinical heterogeneity and identifies several candidate genes as molecular mediators. Oncogene 23: 1575–1585, 2004.[CrossRef][Web of Science][Medline]
  10. Clyne CD, Zhang Y, Slutsker L, Mathis JM, White PC, Rainey WE. Angiotensin II and potassium regulate human CYP11B2 transcription through common cis-elements. Mol Endocrinol 11: 638–649, 1997.[Abstract/Free Full Text]
  11. Davis IJ, Lau LF. Endocrine and neurogenic regulation of the orphan nuclear receptors Nur77 and Nurr-1 in the adrenal glands. Mol Cell Biol 14: 3469–3483, 1994.[Abstract/Free Full Text]
  12. de Fraipont F, El Atifi M, Cherradi N, Le Moigne G, Defaye G, Houlgatte R, Bertherat J, Bertagna X, Plouin PF, Baudin E, Berger F, Gicquel C, Chabre O, Feige JJ. Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 90: 1819–1829, 2005.[Abstract/Free Full Text]
  13. Denner K, Rainey WE, Pezzi V, Bird IM, Bernhardt R, Mathis JM. Differential regulation of 11 beta-hydroxylase and aldosterone synthase in human adrenocortical H295R cells. Mol Cell Endocrinol 121: 87–91, 1996.[CrossRef][Web of Science][Medline]
  14. Else T, Hammer GD. Genetic analysis of adrenal absence: agenesis and aplasia. Trends Endocrinol Metab 16: 458–468, 2005.[CrossRef][Web of Science][Medline]
  15. Everett LM, Crabb DW. Sensitivity of virally-driven luciferase reporter plasmids to members of the steroid/thyroid/retinoid family of nuclear receptors. J Steroid Biochem Mol Biol 70: 197–201, 1999.[CrossRef][Web of Science][Medline]
  16. Fallo F, Pezzi V, Barzon L, Mulatero P, Veglio F, Sonino N, Mathis JM. Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas. Eur J Endocrinol 147: 795–802, 2002.[Abstract]
  17. Farr A, Roman A. A pitfall of using a second plasmid to determine transfection efficiency. Nucleic Acids Res 20: 920, 1992.[Free Full Text]
  18. Fernandez PM, Brunel F, Jimenez MA, Saez JM, Cereghini S, Zakin MM. Nuclear receptors Nor1 and NGFI-B/Nur77 play similar, albeit distinct, roles in the hypothalamo-pituitary-adrenal axis. Endocrinology 141: 2392–2400, 2000.[Abstract/Free Full Text]
  19. Giguere V. Orphan nuclear receptors: from gene to function. Endocr Rev 20: 689–725, 1999.[Abstract/Free Full Text]
  20. Giordano TJ, Thomas DG, Kuick R, Lizyness M, Misek DE, Smith AL, Sanders D, Aljundi RT, Gauger PG, Thompson NW, Taylor JM, Hanash SM. Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. Am J Pathol 162: 521–531, 2003.[Abstract/Free Full Text]
  21. Ho CK, Strauss JF 3rd. Activation of the control reporter plasmids pRL-TK and pRL-SV40 by multiple GATA transcription factors can lead to aberrant normalization of transfection efficiency. BMC Biotechnol 4: 10, 2004.[CrossRef][Medline]
  22. Holland OB, Mathis JM, Bird IM, Rainey WE. Angiotensin increases aldosterone synthase mRNA levels in human NCI-H295 cells. Mol Cell Endocrinol 94: R9–R13, 1993.[CrossRef][Web of Science][Medline]
  23. Honkaniemi J, Zhang JS, Longo FM, Sharp FR. Stress induces zinc finger immediate early genes in the rat adrenal gland. Brain Res 877: 203–208, 2000.[CrossRef][Web of Science][Medline]
  24. Horvath A, Mathyakina L, Vong Q, Baxendale V, Pang AL, Chan WY, Stratakis CA. Serial analysis of gene expression in adrenocortical hyperplasia caused by a germline PRKAR1A mutation. J Clin Endocrinol Metab 91: 584–596, 2006.[Abstract/Free Full Text]
  25. Huszar T, Mucsi I, Terebessy T, Masszi A, Adamko S, Jeney C, Rosivall L. The use of a second reporter plasmid as an internal standard to normalize luciferase activity in transient transfection experiments may lead to a systematic error. J Biotechnol 88: 251–258, 2001.[CrossRef][Web of Science][Medline]
  26. Kiefer SM, Ohlemiller KK, Yang J, McDill BW, Kohlhase J, Rauchman M. Expression of a truncated Sall1 transcriptional repressor is responsible for Townes-Brocks syndrome birth defects. Hum Mol Genet 12: 2221–2227, 2003.[Abstract/Free Full Text]
  27. Kigoshi T, Uchida K, Morimoto S. Elongation factor 2 as the major substrate for Ca2+/calmodulin-dependent protein kinase in rat adrenal glomerulosa cells. J Steroid Biochem 32: 381–385, 1989.[CrossRef][Web of Science][Medline]
  28. Lehoux JG, Fleury A, Ducharme L. The acute and chronic effects of adrenocorticotropin on the levels of messenger ribonucleic acid and protein of steroidogenic enzymes in rat adrenal in vivo. Endocrinology 139: 3913–3922, 1998.[Abstract/Free Full Text]
  29. Li C, Wong WH. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci USA 98: 31–36, 2001.[Abstract/Free Full Text]
  30. Lu L, Suzuki T, Yoshikawa Y, Murakami O, Miki Y, Moriya T, Bassett MH, Rainey WE, Hayashi Y, Sasano H. Nur-related factor 1 and nerve growth factor-induced clone B in human adrenal cortex and its disorders. J Clin Endocrinol Metab 89: 4113–4118, 2004.[Abstract/Free Full Text]
  31. Ma Y, Chai L, Cortez SC, Stopa EG, Steinhoff MM, Ford D, Morgan J, Maizel AL. SALL1 expression in the human pituitary-adrenal/gonadal axis. J Endocrinol 173: 437–448, 2002.[Abstract]
  32. Martin LJ, Tremblay JJ. The human 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase type 2 promoter is a novel target for the immediate early orphan nuclear receptor Nur77 in steroidogenic cells. Endocrinology 146: 861–869, 2005.[Abstract/Free Full Text]
  33. Martinez-Gonzalez J, Badimon L. The NR4A subfamily of nuclear receptors: new early genes regulated by growth factors in vascular cells. Cardiovasc Res 65: 609–618, 2005.[CrossRef][Web of Science][Medline]
  34. Matuszyk J, Ziolo E, Cebrat M, Kochel I, Strzadala L. Nurr1 affects pRL-TK but not phRG-B internal control plasmid in genetic reporter system. Biochem Biophys Res Commun 294: 1036–1039, 2002.[CrossRef][Web of Science][Medline]
  35. Mukai K, Mitani F, Agake R, Ishimura Y. Adrenocorticotropic hormone stimulates CYP11B1 gene transcription through a mechanism involving AP-1 factors. Eur J Biochem 256: 190–200, 1998.[Web of Science][Medline]
  36. Mukai K, Mitani F, Shimada H, Ishimura Y. Involvement of an AP-1 complex in zone-specific expression of the CYP11B1 gene in the rat adrenal cortex. Mol Cell Biol 15: 6003–6012, 1995.[Abstract]
  37. Osborne SA, Tonissen KF. pRL-TK induction can cause misinterpretation of gene promoter activity. Biotechniques 33: 1240–1242, 2002.[Web of Science][Medline]
  38. Penhoat A, Naville D, El Mourabit H, Buronfosse A, Durand P, Begeot M. Functional expression of the human ACTH receptor gene. Endocr Res 26: 549–557, 2000.[Web of Science][Medline]
  39. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45, 2001.[Abstract/Free Full Text]
  40. Rainey WE, Bird IM, Mason JI. The NCI-H295 cell line: a pluripotent model for human adrenocortical studies. Mol Cell Endocrinol 100: 45–50, 1994.[CrossRef][Web of Science][Medline]
  41. Rainey WE, Bird IM, Sawetawan C, Hanley NA, McCarthy JL, McGee EA, Mason JI. Regulation of human adrenal carcinoma cell (NCI-H295) production of C19 steroids. J Clin Endocrinol Metab 77: 731–737, 1993.[Abstract]
  42. Rainey WE, Carr BR, Wang ZN, Parker CR Jr. Gene profiling of human fetal and adult adrenals. J Endocrinol 171: 209–215, 2001.[Abstract]
  43. Rainey WE, Saner K, Schimmer BP. Adrenocortical cell lines. Mol Cell Endocrinol 228: 23–38, 2004.[CrossRef][Web of Science][Medline]
  44. Romero DG, Plonczynski M, Vergara GR, Gomez-Sanchez EP, Gomez-Sanchez CE. Angiotensin II early regulated genes in H295R human adrenocortical cells. Physiol Genomics 19: 106–116, 2004.[Abstract/Free Full Text]
  45. Romero DG, Plonczynski MW, Gomez-Sanchez EP, Yanes LL, Gomez-Sanchez CE. RGS2 is regulated by angiotensin II and functions as a negative feedback of aldosterone production in H295R human adrenocortical cells. Endocrinology 147: 3889–3897, 2006.[Abstract/Free Full Text]
  46. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386, 2000.[Medline]
  47. Schimmer BP, Cordova M, Cheng H, Tsao A, Goryachev AB, Schimmer AD, Morris Q. Global profiles of gene expression induced by adrenocorticotropin in Y1 mouse adrenal cells. Endocrinology 147: 2357–2367, 2006.[Abstract/Free Full Text]
  48. Seifert M, Scherf M, Epple A, Werner T. Multievidence microarray mining. Trends Genet 21: 553–558, 2005.[CrossRef][Web of Science][Medline]
  49. Sirianni R, Seely JB, Attia G, Stocco DM, Carr BR, Pezzi V, Rainey WE. Liver receptor homologue-1 is expressed in human steroidogenic tissues and activates transcription of genes encoding steroidogenic enzymes. J Endocrinol 174: R13–R17, 2002.[Abstract]
  50. Staels B, Hum DW, Miller WL. Regulation of steroidogenesis in NCI-H295 cells: a cellular model of the human fetal adrenal. Mol Endocrinol 7: 423–433, 1993.[Abstract/Free Full Text]
  51. Thavathiru E, Das GM. Activation of pRL-TK by 12S E1A oncoprotein: drawbacks of using an internal reference reporter in transcription assays. Biotechniques 31: 528–530, 532, 2001.[Web of Science][Medline]
  52. Velazquez-Fernandez D, Laurell C, Geli J, Hoog A, Odeberg J, Kjellman M, Lundeberg J, Hamberger B, Nilsson P, Backdahl M. Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy. Surgery 138: 1087–1094, 2005.[CrossRef][Web of Science][Medline]
  53. Viard I, Hall SH, Jaillard C, Berthelon MC, Saez JM. Regulation of c-fos, c-jun and jun-B messenger ribonucleic acids by angiotensin-II and corticotropin in ovine and bovine adrenocortical cells. Endocrinology 130: 1193–1200, 1992.[Abstract/Free Full Text]
  54. Watanabe G, Lee RJ, Albanese C, Rainey WE, Batlle D, Pestell RG. Angiotensin II activation of cyclin D1-dependent kinase activity. J Biol Chem 271: 22570–22577, 1996.[Abstract/Free Full Text]
  55. Wilson TE, Mouw AR, Weaver CA, Milbrandt J, Parker KL. The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase. Mol Cell Biol 13: 861–868, 1993.[Abstract/Free Full Text]
  56. Zhang P, Mellon SH. Multiple orphan nuclear receptors converge to regulate rat P450c17 gene transcription: novel mechanisms for orphan nuclear receptor action. Mol Endocrinol 11: 891–904, 1997.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
X. Hu, J. D. Dietz, C. Xia, D. R. Knight, W. T. Loging, A. H. Smith, H. Yuan, D. A. Perry, and J. Keiser
Torcetrapib Induces Aldosterone and Cortisol Production by an Intracellular Calcium-Mediated Mechanism Independently of Cholesteryl Ester Transfer Protein Inhibition
Endocrinology, May 1, 2009; 150(5): 2211 - 2219.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
E. F Nogueira, Y. Xing, C. A V Morris, and W. E Rainey
Role of angiotensin II-induced rapid response genes in the regulation of enzymes needed for aldosterone synthesis
J. Mol. Endocrinol., April 1, 2009; 42(4): 319 - 330.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
M. Bielinska, H. Parviainen, S. Kiiveri, M. Heikinheimo, and D. B. Wilson
REVIEW PAPER: Origin and Molecular Pathology of Adrenocortical Neoplasms
Vet. Pathol., March 1, 2009; 46(2): 194 - 210.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
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
Physiol Genomics, December 19, 2007; 32(1): 117 - 127.
[Abstract] [Full Text] [PDF]

Home page
 J Mol EndocrinolHome page
E. F Nogueira, C. A Vargas, M. Otis, N. Gallo-Payet, W. B Bollag, and W. E Rainey
Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells
J. Mol. Endocrinol., December 1, 2007; 39(6): 365 - 374.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Table
Right arrow All Versions of this Article:
30/1/26    most recent
00187.2006v1
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 Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Romero, D. G.
Right arrow Articles by Gomez-Sanchez, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Romero, D. G.
Right arrow Articles by Gomez-Sanchez, C. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.