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 11β-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 11β-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
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 11β-hydroxylase.
MATERIALS AND METHODS
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 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 11β-hydroxylase Taqman primers and probes have been previously reported (16), except that 11β-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.
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.
Reporter plasmids carrying human 11β-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).
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 11β-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.
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).
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, 11β-hydroxylase and aldosterone synthase, were quantified using a highly specific Taqman real-time RT-PCR assay. 11β-Hydroxylase mRNA was upregulated by ANG II and forskolin, whereas aldosterone synthase mRNA was upregulated by ANG II and potassium (Table 3).
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 11β-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 11β-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 11β-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 11β-hydroxylase (n = 2; BCL11B, HMGA1) or increased expression of 11β-hydroxylase (n = 1; EGR1). Figure 1C shows the ratio of aldosterone synthase to 11β-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 11β-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.
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 11β-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.
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, 11β-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, β-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 (β-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 11β-hydroxylase gene expression and that cotransfection of c-fos or FOSB increases the expression of an 11β-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, 11β-hydroxylase, 3β-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 11β-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 11β-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 11β-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.
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).
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:).
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
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