Angiotensin II-regulated transcription regulatory genes in adrenal steroidogenesis

Abstract

Transcription regulatory genes are crucial modulators of cell physiology and metabolism whose intracellular levels are tightly controlled in response to extracellular stimuli. We previously reported a set of 29 transcription regulatory genes modulated by angiotensin II in H295R human adrenocortical cells and their roles in regulating the expression of the last and unique enzymes of the glucocorticoid and mineralocorticoid biosynthetic pathways, 11β-hydroxylase and aldosterone synthase, respectively, using gene expression reporter assays. To study the effect of this set of transcription regulatory genes on adrenal steroidogenesis, H295R cells were transfected by high-efficiency nucleofection and aldosterone and cortisol were measured in cell culture supernatants under basal and angiotensin II-stimulated conditions. BCL11B, BHLHB2, CITED2, ELL2, HMGA1, MAFF, NFIL3, PER1, SERTAD1, and VDR significantly stimulated aldosterone secretion, while EGR1, FOSB, and ZFP295 decreased aldosterone secretion. BTG2, HMGA1, MITF, NR4A1, and ZFP295 significantly increased cortisol secretion, while BCL11B, NFIL3, PER1, and SIX2 decreased cortisol secretion. We also report the effect of some of these regulators on the expression of endogenous aldosterone synthase and 11β-hydroxylase under basal and angiotensin II-stimulated conditions. In summary, this study reports for the first time the effects of a set of angiotensin II-modulated transcription regulatory genes on aldosterone and cortisol secretion and the expression levels of the last and unique enzymes of the mineralocorticoid and glucocorticoid biosynthetic pathways. Abnormal regulation of mineralocorticoid or glucocorticoid secretion is involved in several pathophysiological conditions. These transcription regulatory genes may be involved in adrenal steroidogenesis pathologies; thus they merit additional study as potential candidates for therapeutic intervention.

one of the most basic levels in the regulation of cellular physiology is the level of control of gene transcription. Gene products with “Transcription Regulator Activity” are defined by the Gene Ontology Database as any gene products that play a role in regulating transcription, which may bind a promoter or enhancer DNA sequence or interact 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 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 (10, 13, 25, 27).

We previously reported (46) a high-throughput screening of the transcription regulatory genes modulated by ANG II, potassium, and forskolin in human H295R adrenocortical cells and their role in steroidogenic enzyme gene expression. We reported that ANG II modifies the expression level of 29 transcription regulatory genes, increasing and decreasing the expression of 25 and 4 genes, respectively. Forskolin, an adenylate cyclase activator that mimics ACTH-mediated responses in this adrenal cell line, and extracellular potassium also regulate many of these transcription regulatory genes, suggesting some degree of convergence in the intracellular signaling pathways triggered by these aldosterone secretagogues and ANG II. Reporter plasmids under the control of the human aldosterone synthase and 11β-hydroxylase promoters were used to analyze the effect of these ANG II-modulated transcription regulatory genes on the expression of the last and unique enzymes of the mineralo- and glucocorticoid biosynthetic pathways, aldosterone synthase and 11β-hydroxylase, in H295R cells. Most of the ANG II-upregulated transcription regulatory genes increase the expression of both steroidogenic enzymes. Even more, some of them (NFIL3, NR4A1, NR4A2, NR4A3) show a strong preference for upregulating the expression of aldosterone synthase compared with 11β-hydroxylase.

Since mineralo- and glucocorticoid biosynthesis are multistep pathways that not only involve a series of sequential enzymatic steps but also are regulated by many intracellular signaling pathways (9, 50), we wanted to study the effect of a set of ANG II-modulated transcription regulatory genes in the mineralo- and glucocorticoid biosynthetic pathway output, the secretion of aldosterone and cortisol. Using high-efficiency transfection by nucleofection, we analyzed the effect of 24 ANG II-modulated transcription regulatory genes on the secretion of aldosterone and cortisol under basal and ANG II-stimulated conditions in the human adrenocortical cell line H295R. We then selected five transcription regulatory genes and analyzed their effects on gene expression levels of endogenous aldosterone synthase and 11β-hydroxylase. In summary, we report for the first time the effect of a set of ANG II-modulated transcription regulatory genes on aldosterone and cortisol secretion by H295R human adrenocortical cells.

MATERIALS AND METHODS

Cell culture.

H295R human adrenocortical cells (6) were cultured in H295R complete medium containing DMEM-F-12 (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 (43).

Transfection and steroid secretion.

H295R cells were transfected with Nucleofector technology (Amaxa Biosystems) as previously reported (48). Briefly, three million log phase cells were resuspended in 100 μl of Nucleofector Solution R, mixed with 3 μg of plasmid DNA, and electroporated with the proprietary program P-20. Cells were allowed to recover for 15 min in RPMI 1640 medium at 37°C and then plated in 24-well plates with 1 ml of complete medium per well. Cells were cultured for 16 h. The medium was then removed, and the cells were incubated with prewarmed medium with or without 10 nM ANG II (American Peptide, Sunnyvale, CA) for 24 h. At the end of the incubation period, cell culture supernatants were saved for aldosterone and cortisol determination by ELISA as previously reported (22, 47). Cells were lysed with M-PER lysis buffer (Pierce, Rockford, IL), and protein concentration was measured with the Coomassie Plus kit (Pierce). Steroid secretion was standardized by total cellular protein. Results are expressed as percentage compared with transfections with a control plasmid.

RNA extraction and real-time RT-PCR.

Total RNA was extracted, DNAase treated, quantified, and reverse transcribed as previously described (43). Aldosterone synthase mRNA expression was quantified with the Taqman Gene expression assay master mix (Applied Biosystems, Foster City, CA) and specific aldosterone synthase primers (Hs01597732_a1, Applied Biosystems) according to manufacturer-suggested protocols. 11β-Hydroxylase mRNA expression was quantified by Taqman technique as previously reported (23) with the following specific primers: forward 5′-GGCAGAGGCAGAGATGCTG-3′, reverse 5′-CTCTTGGGTTAGTGTCTCCACCT-3′, probe 5′-HEX-TGCTGCACCATGTGCTGAAACACCT-BH1–3′. GAPDH mRNA expression was quantified as previously reported (43). 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.

Plasmids.

Mammalian expression plasmids expressing human or mouse genes under a cytomegalovirus promoter have been reported previously (46). All expression plasmids were obtained from Open Biosystems (Huntsville, AL) except for NR4A3, which was purchased from Origene Technologies (Rockville, MD). We previously reported (46) that this set of transcription regulatory protein-overexpressing plasmids effectively increased the expression of each of the genes as determined by RT-PCR.

Statistical analysis.

All results are expressed as means ± SE. Multiple groups were analyzed by two-way ANOVA followed by Student-Newman-Keuls multiple comparison test. Differences were considered statistically significant at P < 0.05. Statistical calculations were performed with SigmaPlot version 11 (Systat Software, San Jose, CA).

RESULTS

Angiotensin II-modulated transcription regulatory genes and steroidogenesis.

To determine the role of ANG II-modulated transcription regulatory genes in adrenal steroidogenesis, H295R human adrenocortical cells were transfected with plasmids overexpressing 24 different transcription regulatory genes (listed in Table 1) and incubated in the presence or absence of ANG II, and then aldosterone and cortisol secretion levels were quantified in cell culture supernatants.

Table 1.

Transcription regulatory genes

Gene Symbol	Gene Name	Previous Symbols	Aliases
BCL11B	B-cell CLL/lymphoma 11B		CTIP-2, CTIP2, hRIT1-α
BHLHB2	Basic helix-loop-helix family, member 2	STRA13	DEC1, bHLHe40
BTG2	BTG family, member 2		PC3, TIS21, MGC126063, MGC126064
CITED2	Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2		MRG1
EGR1	Early growth response 1		TIS8, G0S30, NGFI-A, KROX-24, ZIF-268, AT225, ZNF225
EGR2	Early growth response 2	KROX20
ELL2	Elongation factor, RNA polymerase II, 2
FOS	FBJ murine osteosarcoma viral oncogene homolog		c-fos, AP-1
FOSB	FBJ murine osteosarcoma viral oncogene homolog B		G0S3, GOSB, GOS3, AP-1, MGC42291, DKFZp686C0818
HDAC5	Histone deacetylase 5		KIAA0600, NY-CO-9, FLJ90614
HMGA1	High-mobility group AT-hook 1	HMGIY
MAFF	v-maf musculoaponeurotic fibrosarcoma oncogene homolog F		hMafF
MITF	Microphthalmia-associated transcription factor		WS2A, MI, bHLHe32
NFIL3	Nuclear factor, interleukin 3 regulated	IL3BP1	E4BP4, NFIL3A, NF-IL3A
NR4A1	Nuclear receptor subfamily 4, group A, member 1	HMR, GFRP1	TR3, N10, NAK-1, NGFIB, NUR77
NR4A2	Nuclear receptor subfamily 4, group A, member 2	NURR1	TINUR, NOT, RNR1, HZF-3
NR4A3	Nuclear receptor subfamily 4, group A, member 3		CSMF, CHN, NOR1, MINOR
PER1	Period homolog 1	PER	RIGUI
RUNX1T1	runt-related transcription factor 1; translocated to, 1	AML1T1, CBFA2T1	CDR, ETO, MTG8, ZMYND2
SALL1	sal-like 1	TBS	Hsal1, ZNF794
SERTAD1	SERTA domain containing 1		SEI1, TRIP-Br1
SIX2	SIX homeobox 2
VDR	Vitamin D (1,25-dihydroxyvitamin D3) receptor		NR1I1
ZNF295	Zinc finger protein 295		KIAA1227, ZBTB21

The genes under study were selected from our previous study in which we showed that these 24 transcription regulatory genes were modulated at the mRNA expression level by ANG II in H295R cells. Furthermore, most of these genes significantly regulated aldosterone synthase and/or 11β-hydroxylase reporter genes. High-efficiency transfection was performed by nucleofection technology, which has a transfection efficiency higher than 50% in H295R cells under the conditions described in materials and methods. This approach proved to be crucial to overcome the basal steroidogenesis of this cell line that otherwise would obscure the effect of specific genes if the transfection efficiency were as low as that achieved with traditional transfection methods. The transcription regulatory gene expression plasmids used in this study have been previously described and documented to increase the expression levels of each of the overexpressed genes (46). A submaximal stimulatory concentration of ANG II (10 nM) was chosen to avoid saturating the steroidogenic response of the adrenocortical cells. All transfections were performed with three different plasmid preparations, to avoid any plasmid DNA preparation-specific confounding effect. H295R cells is a widely used in vitro cell system in the study of adrenal cell physiology since its regulation and steroid secretion mimic those of freshly isolated adrenal cells (40, 41).

Figure 1 shows aldosterone (Fig. 1A) and cortisol (Fig. 1B) secretion by H295R cells as a percentage of control cells under basal and ANG II stimulatory conditions. We found that overexpression of nine transcription regulatory genes either increases (BCL11B, BHLHB2, CITED2, ELL2, MAFF, NFIL3, PER1, SERTAD1) or decreases (EGR1) aldosterone secretion under basal conditions. When H295R cells were treated with a submaximal dose of ANG II, overexpression of eight transcription regulatory genes was observed to either increase (BHLHB2, HMGA1, NFIL3, SERTAD1, VDR) or decrease (EGR1, FOSB, ZNF295) ANG II-mediated aldosterone secretion.

Fig. 1.

Effect of transcription regulatory genes on aldosterone (A) and cortisol (B) secretion. H295R cells were transfected with transcription regulatory gene-expressing plasmids or control plasmid. Cells were incubated under basal or angiotensin II (ANG II; 10 nM)-stimulated conditions for 24 h. Aldosterone and cortisol were measured by ELISA in cell culture supernatants. Data are expressed as fold induction compared with control plasmid. Transfection experiments were performed in duplicate with 3 independent plasmid DNA preparations at least 3 times. *P < 0.05 vs. Control.

We measured cortisol secretion in the same cell culture supernatant. We observed that overexpression of eight transcription regulatory genes either increased (BTG2, HMGA1, MITF, NR4A1, ZFP295) or decreased (BCL11B, NFIL3, SIX2) cortisol secretion under basal conditions. When H295R cells were treated with a submaximal dose of ANG II, overexpression of seven transcription regulatory genes was observed to either increase (BTG2, HMGA1, MITF, ZFP295) or decrease (NFIL3, PER1, SIX2) ANG II-mediated cortisol secretion.

Angiotensin II-modulated transcription regulatory genes and endogenous steroidogenic enzyme expression.

When we analyzed transcription regulatory gene-mediated aldosterone or cortisol secretion (see Fig. 1) and our previously reported data on the effect of these genes in reporter gene expression studies of aldosterone synthase or 11β-hydroxylase (46), we observed that there was not always agreement (see Table 2 and discussion). Furthermore, the effects were opposite for some genes. For example, BHLHB2 significantly decreased aldosterone synthase expression quantified with a reporter gene (46) but significantly increased basal and ANG II-mediated aldosterone secretion (Fig. 1). We selected five genes, four of them (BHLHB2, HMGA1, SERTAD1, and VDR) presenting contradictory results and NFIL3, which presented the most potent aldosterone synthase expression and aldosterone secretion effect, for further studies of their effects on endogenous aldosterone synthase and 11β-hydroxylase expression. We performed high-efficiency transfections with all five of these transcription regulatory genes using nucleofection technology and quantified endogenous aldosterone synthase and 11β-hydroxylase expression under basal and ANG II stimulatory conditions at 3 and 12 h after hormone treatment (Figs. 2 and 3). Incubation times were selected to reflect either 1) early regulated genes at 3 h of incubation or 2) maximal endogenous ANG II-stimulated aldosterone synthase expression at 12 h as we have previously reported (43). Figures 2 and 3 show, respectively, the endogenous aldosterone synthase and 11β-hydroxylase mRNA expression level for cells transfected with these five selected transcription regulatory genes or a control plasmid under basal or submaximal ANG II (10 nM) stimulatory conditions 3 and 12 h after hormone stimulation.

Table 2.

Summary of angiotensin II-modulated transcription regulatory genes' role in steroidogenic enzyme gene expression and steroid secretion

Gene	ANG II/Control mRNA*	Aldosterone Synthase Reporter*	11β-Hydroxylase Reporter*	Aldosterone Secretion		Cortisol Secretion
				Basal	ANG II	Basal	ANG II
BCL11B	↑		↓	↑		↓
BHLHB2	↑	↓	↓	↑	↑
BTG2	↑	↓	↓			↑	↑
CITED2	↑	↑	↑	↑
EGR1	↑		↑	↓	↓
EGR2	↑	↑	↑
ELL2	↑	↑	↑	↑
FOS	↑	↑	↑
FOSB	↑	↑	↑		↓
HDAC5	↑	↑	↑
HMGA1	↑		↓		↑	↑	↑
MAFF	↑	↑	↑	↑
MITF	↑	↑	↑			↑	↑
NFIL3	↑	↑	↑	↑	↑	↓	↓
NR4A1	↑	↑	↑			↑
NR4A2	↑	↑	↑
NR4A3	↑	↑	↑
PER1	↑	↑	↑	↑			↓
RUNX1T1	↓
SALL1	↑	↓	↓
SERTAD1	↑			↑	↑
SIX2	↓					↓	↓
VDR	↑	↑	↑		↑
ZNF295	↑				↓	↑	↑

Absence of an arrow indicates “no change.”

^*Data from Romero et al. (46).

Fig. 2.

Effect of transcription regulatory genes on endogenous aldosterone synthase expression. H295R cells were transfected with transcription regulatory gene-expressing plasmids or control plasmid and stimulated with ANG II (10 nM) for 3 or 12 h, and then aldosterone synthase expression was quantified by real-time RT-PCR. Transfection experiments were performed in triplicate with 3 independent plasmid DNA preparations at least 3 times. *P < 0.05 vs. Control. AU, arbitrary unit.

Fig. 3.

Effect of transcription regulatory genes on endogenous 11β-hydroxylase expression. H295R cells were transfected with transcription regulatory gene-expressing plasmids or control plasmid and stimulated with ANG II (10 nM) for 3 or 12 h, and then 11β-hydroxylase expression was quantified by real-time RT-PCR. Transfection experiments were performed in triplicate with 3 independent plasmid DNA preparations at least 3 times. *P < 0.05 vs. Control.

As expected, in H295R cells transfected with the control plasmid ANG II significantly increased endogenous aldosterone synthase mRNA expression 12 h after ANG II addition, while it did not significantly modify its expression after 3 h of treatment. BHLHB2 was the only transcription regulatory gene to cause a significant decrease in endogenous aldosterone synthase expression after 12 h of ANG II stimulation. The other four transcription regulatory genes (HMGA1, NFIL3, SERTAD1, and VDR) increased endogenous aldosterone synthase expression, although the degree of stimulation and the conditions (time and hormone stimulation status) differed for each gene. The most significant effect on endogenous aldosterone synthase mRNA expression was observed after 3 h of treatment with NFIL3, SERTAD1, and VDR under both basal and ANG II stimulatory conditions. HMGA1 only upregulated endogenous aldosterone synthase mRNA expression upon ANG II stimulation.

In H295R cells transfected with the control plasmid, ANG II significantly increased endogenous 11β-hydroxylase mRNA expression 3 and 12 h after ANG II addition, although to a much lower extent than the effect on aldosterone synthase. NFIL3 showed a significant stimulatory effect on endogenous 11β-hydroxylase mRNA expression after 12 of treatment under basal and ANG II stimulatory conditions. Neither BHLHB2 nor HMGA1 showed a significant regulatory effect on endogenous 11β-hydroxylase mRNA expression. Both SERTAD1 and VDR significantly decreased both basal and ANG II-stimulated endogenous 11β-hydroxylase mRNA expression.

DISCUSSION

We report for the first time a high-throughput functional screening of the steroidogenic activity of transcription regulatory genes modulated by ANG II with which we identified several new transcription regulatory genes that have not been previously known to regulate aldosterone and cortisol secretion. We also report the effects of five of these transcription regulatory genes on the expression levels of endogenous aldosterone synthase and 11β-hydroxylase under basal and ANG II-stimulated conditions.

Table 2 shows the effect (increase, decrease, or no change) of ANG II on the mRNA expression levels of all the transcription regulatory genes under study, their effect on the expression levels of aldosterone synthase and 11β-hydroxylase quantified with reporter genes, and their effect on aldosterone and cortisol secretion under basal and ANG II-stimulated conditions. The effect of ANG II on transcription regulatory gene expression levels and the effect of these transcription regulatory genes on steroidogenic enzyme reporter gene expression were previously reported (46).

Comparison of the effects of the ANG II-modulated transcription regulatory genes on aldosterone synthase reporter gene expression and on basal aldosterone secretion indicate that only nine genes (37.5%) show concordance in their effects. On the contrary, most of the genes (15/24) show either opposite or discordant (either increase or decrease in one variable and no effect in the other) effects. The nonconcordance is even more pronounced when 11β-hydroxylase reporter gene expression and cortisol secretion are compared. Only 6 of the 24 genes (25%) show similar effects on both variables. While this disagreement between the effect of these genes on the expression of the last and unique enzymes of the mineralo- and glucocorticoid biosynthetic pathways and steroid secretion does not invalidate reporter gene expression studies, it clearly demonstrates that extreme care should be taken in interpreting key steroidogenic enzyme reporter gene expression studies, as they do not always translate into increased secretion of the final biosynthetic product, either aldosterone or cortisol. A clear example of this point is the three NGFI-B nuclear orphan receptor superfamily members (NR4A1, NR4A2, and NR4A3), which significantly upregulated aldosterone synthase reporter gene expression but had no significant effect on aldosterone secretion by H295R cells.

The three members of the NGFI-B nuclear orphan receptor superfamily, NR4A1 (Nur77, NGFI-B), NR4A2 (Nurr1), and NR4A3 (Nor1) (20, 33), are highly expressed in the adrenal cortex (4, 11, 15, 56). We and others have reported that NGFI-B family members are upregulated by ANG II in H295R cells (4, 37, 43, 46, 51) and freshly isolated rat or bovine zona glomerulosa adrenal cells (36, 45, 51). NGFI-B family members have been reported to regulate the expression of several steroidogenic enzymes, including aldosterone synthase, 11β-hydroxylase, 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase, and 21-hydroxylase in the adrenal gland (3, 4, 15, 32, 46, 56, 57). However, all of these studies, with the exception of one reported by Bassett et al. (3) on the effect of NR4A1 on 3β-hydroxysteroid dehydrogenase expression, used reporter genes. Bassett et al. (3) reported that NR4A1 overexpression in human cultured fetal zone adrenal cells increases endogenous 3β-hydroxysteroid dehydrogenase expression and cortisol secretion, as we confirmed in the present report (see Fig. 1). All studies of aldosterone secretion regulation by NGFI-B family members, including our previous study (46), have been performed with reporter plasmids. In the present study we observed that NR4A1 and NR4A2 slightly increase cortisol secretion under basal conditions but none of the three NGFI-B family members significantly modified aldosterone secretion under either basal or ANG II stimulatory conditions. Similar results were observed when H295R cells were transduced with lentiviruses overexpressing any of the three NGFI-B family members (unpublished data). Recently, Nogueira et al. (37) reported that overexpression of a dominant-negative mutant of NR4A1 decreased endogenous aldosterone synthase expression and aldosterone secretion in ANG II-stimulated H295R cells. These latest results and those presented in this report may suggest that NGFI-B family members are indeed involved in ANG II-stimulated aldosterone secretion regulation. However, since NGFI-B family members have redundant roles and the expression of all of them is upregulated by ANG II stimulation, the overexpression of a single member may not significantly alter the final steroidogenic output of adrenal cells.

Ten transcription regulatory genes (BCL11B, BHLHB2, CITED2, ELL2, HMGA1, MAFF, NFIL3, PER1, SERTAD1, and VDR) were upregulated by ANG II and increased aldosterone secretion under basal, ANG II-stimulated, or both conditions. Of these, CITED2, HMGA1, and PER1 have already been studied in the adrenal gland, and a role for them in adrenal steroidogenesis has been suggested.

CITED2 (Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2) is a transcriptional coregulator of the cAMP response element binding protein (CREB) binding protein (CBP) and p300 (5). CITED2 was originally implicated in adrenal development because CITED2-null embryos die during gestation with profound developmental abnormalities, including cardiac malformations, neural crest defects, and adrenal agenesis (2). CITED2 is expressed as early as 7 wk of gestation within the definitive zone of the human fetal adrenal gland (16, 24). CITED2 regulates adrenal gland morphogenesis by interacting with the transcription factor Wt1 to stimulate expression of the nuclear hormone receptor Sf-1 in the adrenogonadal primordium prior to the separation between gonad and adrenal cortex (53). In the adult adrenal gland, CITED2 is expressed in the zona glomerulosa and zona reticularis (24). Furthermore, CITED2 was found to be expressed in 75% of the adrenocortical carcinomas analyzed as well as in H295R cells (24). CITED2 expression is upregulated by basic fibroblast growth factor, forskolin, and overexpression of steroidogenic factor-1 (SF-1) in H295R cells (16, 24). Our results demonstrate that CITED2 increases aldosterone synthase expression (46) and basal levels of aldosterone secretion in H295R cells by almost 70%, suggesting a role of CITED2 not only in adrenal development but also in adulthood, in agreement with its expression in the zona glomerulosa of the adult adrenal gland.

HMGA1 belongs to the “high-mobility group” (HMGA) protein family. HMGA proteins are architectural transcription factors that both positively and negatively regulate the transcription of a variety of genes. They do not display direct transcriptional activation capacity but regulate gene expression by changing the DNA conformation by binding to AT-rich regions in the DNA and/or direct interaction with several transcription factors (8). HMGA1 is expressed in the fetal and adult human adrenal gland (19). Our results indicate that HMGA1 increases ANG II-stimulated endogenous aldosterone synthase expression after 3 h of stimulation and ANG II-stimulated aldosterone secretion. HMGA1 stimulatory effect on endogenous aldosterone synthase expression was only observed under ANG II stimulatory conditions, in agreement with our previous results (46), where HMGA1 did not modify aldosterone synthase reporter gene expression under basal conditions. Surprisingly, HMGA1 had a potent stimulatory effect on cortisol secretion under basal and ANG II-stimulated conditions despite significantly decreasing 11β-hydroxylase reporter gene expression (46).

PER1 (Period 1) is a transcription factor that belongs to the circadian clock gene family. PER1 is expressed in all cortical layers of the mouse and rat adrenal gland, and its levels of expression follow a daily circadian rhythm (7, 14, 21). A transgenic mouse expressing luciferase under the control of Per1 promoter nicely demonstrated that PER1 is increased during the light phase and that light-mediated Per1 induction is ACTH independent and maximal in the zona glomerulosa of the mouse adrenal gland (26). Furthermore, Per1 is expressed in Rhesus macaque adrenal gland and presents a daily cycle of expression peaking at early morning (30). Our results indicate that PER1 upregulates basal aldosterone secretion, suggesting that this transcription regulatory gene may be involved in the circadian cycle of aldosterone secretion.

Three transcription regulatory genes (EGR1, FOSB, and ZNF295) were upregulated by ANG II but decreased aldosterone secretion under basal, ANG II-stimulated, or both conditions. This is a very interesting set of genes that may exert negative feedback on ANG II-mediated aldosterone secretion as we have previously reported (44, 49) with two members of the regulators of G protein signaling (RGS) family, RGS2 and RGS4.

EGR1 (early growth response gene 1) is upregulated by ANG II in human H295R adrenocortical cells and primary cultures of rat and bovine glomerulosa cells (36, 43). Nogueira et al. (37) reported that EGR1 overexpression in H295R cells increases the reporter activity of CYP21A2 but significantly (50%) decreases the reporter activity of CYP11B2. Our results indicate that EGR1 decreases both basal and ANG II-stimulated aldosterone secretion, a decrease that may be mediated, at least in part, by a decrease in CYP11B2 expression. Although mainly known as a transcriptional activator, EGR1 also has potent transcriptional repressor activity mapped upstream of the zinc finger domain (17) in many experimental models (12, 31, 52). Furthermore, whole genome gene expression analysis of EGR1-null mouse embryonic fibroblasts (MEFs) indicates that of the 266 genes whose expression was regulated compared with wild-type MEFs, almost 60% of them were upregulated, suggesting that EGR1 may mainly function as a transcriptional repressor (28). These results may explain the inhibitory effect of EGR1 on aldosterone secretion and suggest a negative feedback role for EGR1 in mineralocorticoid secretion.

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 (42, 54). Furthermore, ACTH treatment in vivo increases both c-Fos and FOSB mRNA in rat adrenal zona glomerulosa and zona fasciculata (29), and a c-Fos reporter gene is activated by ANG II in H295R cells (55). Rincon Garriz et al. (42) reported that c-fos binds to the StAR protein proximal promoter forming a heterodimer with c-Jun. Furthermore, overexpression of a dominant-negative mutant of c-Fos decreased ANG II-mediated upregulation of StAR protein expression and aldosterone secretion. Under our experimental conditions, FOS did not modify either basal or ANG II-stimulated aldosterone secretion. The difference between our results and those of Rincon Garriz et al. may be due to the fact that in our experimental conditions we overexpressed a functional FOS that may have titrated JUN proteins and consequently fewer functional heterodimers would be able to bind, for example, to the StAR protein promoter and activate its transcription.

When we analyzed the effect of five transcription regulatory genes on endogenous aldosterone synthase expression, we found some surprising and puzzling results. For example, BHLHB2 decreased endogenous and reporter plasmid aldosterone synthase expression; however, it potently stimulated aldosterone secretion under basal and ANG II-stimulated conditions, suggesting that the net steroidogenic effect of a gene (i.e., BHLHB2) cannot easily be extrapolated from gene expression studies in a complex metabolic pathway such as that of steroidogenesis, even when studying key steroidogenic enzymes such as aldosterone synthase.

SERTAD1 was a strong inducer of endogenous aldosterone synthase expression and significantly increased basal and ANG II-mediated aldosterone secretion. The lack of effect of SERTAD1 on aldosterone synthase reporter gene expression highlights a very important point. It is very difficult to define the extent of a gene promoter since transcription regulatory regions may reside not only several kilobases upstream of the transcription initiation site but even downstream or in the coding region of the gene, as clearly observed in the ENCODE project (18).

NFIL3 and VDR showed a very good correlation at endogenous and reporter gene aldosterone synthase expression and aldosterone secretion, suggesting that expression levels of key biosynthetic enzymes may be a good first approach, but further validation at the metabolic level (i.e., aldosterone secretion) should be performed to study the effect of particular genes in adrenal gland steroidogenesis.

Five transcription regulatory genes (BTG2, HMGA1, MITF, NR4A1, and ZFP295) were upregulated by ANG II and increased cortisol secretion under basal, ANG II-stimulated, or both conditions. Three transcription regulatory genes (BCL11B, NFIL3, and PER1) were upregulated by ANG II and decreased cortisol secretion under basal, ANG II-stimulated, or both conditions. Further studies are required to analyze whether the slight inhibitory effect of NFIL3 on cortisol secretion is due to the diversion of substrate to the synthesis of aldosterone because of its strong stimulatory effect on aldosterone secretion.

Mukai et al. (34, 35) 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. In agreement with these studies, we previously reported (46) that FOS and FOSB increase the expression of an 11β-hydroxylase reporter gene. However, neither FOS nor FOSB significantly modified cortisol secretion under basal or ANG II stimulatory conditions, highlighting again that extreme care should be used when extrapolating reporter gene studies to the actual modulation of steroid secretion.

A similar lack of correlation between the effect of the five transcription regulatory genes on reporter gene expression and endogenous gene expression and steroid synthesis was observed with 11β-hydroxylase mRNA expression as with aldosterone synthase mRNA expression. BHLHB2 and HMGA1 both decreased 11β-hydroxylase reporter gene expression; however, neither significantly altered endogenous 11β-hydroxylase mRNA levels. Furthermore, HMGA1 significantly increased cortisol secretion under both basal conditions and ANG II stimulation. NFIL3 decreased endogenous and reporter gene 11β-hydroxylase expression, producing a small, though significant inhibitory effect on cortisol secretion. Finally, expression of SERTAD1 and VDR potently inhibited endogenous 11β-hydroxylase mRNA expression, but neither gene decreased 11β-hydroxylase reporter gene expression or cortisol secretion.

Human H295R adrenocortical cells were used as the experimental model because this is the only well-characterized 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, 41). Although the H295R cell is a well-established human adrenocortical cell model that has greatly advanced the adrenal cell physiology field in the last 20 years, generating more than 400 publications, it is not an ideal adrenocortical cell line experimental model. One disadvantage is that H295R cells function as both adrenal gland zona glomerulosa and zona fasciculata cells. It is possible that this phenotype may have been acquired during transformation, since a new recently described human adrenocortical cell line, HAC15, also has a mixed zona glomerulosa/zona fasciculata steroid secretion pattern and regulation (38). The fact that no adrenal zona glomerulosa- or zona fasciculata-specific cell lines have been described belies the great effort to produce such cell lines. Once produced, such cells would provide better models for the study of adrenal gland zone-specific steroidogenesis and its control.

In summary, we describe the effects of a set of 24 ANG II-regulated transcription regulatory genes on the synthesis of aldosterone and cortisol in H295R human adrenocortical cells. In addition, our data raise concern about the lack of correlation between the effect of specific genes on reporter gene activity of key steroidogenic enzymes and the ultimate physiological effect, steroid secretion, for some genes. Many of the genes reported in the present report may be involved in dysregulation of either glucocorticoid or mineralocorticoid secretion and consequently involved in the pathophysiology of abnormal steroid secretion by the adrenal gland.

GRANTS

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 the University of Mississippi Medical Center Intramural Research Support Program (to D. G. Romero).

DISCLOSURES

The authors have nothing to disclose.