• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of endoArchiveHomepageTES HomepageSubscriptionsSubmissionAbout
Endocrinology. Jul 2009; 150(7): 3110–3117.
Published online Apr 2, 2009. doi:  10.1210/en.2008-1624
PMCID: PMC2703558

Regulation of Aldosterone and Cortisol Production by the Transcriptional Repressor Neuron Restrictive Silencer Factor

Abstract

Aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) regulate aldosterone and cortisol production, respectively. The expression of these enzymes is promoted by calcium influx through Cav3.2, a T-type calcium channel. Neuron-restrictive silencer factor (NRSF) binds to neuron-restrictive silencer element (NRSE) to suppress the transcription of NRSE-containing genes. We found a NRSE-like sequence in human CYP11B2 and CYP11B1 genes as well as the CACNA1H gene of many mammalian species. The CACNA1H gene encodes the α-subunit of Cav3.2. Here we investigated how NRSF/NRSE regulates aldosterone and cortisol synthesis. Inhibition of endogenous NRSF by an adenovirus-expressing dominant-negative NRSF (AD/dnNRSF) increased human CYP11B2 and CYP11B1 mRNA expression, leading to aldosterone and cortisol secretion in human adrenocortical (H295R) cells. In reporter gene experiments, NRSE suppressed luciferase reporters driven by CYP11B2 and CYP11B1 promoters and dnNRSF enhanced them. Moreover, cotransfection of dnNRSF increased luciferase activity of reporter genes after deletion or mutation of NRSE, suggesting that NRSF/NRSE regulates transcription of CYP11B2 and CYP11B1 genes indirectly. AD/dnNRSF augmented mRNA expression of rat CYP11B2 and CYP11B1 genes, neither of which contains a NRSE-like sequence in rat adrenal cells. AD/dnNRSE also significantly increased CACNA1H mRNA in H295R and rat adrenal cells. Efonidipine, a T/L-type calcium channel blocker, significantly suppressed dnNRSF-mediated up-regulation of CYP11B2 and CYP11B1 expression. Moreover, NRSF/NRSE is also involved in angiotensin II- and K+-stimulated augmentation of CYP11B2 and CYP11B1 gene transcription. In conclusion, NRSF/NRSE controls aldosterone and cortisol synthesis by regulating CYP11B2 and CYP11B1 gene transcription mainly through NRSF/NRSE-mediated enhancement of the CACNA1H gene.

Aldosterone plays a central role in various pathological conditions, including hypertension, heart failure (HF), and postinfarction ventricular remodeling (1,2). Increased aldosterone or cortisol in HF patients is an independent predictor of mortality risk (3). Thus, understanding the regulation of aldosterone and cortisol synthesis is important for elucidating the mechanisms underlying these disorders. Aldosterone and cortisol are predominantly synthesized and secreted from the adrenal zona glomerulosa and fasciculata, respectively. Aldosterone synthase (CYP11B2) and 11β-hydroxylase (CYP11B1) are responsible for the synthesis of aldosterone (human and rat) and cortisol (human)/corticosterone (rat), respectively (4,5). Expression of these steroid hydroxylases in the adrenal gland is regulated by angiotensin II (AngII) and extracellular potassium (K+)-induced calcium influx through calcium channels, particularly Cav3.2, a T-type calcium channel (6,7,8,9). These stimuli increase intracellular calcium levels and trigger calcium-calmodulin (CaM)-CaM-dependent protein kinase (CaMK) signaling (10,11,12). CYP11B2 expression is regulated by a number of cis-acting transcription factors such as nerve growth factor-induced clone B (13), chicken ovalbumin upstream promoter-transcription factor I (14), cAMP response element binding protein (CREB), and activating transcription factor-1 (15). Moreover, several transcription factors, including CREB, activating transcription factor-1, and steroidogenic factor-1, bind to the 5′-flanking region of the CYP11B1 gene (16,17). Several of these transcription factors are regulated by calcium-CaM-CaMK signaling. However, the transcriptional regulation of aldosterone and cortisol synthesis remains elusive.

The neuron-restrictive silencer element (NRSE) is a crucial inhibitory DNA element that prevents the expression of neuron-specific genes in nonneuronal and undifferentiated neuronal cells (18). NRSEs are present in a number of neuron-specific genes, including SCG10, the type II sodium channel Nav1.2, and synapsin I (19,20). The neuron-restrictive silencer factor (NRSF) is a Kruppel-type zinc-finger repressor. NRSF binds to NRSEs, thereby suppressing expression of NRSE-containing genes. Previously we demonstrated that NRSF/NRSE system regulates fetal cardiac gene expression of atrial natriuretic peptides and B-type natriuretic peptides in cultured cardiomyocytes (21,22).

NRSE-like sequences are found in intron 8 and exon 9 of the human CYP11B2 and CYP11B1 genes. There are no NRSE-like sequences in the rat CYP11B2 and CYP11B1 genes. Moreover, the human and rat CACNA1H genes, which encode the α-subunit of Cav3.2, contain a functional NRSE sequence (23). T-type calcium channels, Cav3.2 in particular, influence aldosterone and cortisol biosynthesis in adrenal cells (9,24,25).

We used NCI-H295R, a human adrenocortical cell line (H295R cells), and rat adrenal cells isolated from adrenal glands to analyze how production of aldosterone and cortisol/corticosterone is transcriptionally regulated. We here demonstrate the crucial role of NRSF/NRSE system in the transcriptional regulation of CACNA1H and subsequent induction of CYP11B2 and CYP11B1 in human and rat. This system also directly regulates NRSEs in human CYP11B2 and CYP11B1 genes.

Materials and Methods

Reagents and antibodies

Efonidipine was kindly provided by Nissan Chemical (Tokyo, Japan). Other reagents and antibodies are listed in the supplemental text 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org.

Plasmids and recombinant adenovirus (AD)

Plasmids encoding Myc-tagged dominant-negative NRSF (dnNRSF) were kindly provided by David J. Anderson (California Institute of Technology, Pasadena, CA). The recombinant AD encoding Myc-tagged dnNRSF was purified and concentrated as described previously (21).

Cell culture

The NCI-H295R human adrenocortical cell line (26) was obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM/F12 medium containing 2% Ultroser SF (Biosepra, Cergy St. Christophe, France), 1% insulin/transferrin/selenium, penicillin, and streptomycin (9). Cells were maintained at 37 C in a humid atmosphere containing 95% air-5% CO2.

Rat adrenal cells were obtained from adrenal glands of female Long Evans rats weighing 200–250 g and isolated according to the modified method previously described (27). Briefly, isolation and cell dissociation were performed in MEM (supplemented with penicillin and streptomycin). After a 50-min incubation at 37 C with collagenase (2 mg/ml) and deoxyribonuclease (25 μg/ml), cells were disrupted by gentle aspiration with a sterile pipette, filtered, and centrifuged for 10 min at 100 × g. The cell pellet was then resuspended in OPTI-MEM medium (Invitrogen, Carlsbad, CA) supplemented with 2% fetal bovine serum, penicillin, and streptomycin. This protocol was approved by our institutional review board for animal research.

Both cells were maintained at 37 C under a humid atmosphere of 95% air-5% CO2.

AD transfection

H295R and rat adrenal cells were cultured in six-well culture dishes at a density of 5 × 105 or 1 × 106 cells/well. One day after H295R cells plating or 2 d after rat adrenal cells plating, each cell was infected with AD/dnNRSF or a control LacZ AD (AD/LacZ) at variety multiplicity of infection (MOI) for 24 h in low-serum medium (DMEM/F12 medium containing 0.1% Ultroser SF or OPTI-MEM medium containing with 0.2% fetal bovine serum). At 24 h after infection, cells were stimulated with the specified drugs for 12 h as indicated in the figures.

Chromatin immunoprecipitation (ChIP) assay

Chromatin from H295R cells was prepared using a ChIP assay kit (Upstate, Charlottesville, VA). The purified chromatin was immunoprecipitated with normal IgG or anti-Myc antibodies or was not treated with antibody (non) 36 h after infection with AD/dnNRSF or AD/LacZ. The immunoprecipitated product was analyzed in PCRs using the following primer pairs: NRSE from the human CYP11B2 gene (NRSEB2) ChIP primers, 5′-GTAAGGTGGGGCTGGTCAGAAGT-3′ (forward) and 5′-TCTGAAAGTGAGGAGGGGGGACGT-3′ (reverse); controlB2 ChIP primers, 5′-GCAAGCAAGAAGACAGTGGAGGG-3′ (forward) and 5′-TGTAGCTCAGGGTTGCTGACCTG-3′ (reverse); NRSE from the human CYP11B1 gene (NRSEB1) ChIP primers, 5′-CAGGAATGAAACAGGTTGGAGGC-3′ (forward) and 5′-GAGACGTGATTAGTTGATGGCTC-3′ (reverse); and controlB1 ChIP primers, 5′-GGCTGTGAATCCATCTGGTCATG-3′ (forward) and 5′-GATAAAGGGGATATCACCACCG-3′ (reverse). Aliquots of chromatin obtained before immunoprecipitation were also analyzed (input).

Reporter constructs

The 1523-bp proximal promoter of the CYP11B2 gene and the 1100-bp proximal promoter of the CYP11B1 gene were described previously (13,17). These sequences were PCR amplified using a BAC clone as a template (RP11–304E16; BACPAC Resource Center, Oakland, CA). Details are described in the supplemental text 2.

Luciferase assay

For transfection experiments, H295R cells were cultured in 24-well culture dishes at a density of 5 × 104 cells/well and transfected 36 h later. Transfection was performed using 2.0 μl of Lipofectamine (Invitrogen, Carlsbad, CA) and 0.3 μg of reporter plasmid DNA in DMEM/F12 medium for 4 h at 37 C. For cotransfection experiments, various amounts of expression plasmids were included in the transfection reaction; the total amount of DNA was kept constant by adding carrier DNA (empty expression vector). In all experiments, 0.1 μg of pRL-SV40 (Toyo, Tokyo, Japan), an expression plasmid in which the simian virus 40 (SV40) promoter is fused to the Renilla luciferase gene, was cotransfected and used to normalize the luciferase activity. After transfection, cells were incubated in low-serum medium (DMEM/F12 medium containing 0.1% Ultroser SF and antibiotics) for 24 h. Where indicated, transfected cells were stimulated for 12 h. Cells were then lysed and assayed using a luciferase assay system (Promega, Madison, WI) and a luminometer (TR717; Applied Biosystems, Foster City, CA). In each experiment, cell lysate aliquots from duplicate wells were assayed, and the luciferase activities were normalized to that derived from pRL-SV40 luciferase.

RT-PCR

Total RNA (1 μg) extracted from H295R and rat adrenal cells with Trizol (Invitrogen, Carlsbad, CA) was reverse transcribed with SuperScript II and random primers (Invitrogen). The expression levels of specific mRNAs were measured using SYBR green real-time PCRs (QIAGEN, Valencia, CA) with gene-specific primers (supplemental Table 1). Real-time PCR primers and probes for human CYP11B2, human CYP11B1, human NRSF, rat CYP11B2, and rat CYP11B1 were used for TaqMan probe assays (assay Hs01597732_m1, Hs01596404_m1, Hs00194498_m1, Rn02396730_g1, Rn02607234_g1; Applied Biosystems). RT-PCRs were conducted in an ABI Prism Sequence Detection System 7700 (Applied Biosystems).

Western blot analysis

Details are available in supplemental text 3.

EMSA

Nuclear extract from H295R cells was prepared as previously described (21). Double-stranded oligonucleotides containing two copies of the NRSEB2 (5′-GCCTCTGTCCTAGGTGCTGAA-3′), a mutant variant of NRSEB2 (5′-GCCTCTGTAATACGTGCTGAA-3′), the NRSEB1 (5′-GCTTCTGTCCTAGGTGCTGAA-3′), or a mutant variant of NRSEB1 (5′-GCTTCTGTAATACGTGCTGAA-3′) were synthesized and used as probes for EMSAs. DNA-protein binding reactions were carried out in a 20-μl final volume of reaction buffer containing 20 mmol/liter HEPES (pH 7.9), 125 mmol/liter KCl, 5 mmol/liter MgCl2, 10% glycerol, 125 μg/ml polydeoxyinosinic-deoxycytidylic acid, and 1 mmol/liter dithiothreitol. The nuclear extract (15 μg protein) was added to the reaction buffer and preincubated for 10 min on ice. Radiolabeled DNA probe was then added, and the nuclear extract was incubated for another 30 min at room temperature. Samples were separated on a 6% retardation gel (Invitrogen) at 300 V in 0.5× Tris-borate EDTA buffer for 20 min. For competition assays, a 50-fold excess of double-stranded NRSEB2, NRSEB1, the mutated version of NRSEB2, the mutated version of NRSEB1, a consensus NRSE, or the CREB sequence (supplemental Table 1) was incubated in reaction mixture with a radiolabeled NRSEB2 or NRSEB1 probe as described above. NRSF antibodies were used in the gel shift experiment.

RIA and ELISA

Aldosterone and cortisol secretion was assayed as previously described (9). Details are provided in the supplemental text 4.

Statistical analysis

Results are expressed as the means ± sd. Student’s t tests were used to analyze differences between two groups. Within-group differences and between-group differences after treatments were assessed with one-way ANOVA and two-way ANOVA, respectively. Significant differences were defined as P < 0.05.

Results

NRSE-like sequences are present in the human CYP11B2 (hCYP11B2) and CYP11B1 (hCYP11B1) genes

A homology search for NRSE-like sequences identified NRSE-like sequences within the genomic sequence corresponding to intron 8 and exon 9 of the human genes but not in those of other species (supplemental Table 2).

NRSF binds to the NRSEs in the hCYP11B2 and hCYP11B1 genes in H295R cells

Western blots showed that NRSF was localized in the nuclei of H295R and primary rat adrenal cells in a manner similar to that observed in HeLa cells, which express NRSF abundantly (Fig. 1A1A).). To test whether the NRSF binds to the NRSEs in hCYP11B2 (NRSEB2) and hCYP11B1 (NRSEB1) genes, we performed an EMSA with nuclear extracts from H295R cells. A shifted band was observed when the putative NRSEB2 sequence was used as the radiolabeled probe (Fig. 11,, B and D, lane 2). No band shift was detected when the binding reaction included excess molar amounts of unlabeled NRSEB2 (Fig. 11,, B and D, lane 3), consensus NRSE (Fig. 1B1B,, lane 5), or NRSEB1 (Fig. 1D1D,, lane 5). In contrast, unlabeled probe corresponding to mutated NRSEB2 (Fig. 11,, B and D, lane 4) or CREB-binding sequences (Fig. 1B1B,, lane 6) had no effect on the band shift. In addition, the band was supershifted in reactions containing antibodies specific for NRSF (Fig. 1C1C,, lane 4, arrowhead). We obtained similar results using radiolabeled NRSEB1 probe (Fig. 1D1D,, lanes 7–10). As shown in the EMSA, NRSF binds to both NRSEs.

Figure 1
NRSF binds to NRSEs in the human CYP11B2 (NRSEB2) and CYP11B1 (NRSEB1) genes in H295R cells. Panel A, Nuclear proteins from H295R and rat adrenal cells were immunoblotted with the antibodies indicated at the left. HeLa cells were used as a positive control ...

NRSE functions in the hCYP11B2 and hCYP11B1 genes in H295R cells

To test whether the NRSF/NRSE system regulates aldosterone and cortisol biosynthesis in H295R cells, we examined the effects of dnNRSF, which is lacking the repressor domain but still contains the DNA-binding domain (28). Aldosterone and cortisol secretion in human cells increased in response to NRSF inhibition with an AD/dnNRSF (Fig. 22,, A and B). The mRNA levels of hCYP11B2 and hCYP11B1 were also markedly increased after transfection with AD/dnNRSF (Fig. 22,, C and D).

Figure 2
NRSE regulates hCYP11B2 and hCYP11B1 gene expression in H295R cells. A and B, Effect of inhibiting endogenous NRSF using AD/dnNRSF on aldosterone (A) and cortisol (B) secretion from H295R cells. C and D, hCYP11B2 (C) and hCYP11B1 (D) mRNA were analyzed ...

In a ChIP assay, Myc-tagged dnNRSF bound to NRSEB2 and NRSEB1 in human cells (supplemental Fig. 1, A and B). Chromatin DNA fragments coimmunoprecipitated by anti-Myc antibodies but not control IgG or no antibody were amplified using primers specific for the NRSEs in hCYP11B2 and hCYP11B1. The coimmunoprecipitated DNA fragment was not amplified by control primers corresponding to 5′-flanking sequences from the hCYP11B2 and hCYP11B1 genes.

To examine whether NRSEB2 and NRSEB1 function in the transcriptional regulation of hCYP11B2 and hCYP11B1 genes, we constructed a reporter plasmid consisting of the hCYP11B2 or hCYP11B1 promoter and the luciferase gene (CYP11B2/Luc or CYP11B1/Luc) followed by wild-type NRSEB2 (CYP11B2/Luc/NRSEB2) or NRSEB1 (CYP11B1/Luc/NRSEB1). Plasmids in which wild-type NRSEB2 or NRSEB1 was replaced with mutated NRSEB2 or mutated NRSEB1 (CYP11B2/Luc/mtNRSEB2 or CYP11B1/Luc/mtNRSEB1, respectively) were also constructed to confirm the functional reliability of NRSE. The luciferase activity of CYP11B2/Luc/NRSEB2 was reduced by about 35% compared with that of the control CYP11B2/Luc reporter gene (Fig. 2E2E).). Replacement of NRSEB2 with mutated NRSEB2 recovered the NRSE-dependent reduction of promoter activity, as demonstrated by comparison of CYP11B2/Luc/NRSEB2 and CYP11B2/Luc/mtNRSEB2. Similar results were obtained when CYP11B1/Luc/NRSEB1 and CYP11B1/Luc/mtNRSEB1 were compared (Fig. 2F2F).). Furthermore, thymidine kinase promoter-dependent gene regulation was inhibited in the presence of either NRSEB2 or NRSEB1 (supplemental Fig. 2). These results suggest that NRSEB2 and NRSEB1 function directly in the transcriptional regulation of hCYP11B2 and hCYP11B1 genes in human adrenal cells.

NRSF/NRSE-mediated transcriptional regulation of human CACNA1H (hCACNA1H) is involved in hCYP11B2 and hCYP11B1 expression in H295R cells

To confirm that NRSEB2 and NRSEB1 suppress hCYP11B2 and hCYP11B1 luciferase activity, respectively, an expression plasmid encoding dnNRSF was cotransfected with the reporter genes. Reporter activity was three times greater in CYP11B2/Luc after dnNRSF expression (Fig. 3A3A).). The effect of dnNRSF on reporter activity in CYP11B2/Luc/NRSEB2 was greater than recovery of NRSE-dependent inhibition and was similar to that observed in CYP11B2/Luc (Fig. 3A3A).). Furthermore, dnNRSF affected reporter activity, even in CYP11B2/Luc/mtNRSEB2 (Fig. 3A3A).). We observed similar results in the hCYP11B1 promoter-dependent reporter assay (Fig. 3B3B).). These data clearly indicate that the contribution of indirect regulation by NRSF/NRSE system is much greater than direct regulation of hCYP11B2 and hCYP11B1 via NRSEB2 and NRSEB1.

Figure 3
NRSF/NRSE-mediated transcriptional regulation of hCACNA1H is involved in hCYP11B2 and hCYP11B1 expression in H295R cells. A and B, H295R cells were transfected with the plasmids indicated at the left together with dnNRSF or control plasmid. The effect ...

We have previously shown that hCACNA1H contains a NRSE (23). The transcriptional product of CACNA1H, Cav3.2, is thought to be involved in aldosterone and cortisol production (9). We hypothesized that indirect regulation of hCYP11B2 and hCYP11B1 genes via T-type calcium channel-mediated (Cav3.2 mediated) calcium-CaM-CaMK contributes to hCYP11B2 and hCYP11B1 expression. To test this hypothesis, we investigated the role of NRSF in transcriptional regulation of the hCACNA1H gene. hCACNA1H mRNA was up-regulated by AD/dnNRSF in H295 cells (Fig. 3C3C),), but AD/dnNRSF had no effect on human CACNA1G (hCACNA1G) or human CACNA1C (hCACNA1C) mRNA, neither of which contain a NRSE sequence (Fig. 33,, D and E). These genes encode the α-subunit of Cav3.1, a T-type calcium channel, and the α-subunit of Cav1.2, an L-type calcium channel, respectively.

To verify the contribution of calcium channel-mediated regulation to dnNRSF-promoted hCYP11B2 and hCYP11B1 expression, we examined the effect of efonidipine, a dual T/L-type calcium channel blocker, on reporter activity in CYP11B2/Luc, CYP11B2/Luc/NRSEB2, and CYP11B2/Luc/mtNRSEB2 in the presence and absence of dnNRSF. Efonidipine (0.3 μm) completely inhibited the dnNRSF-induced reporter activity of CYP11B2/Luc and CYP11B2/Luc/mtNRSEB2 (Fig. 3F3F).). The promoter activity of CYP11B2/Luc/NRSEB2 in response to a combination of dnNRSF and efonidipine was significantly greater than that observed with efonidipine alone (Fig. 3F3F).). These differences indicate that NRSF/NRSE system directly mediates the increase in promoter activity in humans, although, as expected, this effect was smaller than the calcium-mediated effect. Nifedipine (0.3 μm), an L-type calcium channel blocker, did not reduce the dnNRSF-induced activity of the three CYP11B2 promoter reporters (data not shown). Similar results were obtained for hCYP11B1-regulated reporter expression (Fig. 3G3G).). Collectively, these data suggest that NRSF/NRSE mainly regulates the expression of hCYP11B2 and hCYP11B1 in a manner dependent on calcium influx via T-type calcium channels.

Blocking T-type calcium channels inhibits the dnNRSF-induced increases in hCYP11B2 and hCYP11B1 mRNA expression in H295R cells

We assumed that CaMK activation via calcium influx through Cav3.2 might enhance the promoter activity of the hCYP11B2 and hCYP11B1 genes containing upstream cis elements responsive to CaMK signaling. Thus, we investigated the effects of efonidipine, nifedipine, and KN93 (a CaMK I, II, and IV inhibitor) on the dnNRSF-induced increase in aldosterone and cortisol secretion and the dnNRSF-regulation of hCYP11B2 and hCYP11B1 mRNA expression in H295R cells. Increased aldosterone and cortisol secretion was suppressed by either efonidipine or KN93 in a dose-dependent manner (Fig. 44,, A and B). Nifedipine, which was administered at concentrations greater than those of efonidipine, did not affect secretion of either aldosterone or cortisol (Fig. 44,, A and B). Moreover, dnNRSF-induced increases in hCYP11B2 and hCYP11B1 mRNAs were suppressed by efonidipine and KN93 but not by nifedipine (Fig. 44,, C and D). These data suggest that a T-type calcium channel, probably Cav3.2, is responsible for dnNRSF-induced hCYP11B2 and hCYP11B1 expression.

Figure 4
A T-type calcium channel blocker and a CaMK inhibitor strongly inhibit dnNRSF-induced aldosterone and cortisol secretion by reducing hCYP11B2 and hCYP11B1 mRNA levels in H295R cells. A–D, H295R cells infected with AD/dnNRSF (20 MOIs) were treated ...

NRSF/NRSE-mediated transcriptional regulation of rat CACNA1H (rCACNA1H) is involved in aldosterone and corticosterone production in rat adrenal cells

To confirm whether the effect of NRSF/NRSE-mediated steroidogenesis was widely adapted in the other species, we performed similar experiments using in freshly rat isolated adrenal cells. Although rat CYP11B2 (rCYP11B2) and rat CYP11B1 (rCYP11B1) genes do not have NRSEs, aldosterone and corticosterone synthesis were enhanced by AD/dnNRSF in rat adrenal cells due to increased rCYP11B2 and rCYP11B1 mRNA expression (Fig. 55,, A–D). rCACNA1H gene has a NRSE (supplemental Table 2). As expected, rCACNA1H mRNA was up-regulated by AD/dnNRSF in rat adrenal cells (Fig. 5E5E),), whereas rCACNA1G and rCACNA1C mRNA were not (supplemental Fig. 3, A and B). Increased expression of Cav3.2 in rat adrenal cells was confirmed by immunoblot analysis using a Cav3.2-specific antibody (Fig. 5F5F).). These results suggest that the transcriptional regulation of CACNA1H by NRSF/NRSE is involved in aldosterone and corticosterone synthesis in rat isolated adrenal cells as well as human H295R cells.

Figure 5
dnNRSF increases aldosterone and corticosterone synthesis presumably by up-regulating CACNA1H gene expression in rat adrenal cells. A and B, The effect of AD/dnNRSF (20 MOIs) on aldosterone (A) and corticosterone (B) secretion in rat adrenal cells. C ...

NRSF/NRSE system mediates AngII- and K+-induced increases in hCACNA1H, hCYP11B2, and hCYP11B1 mRNA expression in H295R cells

AngII and K+ stimulate hCYP11B2 and hCYP11B1 mRNA expression via the calcium-CaM-CaMK pathway (10,11,12). We investigated NRSF/NRSE-mediated regulation of hCACNA1H, hCYP11B2, and hCYP11B1 gene expression in response to AngII or K+. In the absence of dnNRSF, the expression of hCACNA1H mRNA was enhanced by AngII (100 nm) and K+ (10 mm) by approximately 2.4- and 1.9-fold, respectively. However, in the presence of dnNRSF, hCACNA1H expression was generally unaffected (Fig. 6A6A).). Similarly, in the absence of dnNRSF, stimulation with AngII or K+ increased the expression of the hCYP11B2 gene by 18- and 15-fold, respectively. In the presence of dnNRSF, however, AngII and K+ increased the hCYP11B2 mRNA by less than 1.5-fold (Fig. 6B6B).). Additionally, in the absence of dnNRSF, stimulation with AngII or K+ increased the expression of the hCYP11B1 gene by 2.0- and 2.4-fold, respectively, whereas hCYP11B1 mRNA was increased by approximately 1.3-fold in the presence of dnNRSF (Fig. 6C6C).). Collectively, these results suggest that AngII and K+ augment hCACNA1H, hCYP11B2, and hCYP11B1 mRNA expression through NRSF/NRSE-dependent pathway, at least in part.

Figure 6
Inhibition of NRSF contributes to AngII- and K+-induced increases in hCACNA1H, hCYP11B2, and hCYP11B1 gene expression in H295R cells. A–C, AD/LacZ and AD/dnNRSF (20 MOIs)-infected H295R cells were stimulated by AngII (100 nm) or K+ ...

Discussion

The present study demonstrates that NRSF/NRSE is involved in aldosterone and cortisol/corticosterone synthesis definitely by regulating CYP11B2 and CYP11B1 gene transcription through NRSF/NRSE-mediated enhancement of CACNA1H gene expression in human H295R and rat adrenal cells and partly by NRSF/NRSE-mediated direct enhancement of CYP11B2 and CYP11B1 gene transcription in human H295R cells.

Among the key molecules in aldosterone and cortisol/corticosterone synthesis, hCYP11B2, hCYP11B1, and hCACNA1H genes have NRSE-like sequences in their transcriptional regulatory regions. However, in a number of other mammalian species including rats, only the CACNA1H gene has a NRSE-like sequence. In the present study, AD/dnNRSF increased expression of CYP11B2 and CYP11B1 mRNA and augmented secretion of aldosterone and cortisol/corticosterone in both human H295R cells and rat adrenal cells, clearly indicating that NRSF/NRSE indirectly regulates CYP11B2 and CYP11B1 gene expression. NRSF/NRSE increases expression of the CACNA1H gene in mouse cardiomyocytes, and NRSF binds to the NRSE in the CACNA1H gene by EMSA (23). In the present study, AD/dnNRSF augmented CACNA1H mRNA. Given that calcium influx through Cav3.2 calcium channels augments CYP11B2 and CYP11B1 gene transcription through the CaM-CaMK-dependent pathway, it is likely that NRSF/NRSE-mediated enhancement of CACNA1H gene expression leads to up-regulation of CYP11B2 and CY11B1 mRNA in human H295R cells and rat adrenal cells. The repressor effect of NRSEB2 and NRSEB1 on reporter genes driven by hCYP11B2 and hCYP11B1 promoters is less than 40%; thus, NRSF/NRSE regulates hCYP11B2 and hCYP11B1 gene transcription mainly through the indirect pathway. However, in reporter gene analyses, EMSA and ChIP assay of human H295R cells reveals that NRSF/NRSE system directly regulates transcription of hCYP11B2 and hCYP11B1.

AngII and extracellular K+ stimulate aldosterone and cortisol synthesis by inducing expression of CYP11B2 and CYP11B1. AngII and K+ also induce expression of several types of calcium channels including T-type calcium channels (9). These stimuli increase intracellular calcium and trigger CaM-CaMK signaling. In fact, we recently reported that a dual T/L-type calcium channel blocker dose-dependently inhibits AngII- or K+-induced aldosterone and cortisol secretion (9). However, it is not fully understood how AngII and K+ induce CYP11B2 and CYP11B1 gene expression. In this study, we have demonstrated that AngII- or K+-induced expression of CACNA1H mRNA (Cav3.2) is regulated by the NRSF/NRSE system. Thus, induction of CaM-CaMK via calcium influx through T-type calcium channels may partially account for AngII- or K+-induced aldosterone and cortisol synthesis.

The NRSEs that exist in the intron-exon boundary function as suppressor elements. Several cis-acting enhancer elements of the hCYP11B2 gene, including cAMP response element, nerve growth factor-induced clone B response element-1, and Adrenal 5, have been identified in the 5′-flanking region of the hCYP11B2 gene (13,14,15). NRSE might be the first suppressor element that functions within the hCYP11B2 and hCYP11B1 genes.

The repressor function of the NRSF/NRSE system on gene regulation might be generally ineffective in HF patients. Aldosterone is a clinical marker of HF and AngII is also increased in HF. The increase in both aldosterone and AngII reflects a compensatory response in tissue and systemic renin-angiotensin systems. AngII might cancel the suppressor activity of NRSF in aldosterone synthesis. Previously we demonstrated that NRSF/NRSE system mediates the induction of fetal cardiac genes in cardiomyocytes in response to hypertrophic stimuli (21,22). Mice overexpressing dnNRSF in cardiomyocytes exhibit cardiac phenotypes that resemble dilated cardiomyopathy, and they die of fatal ventricular arrhythmia. Consistently, NRSE-containing genes, such as atrial natriuretic peptides, B-type natriuretic peptides, and α-skeletal actin are all up-regulated in HF. These cancelation of the NRSF/NRSE system might modify the pathophysiological conditions in the HF.

It is unknown how NRSE-mediated repression of NRSE-containing genes (CACNA1H, CYP11B2, and CYP11B1) is regulated. Given that NRSF recruits mSin3 as well as class I and class II histone deacetylase complexes to repress gene transcription (29,30), it is likely that NRSF represses these genes using similar mechanisms. It is also unclear how AngII and K+ cancel the NRSE-mediated repression. Neither AngII nor K+ down-regulates NRSF mRNA or protein expression (supplemental Fig. 4). Therefore, the lack of NRSF/NRSE-mediated suppression cannot be ascribed to a reduction of NRSF. Endothelin or outside-in fibronectin signaling inhibits the binding of NRSF to NRSEs in cultured cardiomyocytes (22). The contribution of this mechanism and the significance of species-specific presence of NRSE in hCYP11B2 and hCYP11B1 genes require further clarification. However, in the present study, we report a novel role of NRSF/NRSE system in aldosterone and cortisol/corticosterone production in human and rat adrenocortical cells (supplemental Fig. 5).

Supplementary Material

[Supplemental Data]

Footnotes

This work was supported by research grants from the Japanese Ministry of Education, Science, and Culture and the Japanese Ministry of Health and Welfare.

Disclosure Summary: The authors have nothing to disclose.

First Published Online April 2, 2009

Abbreviations: AD, Adenovirus; AngII, angiotensin II; CaM, calmodulin; CaMK, CaM-dependent protein kinase; ChIP, chromatin immunoprecipitation; CREB, cAMP response element binding protein; dnNRSF, dominant-negative NRSF; HF, heart failure; K+, potassium; MOI, multiplicity of infection; NRSE, neuron-restrictive silencer element; NRSF, neuron-restrictive silencer factor; SV40, simian virus 40.

References

  • Packer M 1992 The neurohormonal hypothesis: a theory to explain the mechanism of disease progression in heart failure. J Am Coll Cardiol 20:248–254 [PubMed]
  • Struthers AD 2004 Aldosterone-induced vasculopathy. Mol Cell Endocrinol 217:239–241 [PubMed]
  • Güder G, Bauersachs J, Frantz S, Weismann D, Allolio B, Ertl G, Angermann CE, Störk S 2007 Complementary and incremental mortality risk prediction by cortisol and aldosterone in chronic heart failure. Circulation 115:1754–1761 [PubMed]
  • Mornet E, Dupont J, Vitek A, White PC 1989 Characterization of two genes encoding human steroid 11β-hydroxylase [P-450(11)β]. J Biol Chem 264:20961–20967 [PubMed]
  • Curnow KM, Tusie-Luna MT, Pascoe L, Natarajan R, Gu JL, Nadler JL, White PC 1991 The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Mol Endocrinol 5:1513–1522 [PubMed]
  • Aguilera G, Catt KJ 1986 Participation of voltage-dependent calcium channels in the regulation of adrenal glomerulosa function by angiotensin II and potassium. Endocrinology 118:112–118 [PubMed]
  • Bird IM, Mathis JM, Mason JI, Rainey WE 1995 Ca(2+)-regulated expression of steroid hydroxylases in H295R human adrenocortical cells. Endocrinology 136:5677–5684 [PubMed]
  • Koritz SB 1986 The stimulation by calcium and its inhibition by ADP of cholesterol side-chain cleavage activity in adrenal mitochondria. J Steroid Biochem 24:569–576 [PubMed]
  • Imagawa K, Okayama S, Takaoka M, Kawata H, Naya N, Nakajima T, Horii M, Uemura S, Saito Y 2006 Inhibitory effect of efonidipine on aldosterone synthesis and secretion in human adrenocarcinoma (H295R) cells. J Cardiovasc Pharmacol 47:133–138 [PubMed]
  • Pezzi V, Clyne CD, Ando S, Mathis JM, Rainey WE 1997 Ca(2+)-regulated expression of aldosterone synthase is mediated by calmodulin and calmodulin-dependent protein kinases. Endocrinology 138:835–838 [PubMed]
  • Condon JC, Pezzi V, Drummond BM, Yin S, Rainey WE 2002 Calmodulin-dependent kinase I regulates adrenal cell expression of aldosterone synthase. Endocrinology 143:3651–3657 [PubMed]
  • Wilson JX, Aguilera G, Catt KJ 1984 Inhibitory actions of calmodulin antagonists on steroidogenesis in zona glomerulosa cells. Endocrinology 115:1357–1363 [PubMed]
  • Bassett MH, Suzuki T, Sasano H, White PC, Rainey WE 2004 The orphan nuclear receptors NURR1 and NGFIB regulate adrenal aldosterone production. Mol Endocrinol 18:279–290 [PubMed]
  • Kurihara I, Shibata H, Kobayashi S, Suda N, Ikeda Y, Yokota K, Murai A, Saito I, Rainey WE, Saruta T 2005 Ubc9 and protein inhibitor of activated STAT 1 activate chicken ovalbumin upstream promoter-transcription factor I-mediated human CYP11B2 gene transcription. J Biol Chem 280:6721–6730 [PubMed]
  • Clyne CD, Zhang Y, Slutsker L, Mathis JM, White PC, Rainey WE 1997 Angiotensin II and potassium regulate human CYP11B2 transcription through common cis-elements. Mol Endocrinol 11:638–649 [PubMed]
  • Bassett MH, Zhang Y, Clyne C, White PC, Rainey WE 2002 Differential regulation of aldosterone synthase and 11β-hydroxylase transcription by steroidogenic factor-1. J Mol Endocrinol 28:125–135 [PubMed]
  • Wang XL, Bassett M, Zhang Y, Yin S, Clyne C, White PC, Rainey WE 2000 Transcriptional regulation of human 11β-hydroxylase (hCYP11B1). Endocrinology 141:3587–3594 [PubMed]
  • Schoenherr CJ, Anderson DJ 1995 The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes. Science 267:1360–1363 [PubMed]
  • Mori N, Schoenherr C, Vandenbergh DJ, Anderson DJ 1992 A common silencer element in the SCG10 and type II Na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells. Neuron 9:45–54 [PubMed]
  • Chong JA, Tapia-Ramírez J, Kim S, Toledo-Aral JJ, Zheng Y, Boutros MC, Altshuller YM, Frohman MA, Kraner SD, Mandel G 1995 REST: a mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell 80:949–957 [PubMed]
  • Kuwahara K, Saito Y, Ogawa E, Takahashi N, Nakagawa Y, Naruse Y, Harada M, Hamanaka I, Izumi T, Miyamoto Y, Kishimoto I, Kawakami R, Nakanishi M, Mori N, Nakao K 2001 The neuron-restrictive silencer element-neuron-restrictive silencer factor system regulates basal and endothelin 1-inducible atrial natriuretic peptide gene expression in ventricular myocytes. Mol Cell Biol 21:2085–2097 [PMC free article] [PubMed]
  • Ogawa E, Saito Y, Kuwahara K, Harada M, Miyamoto Y, Hamanaka I, Kajiyama N, Takahashi N, Izumi T, Kawakami R, Kishimoto I, Naruse Y, Mori N, Nakao K 2002 Fibronectin signaling stimulates BNP gene transcription by inhibiting neuron-restrictive silencer element-dependent repression. Cardiovasc Res 53:451–459 [PubMed]
  • Kuwahara K, Saito Y, Takano M, Arai Y, Yasuno S, Nakagawa Y, Takahashi N, Adachi Y, Takemura G, Horie M, Miyamoto Y, Morisaki T, Kuratomi S, Noma A, Fujiwara H, Yoshimasa Y, Kinoshita H, Kawakami R, Kishimoto I, Nakanishi M, Usami S, Saito Y, Harada M, Nakao K 2003 NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function. EMBO J 22:6310–6321 [PMC free article] [PubMed]
  • Lotshaw DP 2001 Role of membrane depolarization and T-type Ca2+ channels in angiotensin II and K+ stimulated aldosterone secretion. Mol Cell Endocrinol 2175:157–171 [PubMed]
  • Chen XL, Bayliss DA, Fern RJ, Barrett PQ 1999 A role for T-type Ca2+ channels in the synergistic control of aldosterone production by ANG II and K+. Am J Physiol 276:F674–F683 [PubMed]
  • Gazdar AF, Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, Chrousos GP, Brennan MF, Stein CA, La Rocca RV 1990 Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res 50:5488–5496 [PubMed]
  • Gallo-Payet N, Chouinard L, Balestre MN, Guillon G 1991 Involvement of protein kinase C in the coupling between the V1 vasopressin receptor and phospholipase C in rat glomerulosa cells: effects on aldosterone secretion. Endocrinology 129:623–634 [PubMed]
  • Chen ZF, Paquette AJ, Anderson DJ 1998 NRSF/REST is required in vivo for repression of multiple neuronal target genes during embryogenesis. Nat Genet 20:136–142 [PubMed]
  • Andrés ME, Burger C, Peral-Rubio MJ, Battaglioli E, Anderson ME, Grimes J, Dallman J, Ballas N, Mandel G 1999 CoREST: a functional corepressor required for regulation of neural-specific gene expression. Proc Natl Acad Sci USA 96:9873–9878 [PMC free article] [PubMed]
  • Roopra A, Sharling L, Wood IC, Briggs T, Bachfischer U, Paquette AJ, Buckley NJ 2000 Transcriptional repression by neuron-restrictive silencer factor is mediated via the Sin3-histone deacetylase complex. Mol Cell Biol 20:2147–2157 [PMC free article] [PubMed]

Articles from Endocrinology are provided here courtesy of The Endocrine Society
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...