Interactive Roles of Ets-1, Sp1, and Acetylated Histones in the Retinoic Acid-dependent Activation of Guanylyl Cyclase/Atrial Natriuretic Peptide Receptor-A Gene Transcription*

Cardiac hormones atrial and brain natriuretic peptides activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which plays a critical role in reduction of blood pressure and blood volume. Currently, the mechanisms responsible for regulating the Npr1 gene (coding for GC-A/NPRA) transcription are not well understood. The present study was conducted to examine the interactive roles of all-trans retinoic acid (ATRA), Ets-1, Sp1, and histone acetylation on the transcriptional regulation and function of the Npr1 gene. Deletion analysis of the Npr1 promoter and luciferase assays showed that ATRA enhanced a 16-fold Npr1 promoter activity and greatly stimulated guanylyl cyclase (GC) activity of the receptor protein in both atrial natriuretic peptide (ANP)-dependent and -independent manner. As confirmed by gel shift and chromatin immunoprecipitation assays, ATRA enhanced the binding of both Ets-1 and Sp1 to the Npr1 promoter. The retinoic acid receptor α (RARα) was recruited by Ets-1 and Sp1 to form a transcriptional activator complex with their binding sites in the Npr1 promoter. Interestingly, ATRA also increased the acetylation of histones H3 and H4 and enhanced their recruitment to Ets-1 and Sp1 binding sites within the Npr1 promoter. Collectively, the present results demonstrate that ATRA regulates Npr1 gene transcription and GC activity of the receptor by involving the interactive actions of Ets-1, Sp1, and histone acetylation.

Cardiac hormones atrial and brain natriuretic peptides activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/ NPRA), which plays a critical role in reduction of blood pressure and blood volume. Currently, the mechanisms responsible for regulating the Npr1 gene (coding for GC-A/NPRA) transcription are not well understood. The present study was conducted to examine the interactive roles of all-trans retinoic acid (ATRA), Ets-1, Sp1, and histone acetylation on the transcriptional regulation and function of the Npr1 gene. Deletion analysis of the Npr1 promoter and luciferase assays showed that ATRA enhanced a 16-fold Npr1 promoter activity and greatly stimulated guanylyl cyclase (GC) activity of the receptor protein in both atrial natriuretic peptide (ANP)-dependent and -independent manner. As confirmed by gel shift and chromatin immunoprecipitation assays, ATRA enhanced the binding of both Ets-1 and Sp1 to the Npr1 promoter. The retinoic acid receptor ␣ (RAR␣) was recruited by Ets-1 and Sp1 to form a transcriptional activator complex with their binding sites in the Npr1 promoter. Interestingly, ATRA also increased the acetylation of histones H3 and H4 and enhanced their recruitment to Ets-1 and Sp1 binding sites within the Npr1 promoter. Collectively, the present results demonstrate that ATRA regulates Npr1 gene transcription and GC activity of the receptor by involving the interactive actions of Ets-1, Sp1, and histone acetylation.
Atrial natriuretic peptide (ANP) 2 is a circulatory hormone, which plays a pivotal role in the regulation of sodium excretion, fluid volume, steroidogenesis, and cell proliferation, important factors in the control of blood pressure and blood volume (1)(2)(3)(4). One of the principal loci involved in the regulatory actions of ANP is the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which produces the intracellular second messenger cGMP, thus plays a central role in the pathophysiology of hypertension and cardiovascular disorders (4 -7). The signaling of ANP/cGMP through its downstream effector proteins, including cGMP-dependent protein kinases, phosphodiesterases, and cyclic nucleotide-gated ion channels, mediates the cellular effects of NPRA (4,8). Gene-targeting and expression studies of Npr1 (coding for GC-A/NPRA) have identified the hallmark significance of this receptor in protecting against renal and cardiac pathophysiological conditions such as inhibiting the cardiac hypertrophic growth and fibrosis, extracellular matrix remodeling, and cell proliferation (9 -13). Earlier studies have demonstrated a significant association of Npr1 gene variants with hypertensive family history, left ventricular mass index, and left ventricular septal wall thickness in human essential hypertension (14,15). It has also been shown that a longer thymine adenine repeat unit in spontaneously hypertensive rats regulates the transcription of the Npr1 gene, thus affecting diastolic blood pressure (16). Little is known about transcriptional regulation of the Npr1 gene, but the activity and expression of NPRA, assessed primarily through ANP-stimulated cGMP accumulation, are mediated by factors, including auto-regulation involving natriuretic peptides and other hormones (17)(18)(19)(20)(21)(22)(23)(24).
All-trans retinoic acid (ATRA), a biologically active vitamin A metabolite, plays an important role in a wide range of biological processes, including vision, cardiovascular events, neoplasia, embryonic development, and cellular differentiation (25,26). It has been suggested that chronic ATRA treatment prevents hypertrophy of intramyocardial and intrarenal arteries and ventricular fibrosis during the development of hypertension (27). Antihypertrophic effect of retinoic acid-mediated signaling has been demonstrated in myocardial cells in response to angiotensin II, endothelin-1, and phenylephrine (28,29). Retinoic acid functions by binding to nuclear receptor proteins, the retinoic acid receptor (RAR), and the retinoid X receptor (RXR) family, which are expressed in a variety of cell types and act as transcription factors upon ligand binding (30,31). Previous studies have shown that retinoic acid receptors also interact with other transcription factors to mediate gene expression (32,33). However, the mechanisms by which retinoic acid modulates these wide range of biological actions are not yet clearly understood.
Little is known about transcriptional regulation of the Npr1 gene and the processes that control Npr1 gene transcription, important for understanding the biological functions of GC-A/ NPRA. The glomerular mesangial cells are an attractive model for investigating a potential interactive role of ANP and ATRA in the regulation of GC-A/NPRA because these cells contain functional receptors for both of these hormones (34 -36). Moreover, it has been reported that the ANP/NPRA signaling cascade is the predominant mechanism mediating the natriuretic, diuretic, and renal hemodynamic responses to acute blood volume expansion (9,11). In the present study, we examined the significance of retinoic acid signaling, which involves Ets-1, Sp1, and histone acetylation in the regulation and functional expression of the Npr1 gene, an important member of GC receptor family, which plays a critical role in the pathophysiology of hypertension and cardiovascular events in disease states.
Plasmids and Promoter Constructs-The promoter-luciferase reporter constructs were generated by cloning PCR-amplified DNA fragments of various lengths of the murine Npr1 gene promoter (37). The cloning of various deletion constructs of the Npr1 promoter upstream of the promoter-less firefly luciferase gene in the pGL3-basic vector has been described earlier (17,38).
Cell Culture and Luciferase Assay-Mouse mesangial cells were isolated and cultured as described previously (35). Cells were grown in DMEM supplemented with 10% FCS and ITS. The cultures were maintained at 37°C in an atmosphere of 5% CO 2 and 95% O 2 , and experiments were performed using cells between 4 to 15 passages. The cells were transfected using Lipofectamine 2000 reagent with 1 g of promoter-reporter con-struct and 0.3 g of pRL-TK, which was used as an internal transfection control. After treatment with ATRA and TTNPB, cells were lysed, and luciferase activity was measured as described previously (38). The results were normalized for the transfection efficiency as relative to light units per Renilla luciferase activity. In overexpression experiments, cells were transfected with expression vectors for RAR␣, RXR␣, Ets-1, or Sp1, and the total DNA content was equalized by inclusion of empty vector. For treatment with ATRA, 24 h after transfection, cells were incubated for 12 h in DMEM containing 0.1% BSA and then further stimulated with increasing concentrations of ATRA or 0.1% dimethyl sulfoxide vehicle (control) for 48 h.
Real-time RT-PCR Assay-Cells were treated with ATRA for 48 h, and total RNA was extracted using an RNeasy mini-kit (Qiagen). First-strand cDNA was synthesized from 1 g of total RNA in a final volume of 20 l using the RT 2 First Strand kit (SABiosciences). Real-time RT-PCR was performed using the Mx3000P real-time PCR system, and data were analyzed with MxPro software (Stratagene, La Jolla, CA). All primers were purchased from SABiosciences. PCR amplifications (in triplicates) were carried out in a 25-l reaction volume using RT 2 real-time TM SYBR Green/ROX PCR Master Mix. The reaction conditions were: 95°C for 10 min; followed by 45 cycles at 95°C for 15 s, and 60°C for 1 min; followed by 1 cycle at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s for the dissociation curve. To monitor assay reproducibility, ␤-actin was amplified from all samples on each plate as a housekeeping gene to normalize expression levels of the Npr1 gene between different samples. The reaction mixture without template cDNA was used as negative controls. Threshold cycle numbers (C T ) were determined with MxPro quantitative PCR software and transformed using the ⌬C T comparative method. Npr1 gene expression value was normalized to expression values of ␤-actin (endogenous control) within each sample. The amount of the Npr1 gene, normalized to ␤-actin and relative to a control, was determined by the comparative Ct method (⌬⌬C T ). In brief, the ⌬C T value was determined by subtracting the average ␤-actin value from the average Npr1 gene value in the same sample. The calculation of ⌬⌬C T involves subtraction of the ⌬C T of the control from the experimental sample. The fold change in Npr1 gene expression was calculated from the 2 (Ϫ⌬⌬CT) . After PCR amplification, a melting curve of each amplicon was determined to verify its accuracy.
Whole Cell Lysate Preparation and Immunoblot Assay-Forty-eight h after treatment with ATRA, cells were lysed, and whole cell lysate was prepared essentially as described earlier (38). The protein concentration of the lysate was estimated using a Bradford protein detection kit (Bio-Rad). Whole cell lysate (50 -80 g) from each sample was mixed with sample loading buffer and separated by using 10% SDS-PAGE. Proteins were electrotransferred onto a polyvinylidene fluoride membrane, which was blocked with 1ϫ Tris-buffered saline-Tween 20 (TBST) containing 5% fat-free milk for 2 h at room temperature and then incubated overnight at 4°C in TBST containing 3% fat-free milk with primary antibodies (1:500 dilution). The membrane was treated with corresponding secondary anti-rabbit, anti-mouse, or anti-chicken horseradish peroxidase-conjugated antibodies (1:5000 dilutions). Protein bands were visualized by enhanced chemiluminescence plus detection system.
Histone Purification-Histone was purified from ATRAtreated cells by using a histone purification mini kit according to the manufacturer's protocols (Active Motif). Briefly, after washing with serum-free medium, cells were scraped in icecold extraction buffer and incubated overnight on a rotating platform at 4°C. Cell extracts were centrifuged at maximum speed for 5 min at 4°C. Supernatant containing the crude histone was transferred to a new tube and neutralized with onefourth volume of 5ϫ neutralization buffer. Crude histone was added on the equilibrated spin column and centrifuged at 500 ϫ g for 3 min at 4°C. The column was washed 3ϫ with wash buffer, and the core histone was eluted by using histone elution buffer. Histone proteins were desalted using Zeba spin columns (Thermo Fisher).
Electrophoretic Mobility Shift Assay-Nuclear extract was prepared from ATRA-treated and untreated cells as described previously (39). EMSA was performed as described previously (40). Briefly, nuclear extract (1.5-2 g of protein) in binding buffer was incubated on ice for 5 min in a total volume of 20 l before addition of the biotin-labeled probe of Ets-1A (40 fmol) or Ets-1B (20 fmol). The reaction for EMSA was allowed to incubate for an additional 25 min at room temperature. In antibody supershift experiments, the nuclear extracts were preincubated with anti-Ets-1 polyclonal antibody for 40 min. Protein⅐DNA complexes were separated on nondenaturing polyacrylamide gel and observed using the LightShift chemiluminescent kit (Pierce).
ChIP and Sequential ChIP-Chromatin immunoprecipitation was performed using the ChIP-IT Express kit (Active Motif) as described previously (41). Briefly, cells (1.5 ϫ 10 7 ) were cross-linked in 1% formaldehyde for 10 min at 22°C, and the reaction was quenched with 0.1 M glycine. Cells were scraped, resuspended in 1 ml of lysis buffer on ice and homogenized with a dounce homogenizer. The homogenate was centrifuged at 5000 rpm for 10 min at 4°C to pellet the nuclei. The pellet was resuspended in 1 ml of digestion buffer and 50 l of enzymatic shearing mixture and incubated at 37°C for 10 min. The reaction was stopped by adding 20 l of 0.5 M EDTA followed by chilling on ice for 10 min. Sheared DNA was centrifuged at 13,000 rpm at 4°C for 10 min, and the supernatant was collected. Ten percent of supernatant was saved as input DNA and processed for further use as a positive control. Immunoprecipitation was performed using protein G magnetic beads and 5 g of antibody of Ets-1, Sp1, RAR␣, Ac H3, Ac H4, or control IgG at 4°C with overnight rotation. Beads were pelleted and washed sequentially once with ChIP buffer 1 and twice with ChIP buffer 2. The bound protein was eluted from the beads by incubation with 10 mM of dithiothreitol at 37°C for 30 min and again immunoprecipitated with a second antibody overnight at 4°C. After washing the magnetic beads, bound protein was eluted by gentle rotation for 15 min in elution buffer AM2 at 22°C. Cross-linking of the protein⅐DNA complex was reversed at 65°C overnight to release DNA. Immunoprecipitated DNA was sequentially treated with RNase A and proteinase K and then purified. The Npr1 promoter region containing Ets-1 and Sp1 binding sites was PCR-amplified using purified DNA as a template and the forward (5Ј-ctctcttgtcgccgaatctg-3Ј) and reverse (5Ј-gtggagagcgagagaacgaga-3Ј) primers. For quantita-tive ChIP assay, real-time PCR was performed with RT 2 realtime TM SYBR Green/ROX PCR Master Mix (SABiosciences) according to the supplier's instructions. The reaction conditions were as follows: 95°C for 10 min; followed by 40 cycles at 95°C for 15 s and 60°C for 1 min; followed by 1 cycle at 95°C for 1 min, 55°C for 30 s, and 95°C for 30 s for the dissociation curve. For ChIP quantitative PCR, 1% input was utilized, and its value was adjusted to 100% for normalization of the results. Each ChIP DNA threshold cycle number (C T ) was normalized to the input DNA fraction C T value to account for differences in chromatin sample preparation. Percent input was calculated as 2 ((⌬CT(normalized ChIP)) .
Plasma Membrane Preparation and Guanylyl Cyclase Activity Assay-The plasma membranes were prepared by suspending cell pellet in 5 volumes of sodium phosphate buffer (10 mM, pH 7.4) containing 250 mM sucrose, 150 mM NaCl, 1 mM PMSF, 5 mM benzamidine, 5 mM EDTA, and 10 g/ml each of leupeptin and aprotinin as described previously (42). Briefly, cells were homogenized and centrifuged at 400 ϫ g for 10 min at 4°C, and the supernatant collected was recentrifuged at 80,000 ϫ g for 1 h at 4°C. The resultant supernatant was discarded, and the pellet was resuspended in 1 ml of HEPES buffer (50 mM, pH 7.4) containing 150 mM NaCl, 1 mM PMSF, 5 mM benzamidine, 5 mM EDTA, and 10 g/ml each of leupeptin and aprotinin and centrifuged at 80,000 ϫ g for 1 h at 4°C. The final pellet was suspended in 200 l of HEPES buffer (pH 7.4). GC activity was assayed as described by Leitman et al. (43) with modifications (42). A 50-g aliquot of plasma membrane was added to 100 l of GC assay buffer containing Tris-Cl buffer (50 mM, pH 7.6), 4 mM MnCl 2 , 2 mM 3-isobutyl-1-methylxanthine, 1 mM BSA, 5 units of creatinine phosphokinase, 7.5 mM creatine phosphate, 0.5 mM GTP, and 0.1 M ANP. The samples were incubated in a water bath at 37°C for 10 min. Reaction was stopped by adding 900 l of 55 mM sodium acetate (pH 6.2), and sample tubes were placed in boiling water bath for 3 min and then on ice for 15 min to stop the reaction. Samples were centrifuged at 13,000 ϫ g for 5 min, supernatant was collected, and the generated cGMP was determined.
cGMP Assay-Twenty-four hours after plating, cells were treated with ATRA for another 24 h and stimulated with ANP at 37°C for 20 min in the presence of 0.2 mM 3-isobutyl-1methylxanthine. Cells were washed three times with PBS and scraped into 0.5 N HCl. Cell suspension was subjected to five cycles of freeze and thaw and then centrifuged at 10,000 rpm for 15 min. The supernatant thus collected was used for the cGMP assay using the direct cGMP correlate-EIA kit according to the manufacturer's protocol.
Transfection of Small Inhibitory RNA-Cells were cultured to 70 -80% confluence in 10% FBS-supplemented antibiotic-free DMEM with ITS and transfected with RAR␣, RXR␣, Ets-1, or Sp1 siRNA (a pool of three target-specific 20 -25-nucleotide sequence siRNAs) using Lipofectamine 2000 reagent. A nontargeting 20 -25-nucleotide sequence siRNA was used as a negative control. Four hours after transfection, fresh medium was added to the plates, and after 24 h cells were treated with ATRA, and luciferase activity was determined.
Statistical Analysis-The results are expressed as mean Ϯ S.E. The statistical significance was evaluated by one-way anal-ysis of variance, followed by Dunnett's multiple comparison tests using PRISM computer software (GraphPad Software, San Diego, CA). A p value of Ͻ 0.05 was considered significant.

RESULTS
The results of the deletion analysis of the Npr1 promoter and luciferase assays showed that the region Ϫ356 to ϩ55 from the transcription start site exhibited a 16-fold increase in luciferase activity in response to ATRA treatment in a dose-dependent manner (Fig. 1, A and B). The incubation of cells with stilbenebased retinoid TTNPB, a RAR-specific agonist, also mimicked ATRA-effect and enhanced Npr1 promoter activity by 6.8-fold (Fig. 1C). There was a 7-fold induction in Npr1 mRNA levels and a 4-fold increase in NPRA protein expression in cells

TABLE 1 Effect of ATRA on intracellular accumulation of cGMP levels and GC activity of GC-A/NPRA
Cells were treated with ATRA (0.5 M) and induced with or without 0.1 M ANP. Intracellular accumulation of cGMP was quantitated by ELISA. GC activity in the plasma membrane preparations of ATRA-treated cells was measured as described under "Experimental Procedures." Values represent the mean Ϯ S.E. of three independent experiments in triplicate. UT, untreated. treated with ATRA compared with untreated cells (Fig. 1, D and  E). To examine whether an increase in NPRA protein expression by ATRA was due to de novo synthesis, the effect of cycloheximide, a protein synthesis inhibitor, was examined. Simultaneous treatment of ATRA and cycloheximide showed that RA-induced expression of NPRA was blocked by cycloheximide (Fig. 1F). The treatment of cells with ATRA and ANP showed an increase in intracellular accumulation of cGMP by 50-fold, and plasma membrane preparations of cells treated with ATRA and ANP exhibited a 90-fold increase in GC activity compared with untreated control cells (Table 1). Cells treated with ATRA and transfected with RXR␣, RAR␣, or both receptors showed an increase in luciferase activity of the Npr1 promoter by 15-, 24-, and 45-fold, respectively, compared with untreated control cells (Fig. 2A). The overexpression of RAR␣ and RXR␣ was confirmed in transfected cells compared with untransfected cells (Fig. 2B). Knockdown of RAR␣ or/and RXR␣ by siRNA reduced the ATRA-mediated activation of Npr1 promoter activity by 61-90% (Fig. 2C). There was a significant reduction in RAR␣ and RXR␣ protein expression in siRNA-transfected cells compared with untransfected control cells (Fig. 2D). Treatment of cells with Ro 41-5253, an RAR␣-specific antagonist, decreased ATRA-induced Npr1 promoter activity by 80% and mRNA levels by 73% (Fig. 2, E  and F).
A schematic map of Npr1 promoter region Ϫ356 to ϩ55 containing Sp1 and Ets-1 binding sites is shown in Fig. 3A. To examine the cooperative interaction between Ets-1 and ATRA, cells were cotransfected with Npr1 promoter and Ets-1 expression plasmid. Treatment with ATRA increased Npr1 promoter activity by 27-fold in Ets-1-transfected cells; however, mutation of Ets-1 binding sites in the Npr1 promoter abolished ATRAmediated increase in luciferase activity (Fig. 3B). The overexpression of Ets-1 was confirmed in transfected cells compared with untransfected cells (Fig. 3C). Knockdown of endogenous Ets-1 by siRNA reduced ATRA-induced Npr1 promoter activity by 78% (Fig. 3D). Similarly, Ets-1 protein expression was also markedly reduced in siRNA-transfected cells (Fig. 3E). ATRA increased the expression of endogenous Ets-1 by almost 4.5-fold, in a dose-dependent manner, compared with untreated cells (Fig. 4A). Treatment with Ro 41-5253 completely blocked the ATRA-induced Ets-1 protein expression in a dose-dependent manner (Fig. 4B). In gel shift assay, the incubation of nuclear extract with Ets-1A and Ets-1B oligonucleotides showed formation of specific nucleoprotein complexes (Fig. 4C, lanes 2 and 6). An enhanced binding was observed with nuclear extract prepared from ATRA-induced cells (Fig. 4C,  lanes 3 and 7). The specificity of the protein⅐DNA complex was confirmed by antibody supershift assays (Fig. 4C, lanes 4 and 8). In vivo quantitative ChIP assay showed that in ATRA-stimulated cells, Ets-1 occupancy of the Npr1 promoter was greatly enhanced compared with untreated cells (Fig. 4D).
To examine the role of Sp1 in ATRA-mediated Npr1 promoter activation, cells were cotransfected with the Npr1 pro- moter and Sp1 expression plasmid. Treatment with ATRA enhanced Npr1 promoter activity by 21-fold in transfected cells compared with 12.5-fold in untransfected cells (Fig. 5A). Overexpression of Sp1 was confirmed in transfected cells (Fig. 5B). In the ATRA-treated cells, quantitative ChIP assay showed an enhanced binding of endogenous Sp1 to the Npr1 promoter containing Sp1 binding sites (Fig. 5C). On the other hand, a marked decrease in Sp1 protein expression was observed in siRNA-transfected cells compared with untransfected control cells (Fig. 5D, upper panel). There was an almost 60% reduction in ATRA-induced Npr1 promoter activity in Sp1 siRNA-transfected cells (Fig. 5D, lower panel). The treatment of cells with mithramycin A, a specific inhibitor that interferes with Sp1 binding sites, attenuated the ATRA-induced Npr1 promoter activity by 75% (Fig. 5E).
Knockdown of Ets-1 blocked RAR␣and RXR␣-mediated induction of Npr1 gene transcription in ATRA-treated cells by almost 60 -70% (Fig. 6A). Similarly, the ablation of endogenous Sp1 protein expression reduced the RAR␣-mediated ATRA effect on Npr1 promoter activity by 70%. Simultaneous knockdown of Ets-1 and Sp1 protein expression dramatically reduced RAR␣and RXR␣mediated Npr1 gene transcription by 90% in ATRA-treated cells (Fig.  6A). To delineate the simultaneous interaction of RAR␣ with Ets-1 or Sp1 in the Npr1 promoter, the sequential ChIP assay was performed. As shown in Fig. 6B (upper  panel), RAR␣ was coimmunoprecipitated with anti-Ets-1 antibody, indicating that RAR␣ forms a complex with Ets-1. In ATRA-treated cells, a higher occupancy of RAR␣ and Ets-1 was observed, whereas knockdown of endogenous Ets-1 significantly attenuated binding of the RAR␣⅐Ets-1 complex to the Npr1 promoter by 85% (Fig. 6B,  middle panel). Presence of RAR␣ in the RAR␣⅐Ets-1 complex was confirmed by direct RAR␣ immunoprecipitation, which showed a 2.5-fold higher occupancy in ATRA-treated cells and a markedly lower occupancy in Ets-1 knockdown cells (Fig.  6B, lower panel). As shown in Fig.  6C (upper panel), RAR␣ was also detected in anti-Sp1 immunoprecipitates confirming that RAR␣ forms a complex with Sp1. Treatment of cells with ATRA showed higher recruitment of RAR␣, which was markedly attenuated by 70% in Sp1 knockdown cells (Fig. 6C, middle panel). In ATRA-treated cells, the direct immunoprecipitation of RAR␣ showed a 3-fold increase in recruitment of RAR␣ to the Npr1 promoter containing Ets-1 and Sp1 sites, and knockdown of endogenous Sp1 markedly lowered its occupancy by 84% (Fig. 6C, lower panel). To examine the ATRAmediated increases in the recruitment of the RAR␣⅐Ets-1⅐Sp1 complex to the Npr1 promoter, the effect of ATRA on histone acetylation was also determined. There was an increased acetylation of histones H3 and H4 in ATRA-treated cells (Fig. 7A). Furthermore, treatment with ATRA showed an increased occupancy of acetylated histones H3 and H4 to the Npr1 promoter containing Ets-1/Sp1 sites (Fig. 7, B and C).

DISCUSSION
The results of the present study demonstrate that ATRA stimulates Npr1 gene transcription and expression in association with Ets-1, Sp1, and histone acetylation. ATRA signifi- cantly induced Npr1 promoter activity in Ets-1-transfected cells, whereas knockdown of Ets-1 dramatically reduced ATRA effects on Npr1 gene transcription. Previously, it has been shown that RAR␣ binds to a functional RARE in the Ets-1 promoter and ATRA induces the expression of Ets-1 mRNA and protein levels (44,45). Consistent with those previous findings, the present result indicate that RAR␣ antagonist inhibited Ets-1 protein expression, thus confirming RAR␣-dependent activation of Ets-1. Similarly, ATRA significantly induced luciferase activity in Sp1-transfected cells. Knockdown of endogenous Sp1 by RNA interference assay or use of mithramycin A, that inhibits binding of Sp1 with its consensus sequence, significantly attenuated the effects of ATRA on Npr1 gene transcription. Data from quantitative ChIP assay showed that treatment with ATRA greatly increased the association of Sp1 with the Npr1 promoter. Previously, it has been shown that RAR␣ func-  tionally interacts with Sp1 to cooperatively activate transcription of interleukin-1␤ and monoamine oxidase B promoter (32,46). Our data show that functional interaction of RAR␣ with Ets-1 and Sp1 is an important mechanism to enhance Npr1 gene transcription. The present results from sequential ChIP assay demonstrate that RAR␣ indirectly associates with Ets-1/ Sp1 binding sites of native chromatin by forming a complex with Ets-1 and Sp1 to modulate Npr1 gene transcription.
Evidence suggests that ATRA treatment induces epigenetic modification at the target loci and regulates gene transcription by DNA demethylation and histone modifications, which include acetylation, methylation, and phosphorylation at the promoter level (47)(48)(49). As a consequence of histone acetylation and decompacted chromatin structure, transcription factors and the basal transcriptional machinery are recruited to the region and activate gene transcription (50 -52). Previous study has shown that an increased acetylation of histones H3 and H4 by retinoic acid regulates gene specific transcriptional changes during early embryonic stem cell differentiation (53). ATRA and histone deacetylase inhibitor have been shown to increase Sp1 binding and acetylation of histones H3 and H4 in the promoter of folate receptor (54). Those previous findings are consistent with our observation indicating that ATRA treatment enhances acetylation of histones H3 and H4 near Ets-1 and Sp1 binding sites in the Npr1 promoter and facilitates increased recruitment of Ets-1 and Sp1 to the Npr1 promoter. The proposed schematic of ATRA interaction with Ets-1 and Sp1 elements in the regulation of Npr1 gene transcription is presented in Fig. 8. The Npr1 promoter region Ϫ356 to ϩ55 contains Ets-1 and Sp1 binding elements; however, the consensus sequence of RARE was not identified by sequence analysis using TFSEARCH (version 1.3) for transcription factor binding sites (55). Therefore, in the absence of functional RAREs, both Ets-1 and Sp1 might serve as the potential targets for ATRA signaling cascade leading to enhanced Npr1 gene transcription. Our data provide a direct evidence for the ATRA-mediated increases in acetylation levels of histones H3 and H4 and enhanced binding of the RAR␣⅐Ets-1⅐Sp1 complex in the Npr1 promoter.
Interestingly, retinoic acid has been shown to exert its effect via nonclassical mechanisms by interacting with other general transcription factors, such as Sp1, activator protein-1, cyclic AMP response element-binding protein (CREB), and Kruppellike factor 4 (32, 33, 56 -58). A cross-talk between retinoic acid receptors and other transcription factors has been implicated as an important regulatory mechanism for gene transcription in various biological functions (32,56,59). Our present results provide the evidence that ATRA interacts with Ets-1 and Sp1 to regulate Npr1 gene transcription in mesangial cells. Ets-1 has been shown to be essential for normal development of mammalian kidneys and for maintenance of glomerular integrity (60,61). Earlier studies have shown that Ets-1 is essential for normal coronary and myocardial development (62). It is implicated that Ets-1 is critical in hematopoiesis and angiogenesis during the earlier stages of embryogenesis, and in later stages, it is important in organ formation and tissue remodeling in kidneys, liver, and vasculature (63). Sp1 has been suggested to regulate multiple housekeeping and growth-related genes, indicating a role in cell growth regulation (64,65). Earlier studies have shown that Sp1 regulates p27 and p21 expression, which play an important role in controlling cardiomyocyte and smooth mus-  The proposed diagram indicates that ATRA increases Npr1 gene transcription and expression in association of Ets-1, Sp1, histone acetylation. ATRA induces Ets-1 protein expression via its receptor RAR␣ and enhances Ets-1 and Sp1 binding to the Npr1 promoter. In the absence of functional RARE, RAR␣ associates with the Ets-1⅐Sp1 complex on the Npr1 promoter and mediates ATRA effects. Furthermore, ATRA acetylates histone H3 and H4 around the Ets-1 and Sp1 binding sites in the Npr1 promoter. Histone acetylation promotes localized unwinding of DNA and allows transcription factors to bind in the region. RA, retinoic acid. The closed upward arrow indicates an increased Ets-1 protein expression; the open upward arrow indicates an increased binding of Ets-1⅐Sp1⅐RAR␣ complex to the Npr1 gene promoter. cle cell hypertrophy (66,67). Recently, Sp1 has been shown to regulate renal protective effect against ischemia/reperfusion injury (68).
In summary, the present results provide direct evidence that ATRA signaling up-regulates Npr1 gene transcription and also stimulates the GC activity of GC-A/NPRA. Our results show that ATRA activates Npr1 gene transcription involving functional interaction of the RAR␣⅐Ets-1⅐Sp1 complex to the Npr1 promoter containing Ets-1/Sp1-binding sites. ATRA also mediates histone modification by increased acetylation of histones H3 and H4 and their recruitment to the Npr1 promoter. Taken together, the present study delineates the novel regulatory mechanisms and provides new insights into the hormonal regulation of Npr1 gene transcription and function, which are critical toward the understanding of the biological functions of GC-A/NPRA for possible therapeutic targets in hypertension and cardiovascular events.