ANP (atrial natriuretic peptide) exerts its biological effects by binding to GC (guanylate cyclase)-A/NPR (natriuretic peptide receptor)-A, which generates the second messenger cGMP. The molecular mechanism mediating Npr1 (coding for GC-A/NPRA) gene regulation and expression is not well understood. The objective of the present study was to elucidate the mechanism by which Ets-1 [Ets (E twenty-six) transformation-specific sequence] contributes to the regulation of Npr1 gene transcription and expression. Chromatin immunoprecipitation and gel-shift assays confirmed the in vivo and in vitro binding of Ets-1 to the Npr1 promoter. Overexpression of Ets-1 enhanced significantly Npr1 mRNA levels, protein expression, GC activity and ANP-stimulated intracellular accumulation of cGMP in transfected cells. Depletion of endogenous Ets-1 by siRNA (small interfering RNA) dramatically decreased promoter activity by 80%. Moreover, methylation of the Npr1 promoter region (−356 to +55) reduced significantly the promoter activity and hypermethylation around the Ets-1 binding sites directly reduced Ets-1 binding to the Npr1 promoter. Collectively, the present study demonstrates that Npr1 gene transcription and GC activity of the receptor are critically controlled by Ets-1 in target cells.
ANP (atrial natriuretic peptide) and BNP (brain natriuretic peptide) principally mediate natriuretic, diuretic, vasorelaxant and antimitogenic responses, largely directed at reducing blood pressure and maintaining fluid volume homoeostasis [1–3]. ANP and BNP bind specifically to GC (guanylate cyclase)-A/NPR (natriuretic peptide receptor)-A, which produces the intracellular second messenger cGMP in response to hormone binding [4–7]. Several studies with Npr1 (the gene coding for GC-A/NPRA) gene-disruption mouse models have revealed the hallmark significance of NPRA in lowering arterial pressure and protecting against renal and cardiac pathophysiological functions [8–11]. It has been reported previously that in Japanese individuals, genetic mutations in the human Npr1 gene promoter conferred increased susceptibility to essential hypertension and left ventricular hypertrophy . It has also been demonstrated that the human Npr1 deletion allele lacking eight nucleotides, which alters the binding sites of AP-2 (activator protein 2) and Zeste, leads to a decreased ability to bind the transcription factor AP-2 and decreased promoter activity compared with the wild-type allele.
Relatively little is known about the regulation of Npr1 gene expression in target cells. Previous studies have demonstrated that the functional interaction of NF-Y (nuclear factor-Y) with Sp1 (stimulating protein 1) is essential for the optimal transcription of the Npr1 gene in vascular smooth muscle cells . The complete genomic nucleotide sequence and promoter region analysis of the murine Npr1 gene indicated that the core transcriptional machinery of the TATA-less Npr1 promoter contains three potential Sp1-binding sites, one inverted CCAAT box, and several putative cis-acting motifs for known transcription factors, including Ets-1 [Ets (E twenty-six) transformation-specific sequence], suggesting that they have a function in regulating Npr1 gene transcription . Previous studies have shown that the basal promoter of Npr1 lies between the region −356 to +55 relative to the TSS (transcription start site) and its transcriptional activity is modulated by GATA-1 (GATA-binding protein 1), Ets-1 and LyF-1 transcription factors . Ets proteins activate or repress the expression of various genes, including c-fos, c-myc, jun B, p53 and bcl2 [16,17]. Ets-1 protein, which is expressed in a variety of cell types, including endothelial cells, mesangial cells, and vascular smooth muscle cells, regulates the transcription of several genes involved in angiogenesis and remodelling of the extracellular matrix [18,19]. Previous studies have shown that the Ets-1 protein is essential for normal coronary and myocardial development [17,20]. Ets-1 also plays a role in kidney development by activating a forkhead-related transcription factor detected during nephrogenesis .
Although Npr1 gene regulation is poorly understood, the activity and expression of NPRA, assessed primarily through ANP-stimulated cGMP accumulation, are regulated by various factors, including hormones, such as endothelin, glucocorticoids, growth factors, and certain physiological and pathophysiological conditions [22–24]. Ang II (angiotensin II) has been shown to repress Npr1 transcriptional activity by binding to its RE (response element), located in the promoter region −1346 to −916 bp from the TSS [25,26]. Previous studies have shown that NPRA expression is regulated by natriuretic peptides  as well as transcriptional repression by cGMP, which is mediated by a cGMP RE in the Npr1 promoter at position −1372 to −1354 bp from the TSS . Indeed, better understanding of the regulation of NPRA expression requires a more extensive functional characterization of its promoter region and the functional significance of the potential cis-elements in this region. The present study demonstrates that Ets-1 plays an integral role in the regulation of Npr1 transcription and GC activity of the receptor through its consensus binding sites present in the Npr1 gene promoter.
MATERIALS AND METHODS
The pGL3-Basic vector, pRL-TK and Dual Luciferase assay system were purchased from Promega. The plasmid isolation kit and RNeasy mini kit for total RNA isolation were obtained from Qiagen. Sequence-specific oligonucleotides were purchased from Midland Certified Reagent Company. Cell culture medium, fetal calf serum, ITS (insulin, transferrin and sodium selenite), Lipofectamine™ 2000, and Superscript One-Step RT-PCR (reverse transcription-PCR) kit were obtained from Invitrogen. PMSF, aprotinin and leupeptin were obtained from Sigma. The HhaI, HpaII and SssI methylases and HhaI, HpaII and BstUI restriction endonucleases were purchased from New England Biolabs. The LightShift Chemiluminescent EMSA (electrophoretic mobility-shift assay) kit was obtained from Pierce and the EZ ChIP (chromatin immunoprecipitation) kit was obtained from Upstate Biotechnology. The direct cyclic GMP correlate-EIA kit was purchased from Assay Designs. Anti-Ets-1 antibody (N-276), anti-rabbit IgG, Ets-1 siRNA (small interfering RNA), control siRNA and Protein A–agarose were purchased from Santa Cruz Biotechnology. Anti-NPRA antibody was obtained from Genway Biotech. The expression vectors for Ets-1 (pEVRF0-Ets-1) and the empty vector (pEVRF0) were kindly provided by Dr Paul Brindle (Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN, U.S.A.) . All other chemicals used were of molecular biology reagent grade.
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 upstream of the promoterless firefly luciferase gene in the pGL3-Basic vector. The cloning of various deletion constructs of the Npr1 promoter and constructs carrying mutations at Ets-1A, Ets-1B or at both sites have been described previously .
Cell transfection and luciferase assay
MMCs (mouse mesangial cells) were isolated and cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) fetal calf serum and ITS as described previously . The MMCs were seeded in 24-well plates and experiments were performed using cells between 4 and 18 passages. Cloned mouse Leydig tumour (MA-10) cells were cultured in modified Waymouth's medium supplemented with 15% (v/v) horse serum as described previously . The cultures were maintained at 37°C in an atmosphere of 5% CO2/95% O2. The cells were transfected using Lipofectamine™ 2000 reagent with 1 μg of the promoter–reporter construct and 300 ng of pRL-TK (containing the Renilla luciferase gene downstream of the thymidine kinase promoter, which was used as an internal transfection control. For co-transfection experiments, expression plasmids of varying concentrations were transfected along with the promoter–reporter construct. Cells were lysed after 48 h and the lysate was used to measure firefly and Renilla luciferase activities using the Promega Dual Luciferase assay kit using a TD 20/20 luminometer (Turner Designs). The results were normalized for the transfection efficiency relative to light units for Renilla luciferase activity. In Ets-1-overexpression experiments, cells were transfected with the Ets-1 expression vector (pEVRF0-Ets-1) or its empty vector (pEVRF0). The total DNA content was equalized by inclusion of the pEVRF0 plasmid. To examine the transfection efficiency, cells were transfected with a pCMV β-galactosidase control plasmid and transfection efficiency was assessed by using the In Situ β-galactosidase staining kit (Stratagene) according to manufacturer's protocol. In MMCs and MA-10 cells, the transfection efficiency was found to be 85% and 90% respectively using Lipofectamine™ 2000.
Cells were transfected with the appropriate expression vector, and at 48 h after transfection, total RNA was extracted using the RNeasy mini kit (Qiagen). Total RNA (1 μg) was reverse-transcribed using the Superscript One-Step RT-PCR kit with the platinum Taq system. The sequences of the primers used for amplification of NPRA and β-actin, as well as PCR conditions, were as described previously . The amplified PCR product increased linearly up to 40 cycles (results not shown). Control experiments were performed with RNA samples but without reverse transcriptase. The specific primers for β-actin were included in the PCR as an internal control. The expected sizes of the amplified NPRA and β-actin PCR products are 456 and 256 bp respectively. After amplification, 15 μl of each PCR mixture was subjected to agarose-gel electrophoresis (1.5% gels) with ethidium bromide (0.5 μg/ml). The gel was digitized and the signal intensities of the corresponding bands were quantified using an Alpha Imaging System (Alpha Innotech).
Whole cell lysate preparation and immunoblot assay
Cells were transfected, and at 48 h after transfection, cells were lysed and whole cell lysates were prepared essentially as described previously . The protein concentration of the lysates was estimated using a Bradford protein detection kit (Bio-Rad). Whole cell lysates (50–80 μg) from each sample were mixed with sample loading buffer and separated by SDS/PAGE (10% gels). Proteins were electrotransferred on to a PVDF membrane. The membrane was blocked with 1×TBST (Tris-buffered saline and 0.1% Tween 20) containing 5% (w/v) non-fat dried skimmed milk powder for 2 h at room temperature (22°C) and incubated overnight at 4°C in TBST containing 3% (w/v) non-fat dried skimmed milk powder with primary antibodies (1:1000 dilution). The membrane was then treated with the corresponding HRP (horseradish peroxidase)-conjugated anti-rabbit, anti-mouse or anti-chicken secondary antibodies (1:5000 dilution). Protein bands were visualized by the ECL (enhanced chemiluminescence) Plus detection system (Amersham Biosciences).
Nuclear extracts were prepared using the method of Dignam et al. . Protein–DNA complexes were detected using 5′-biotin end-labelled double-stranded DNA probes prepared by annealing complementary oligonucleotides. The forward sequences of the wild-type oligonucleotides for Ets-1A and Ets-1B were 5′-CCGCCCGCCTCCGGAACGGCCGGAG-3′ and 5′-TGGGCCAGCCGGACGCCCCTTCTG-3′ respectively. The respective forward sequences for the mutant Ets-1A and Ets-1B binding sites are underlined as follows: 5′-CCCGCCCGCCTCATTCACGGCCGG-3′ and 5′-GGGCCAGCATTCCGCCCCTTCTG-3′. Briefly, nuclear extracts from MMCs (1.5 μg of protein) in binding buffer were incubated on ice for 5 min in a total volume of 20 μl before the addition of the biotin-labelled probe for Ets-1A (40 fmol) or Ets-1B (20 fmol). The reaction was allowed to incubate for an additional 25 min at room temperature. In competition experiments, the nuclear extract was pre-incubated with a 200-fold molar excess of unlabelled double-stranded wild-type or mutant oligonucleotides for 20 min on ice. Protein–DNA complexes were then separated by non-denaturing PAGE and observed using the LightShift Chemiluminescent EMSA kit.
The ChIP assay was performed using the EZ ChIP kit according to the manufacturer's instructions (Upstate Biotechnology). Briefly, at 48 h after transfection, 106 cells were cross-linked in 1% formaldehyde for 10 min at room temperature and the reaction was quenched with 0.1 M glycine. Cells were scraped, pelleted and resuspended in 300 μl of SDS lysis buffer on ice, and then sonicated three times for 10 s each at 30% input, producing fragments of between 500 and 1000 bp in size. The suspension was centrifuged at 10000 g for 10 min at 4°C. The supernatant was diluted 10-fold in ChIP dilution buffer. Immunoprecipitation was performed with Protein G–agarose and 5 μg of the Ets-1 antibody or control IgG. The chromatin solution was pre-cleared by adding Protein G–agarose for 2 h at 4°C and incubated with antibodies at 4°C overnight with rotation. The DNA–protein–antibody complexes were captured on Protein G–agarose beads by incubating for 1 h at 4°C. Beads were pelleted by centrifugation at 4000 g for 1 min and washed sequentially for 5 min each with low-salt buffer, high-salt buffer, lithium chloride wash buffer and 1×Tris-EDTA. The bound protein was eluted twice from the beads by gently rotating for 15 min at room temperature in ChIP elution buffer. After elution of protein–DNA complexes from the beads, cross-linking was reversed at 65°C overnight to release the DNA. The immunoprecipitated DNA was sequentially treated with 10 μg of RNase A and 10 μg of proteinase K and then purified. PCR of the Npr1 promoter region containing the Ets-1-binding sites was carried out using purified DNA as a template. For PCR amplification, the forward primer used was 5′-CTCTCTTGTCGCCGAATCTG-3′ and the reverse primer used was 5′-TCTCGTTCTCTCGCTCTCCAC-3′.
Cells were transfected, and at 48 h after transfection, cells were treated with ANP at 37°C for 20 min in the presence of 0.2 mM 3-isobutyl-1-methylxanthine. Cells were washed three times with PBS and scraped into 0.5 M HCl. Cell suspensions were subjected to five cycles of freezing and thawing, and then centrifuged at 10000 g for 15 min. The supernatant was collected and used for the cGMP assay using the direct cyclic GMP correlate-EIA kit according to the manufacturer's instructions (Assay Designs).
Plasma membrane preparation and GC activity assay
The plasma membrane preparations were prepared by suspending a cell pellet in 5 volumes of 10 mM sodium phosphate buffer (pH 7.4) containing 250 mM sucrose, 150 mM NaCl, 1 mM PMSF, 5 mM benzamidine, 5 mM EDTA and 10 μg/ml each of leupeptin and aprotinin, as described previously . Briefly, cells were homogenized and centrifuged at 400 g for 10 min at 4°C and the supernatant collected was recentrifuged at 30000 rev./min for 1 h at 4°C (70.1 Ti rotor, Beckman). The resultant supernatant was discarded and the pellet was resuspended in 1 ml of 50 mM Hepes buffer (pH 7.4) containing 150 mM NaCl, 1 mM PMSF, 5 mM benzamidine, 5 mM EDTA and 10 μg/ml each of leupeptin and aprotinin, and centrifuged at 30000 rev./min for 1 h at 4°C (70.1 Ti rotor, Beckman). The final pellet was suspended in 200 μl of Hepes buffer (pH 7.4). GC activity was assayed using the method of Leitman et al. , with some modifications . Plasma membrane (50 μg) was added to 100 μl of the GC assay buffer containing 50 mM Tris/HCl buffer (pH 7.6), 4 mM MnCl2, 2 mM 3-isobutyl-1-methylxanthine, 1 mM BSA, 5 units of creatinine phosphokinase, 7.5 mM creatine phosphate, 0.5 mM GTP and 1 μM ANP. The samples were incubated in a water bath at 37°C for 10 min. The reaction was stopped by adding 900 μl of 55 mM sodium acetate (pH 6.2) and sample tubes were placed in a boiling water bath for 3 min and then on ice for 15 min to stop the reaction. Samples were then centrifuged at 13000 g for 5 min, the supernatant was collected, and the quantity of cGMP was determined.
Transfection of siRNA
Cells were cultured to 70%–80% confluence in 10% (v/v) fetal calf serum-supplemented antibiotic-free DMEM with ITS and transfected with Ets-1 siRNA (a pool of three target-specific siRNAs with a sequence of 20–25 nucleotides) using Lipofectamine™ 2000. A non-targeting 20–25 nucleotide sequence siRNA was used as a negative control. At 4 h after transfection, fresh medium was added to the plates and, after 24 h, the medium was replaced. After 48 h, cells were lysed and the clear supernatant was used to measure firefly and Renilla luciferase activities.
Cells were plated on to chamber slides and were transiently transfected with the Ets-1 expression vector or siRNA using Lipofectamine™ 2000. At 24 h after transfection, cells were fixed for 10 min at room temperature in 4% (w/v) paraformaldehyde in PBS, washed in PBS, permeabilized with PBS with 0.2% Triton X-100 for 5 min, and blocked in 2% (w/v) BSA in PBS with 0.1% Tween 20 for 10 min. Cells were then incubated with a polyclonal anti-Ets-1 antibody (1:200 dilution) at 4°C overnight, washed three times for 5 min with PBS with 0.1% Tween 20 and then incubated with an FITC-conjugated anti-rabbit secondary antibody (1:400 dilution) at room temperature for 1 h. Slides were washed three times for 5 min each with PBS and 0.1% Tween 20 before coverslides were mounted on to the slides, using Vectashield mounting medium with DAPI (4′,6-diamidino-2-phenylindole) to visualize nuclei. Immunofluorescence was measured using an Olympus BX51TRF microscope at ×40 magnification. An integrated Magnafire SP Digital Firewire Camera System was used for image processing.
In vitro methylation of the Npr1 promoter
The plasmids pGL3-Basic and the −356/+55 Npr1 promoter construct were methylated in vitro by HhaI, HpaII and SssI methyltransferases following the manufacturer's instructions (New England Biolabs). The completeness of methylation was checked by measuring the extent of protection from digestion by the restriction enzymes HhaI, HpaII and BstUI respectively. Transfection of the plasmids was performed as described above. The wild-type Ets-1A and Ets-1B oligonucleotides used for EMSA were also methylated in vitro by SssI methylase as described above. For the unmethylated control, pGL3-Basic, the −356/+55 Npr1 promoter construct and the Ets-1A and Ets-1B oligonucleotides were mixed with all components required for in vitro methylation except methylases.
Results are means±S.E.M. The statistical significance was evaluated by performing a one-way ANOVA, followed by Dunnett's multiple comparison tests using GraphPad Prism software (GraphPad). A P value of <0.05 was considered to be significant.
The Npr1 promoter construct (−356/+55) containing Ets-1-binding sites exhibited a 60- to 140-fold induction in luciferase activity compared with the pGL3-Basic plasmid when transfected into MMC and MA-10 cells respectively (Figure 1A). Deletion of the region −46 to +55 (containing Ets-1-binding sites) in the construct −356/−46 decreased the promoter activity significantly in both cell lines. To investigate the effect of overexpression of Ets-1 on endogenous Npr1 gene expression in MMCs and MA-10 cells, we analysed the mRNA levels of Npr1 by RT-PCR. Representative levels of Ets-1 enhanced Npr1 mRNA in MMCs and MA-10 cells are shown in Figures 1(B) and 1(C) respectively. There was a 3.5- to 5-fold induction in Npr1 mRNA levels in MMCs and MA-10 cells when transfected with the Ets-1 expression plasmid compared with untransfected controls (Figures 1D and 1E) respectively.
Effect of Ets-1 on
Npr1 gene transcription and expression
We investigated further the binding of endogenously expressed Ets-1 protein to each of the two Ets-1-binding sites present in the Npr1 promoter using a combination of EMSA methods, including competition binding, mutant oligonucleotides and immunoshift assay. Figure 2(A) shows a schematic diagram of the wild-type and mutant Ets-1-binding sites. Incubation of nuclear extracts with wild-type Ets-1A and Ets-1B oligonucleotides resulted in the formation of specific nucleoprotein complexes (Figure 2B, lanes 2 and 10). These complexes were effectively competed by the addition of a 200-fold molar excess of the corresponding unlabelled wild-type probe (Figure 2B, lanes 3 and 11). The addition of unlabelled mutant Ets-1A- and Ets-1B-site oligonucleotides created no competition for DNA–protein binding (Figure 2B, lanes 4 and 12). Furthermore, the incubation of labelled Ets-1A and Ets-1B mutant probes with nuclear extracts did not result in the binding of specific nucleoprotein (Figure 2B, lanes 6 and 14). The specificity of the protein–DNA complex was confirmed by antibody supershift assays. Incubation of nuclear extracts with an anti-Ets-1 antibody before the addition of the probe disrupted the DNA–protein complex, thus ablating the specific band (Figure 2B, lanes 8 and 16). In Figure 2(B), lanes 1, 5, 7, 9, 13 and 15 show the mobility of the probe alone, which was used as a negative control. We used ChIP assay and PCR to determine whether Ets-1 interacts with the Npr1 promoter in a natural chromosome configuration. PCR amplification of the Npr1 promoter region containing the Ets-1-binding sites from immunoprecipitated DNA demonstrated that endogenous Ets-1 binds to the Npr1 promoter in vivo (Figure 2C). Overexpression of Ets-1 showed enhanced binding of Ets-1 to the Npr1 promoter, but no amplification was detected with DNA immunoprecipitated in the absence of the antibody.
In vitro and in vivo binding of Ets-1
To demonstrate the effect of overexpression of Ets-1 protein on Ets-1-inducible Npr1 gene transcription, we used −356/+55 Npr1 promoter constructs with wild-type or mutant versions of either or both of the Ets-1A or Ets-1B sites and these were transiently transfected along with the Ets-1 expression plasmid into MMCs and MA-10 cells (Figures 3A and 3B). Overexpression of Ets-1 with the wild-type construct increased the promoter activity by 11- and 15-fold in MMC and MA-10 cells respectively. There was a significant reduction of approx. 50% in Ets-1-induced promoter activity when either Ets-1 site was mutated compared with the wild-type −356/+55 construct in both of the cell lines (Figures 3A and 3B). Simultaneous mutation of both of the Ets-1 sites did not result in Ets-1-induced Npr1 gene transcription in either cell line (Figures 3A and 3B). The increase in luciferase activity observed after co-transfection with Ets-1 was due to an increase in firefly luciferase activity, whereas there was no significant change in Renilla luciferase activity. The enhanced expression of the Ets-1 protein in transfected MMCs and MA-10 cells was confirmed by Western blot analysis using a polyclonal Ets-1 antibody (Figures 3C and 3D), with β-actin used as a loading control.
Effect of overexpression of Ets-1 on
Npr1 promoter activity
We examined the effect of Ets-1 on the intracellular accumulation of cGMP in both MMCs and MA-10 cells. The treatment of Ets-1-transfected MA-10 cells and MMCs with 100 nM ANP showed an increase in the intracellular accumulation of cGMP by 2-fold and 3.5-fold, respectively, as compared with the untransfected control cells (Figure 4). To investigate the effect of Ets-1 on expression and function of NPRA protein, we performed Western blot and GC activity assay. Figures 5(A) and 5(B) show the representative levels of NPRA protein expression in MMCs and MA-10 cells respectively. NPRA protein expression was induced by 3.5- and 5.5-fold in Ets-1-transfected MMCs and MA-10 cells respectively compared with the empty vector-transfected control cells (Figures 5C and 5D). MA-10 cells showed a higher level of expression of the NPRA protein compared with MMCs. The effect of Ets-1 on GC-A/NPRA signalling was measured using the GC activity assay. Plasma membrane preparations of Ets-1-transfected MMCs and MA-10 cells showed a significant increase in GC activity compared with untransfected controls (Figures 5E and 5F). There was an almost 3-fold increase in GC activity in Ets-1-transfected MMCs when stimulated with ANP compared with untransfected control cells. Similarly, Ets-1-stimulated GC activity was also enhanced by almost 3.5-fold in MA-10 cells compared with unstimulated control cells (Figure 5F).
Effect of Ets-1 on the intracellular accumulation of cGMP
Ets-1-mediated regulation of NPRA expression and GC activity in MMCs and MA-10 cells
Co-transfection of Ets-1 siRNA along with the Npr1 basal promoter reduced luciferase activity by 80% compared with cells transfected with the Npr1 basal promoter alone; however, transfection of control siRNA resulted in no change in luciferase activity (Figure 6A). We assessed the efficiency of siRNA knockdown by Western blot analysis using an anti-Ets-1 antibody. Ets-1 protein expression was reduced markedly in siRNA-transfected cells compared with that in control siRNA-transfected cells (Figure 6B). Immunofluorescence staining with an anti-Ets-1 antibody and an FITC-labelled secondary antibody showed the expression of endogenous Ets-1 in the nuclei of cells transfected with empty vector (Figure 6C, panel 1a). Ets-1 protein was found to be overexpressed in Ets-1-transfected cells; however, Ets-1 siRNA reduced the expression of Ets-1 significantly compared with that in control siRNA-transfected cells (Figure 6C, panels 2a, 3a and 4a). Nuclei staining in the corresponding cells with DAPI is also shown (Figure 6C, panels 1b–4b). An overlay of the Ets-1 protein staining with DAPI confirmed the nuclear localization of Ets-1 (Figure 6C, panels 1c–4c).
The effect of Ets-1 gene silencing on
Npr1 gene transcription and expression using fluorescence microscopy
We investigated further the role of methylation in Npr1 gene transcription and its effect on Ets-1 binding to the Npr1 promoter. By analysing the sequence of the Npr1 promoter between positions −356 and +55 relative to the TSS, we noted that there are 34 CpG sites. To examine the effect of methylation on Npr1 promoter activity, both MMCs and MA-10 cells were transfected with in vitro methylated pGL3-Basic and −356/+55 Npr1 promoter constructs. A schematic representation of −356/+55 Npr1 promoter constructs containing CpG sites recognized by various methylases is shown in Figure 7(A). Luciferase activity of these plasmids was compared with the corresponding unmethylated plasmids. The luciferase activity of the partially HhaI-methylated −356/55 construct was reduced by 70% and 75% in MMCs and MA-10 cells respectively, exhibiting a partial effect of methylation on Npr1 gene transcription (Figures 7B and 7C). Similarly, the partial methylase HpaII reduced the luciferase activity of the −356/+55 construct by almost 80% compared with the unmethylated construct in both MMCs and MA-10 cells. On the other hand, the inhibition of Npr1 gene transcription by methylation was further confirmed by transfection of fully methylated SssI-treated plasmids, which suppressed Npr1 promoter activity by 95% and 98% in MA-10 cells and MMCs respectively (Figures 7B and 7C). Subsequently, EMSA was also performed to investigate whether the methylation of Ets-1 consensus sequences influences the binding of Ets-1 to its recognition sites in the Npr1 promoter. The sequences of the Ets-1A and Ets-1B oligonucleotides used for methylation are shown in Figure 8(A). The unmethylated Ets-1A and Ets-1B oligonucleotides were mixed with nuclear proteins from MMCs and MA-10 cells, which exhibited binding of Ets-1 to the Npr1 promoter (Figure 8B, lanes 2, 5, 8 and 11). Methylation of the wild-type probe at all of the CpG sites present in the Ets-1 recognition site produced significantly weaker shifted bands than the unmethylated probes in both MMCs and MA-10 cells (Figure 8B, lanes 3, 6, 9 and 12).
Effect of methylation on
Npr1 promoter activity
Effect of methylation on
in vitro Ets-1 binding in Npr1 promoter
The results of the present study demonstrate that Npr1 promoter activity is regulated by Ets-1 and show that the endogenously expressed Ets-1 protein associates physically with the Npr1 promoter in vivo and binds to its consensus motifs under in vitro conditions. Overexpression of Ets-1 greatly increased Npr1 mRNA and protein levels and stimulated the GC activity of the receptor in both MMCs and MA-10 cells. However, gene silencing of Ets-1 reduced the basal promoter activity significantly, indicating the critical role of Ets-1 in Npr1 gene transcription. Deletion of Ets motifs along with the TSS present in the region −46 to +55 resulted in a significant decrease in luciferase activity in the −356/−46 construct (Figure 1A). The decrease in luciferase activity may also be attributed to the removal of the initiation sequence CATACTCC present at the TSS in the region −46 to +55. Therefore, to confirm the involvement of the Ets sites in Npr1 gene regulation, we performed in vitro site-directed mutagenesis experiments. The individual contribution of each Ets-1 motif appears to be equivalent, because mutation of either element reduced Ets-1-induced promoter activity by almost 50% compared with the wild-type construct, thus emphasizing the importance of the Ets sites in Npr1 gene transcription. Mutation of both of the sites together abolished Ets-1-dependent Npr1 promoter activity by almost 92% compared with the wild-type control plasmid, further confirming the stimulatory role of the Ets-1 transcription factor in Npr1 gene transcription. A significantly higher level of Npr1 gene transcription and expression was observed in MA-10 cells compared with MMCs. This could be explained by the fact that the MA-10 cell line expresses NPRA at a very high level and has been used as a model system to investigate the receptor-mediated cellular and biochemical responses of ANP . Ets factors share a winged helix–turn–helix DNA-binding domain and interact with the 5′-GGA(T/A)-3′ motif . It has been suggested that Ets family members have a primary function in the formation of the initiation complex on core promoters lacking the TATA sequence and are frequently located in the vicinity of the TSS in various TATA-deficient gene promoters [37,38]. The murine Npr1 gene can be added to this group of genes because it exhibits features which are typical of TATA-less GC-rich promoters and has two Ets-binding motifs close to the TSS . The Ets motifs present in the region −46 to +55 of the Npr1 promoter show a high sequence homology with the Ets-1 consensus sequence RCCGGAA/TGCY, suggesting that this arrangement might serve as a core for recruiting and/or stabilizing transcriptional complexes .
The expression of cell-type-specific genes is tightly regulated by a hierarchical mechanism composed of genetic and epigenetic factors. Epigenetic changes influence gene expression by changes in chromatin structure via modification of DNA (methylation of CpG islands) and/or histones (e.g. methylation, acetylation, and phosphorylation) . A strong correlation between promoter methylation and gene silencing has been extensively demonstrated [41,42]. Our results show the involvement of methylation in Npr1 gene silencing. Thereby, in vitro methylated plasmid constructs exhibited a significant decrease in luciferase activities in the transfected cells. The extent of the decrease in luciferase activity was dependent on the density of methylation in the region −356 to +55 of the Npr1 promoter. Methylation of all 34 sites by SssI methylase dramatically decreased the promoter activity by almost 95–98% in MMCs and MA-10 cells. However, partial methylation by HhaI (seven sites) or HpaII (five sites) methylases reduced the luciferase activity by 70% and 80% respectively in both MMCs and MA-10 cells. Methylation of the HpaII sites reduced the promoter activity more significantly than methylation of the HhaI sites, which can be explained by the fact that two HpaII sites are present within the Ets-1-binding sites that can inhibit the binding of Ets-1 to the promoter. Methylation of the binding sites of various transcription factors, such as AP-2, Sp3 and Sp1/Sp3, has been shown previously to have a direct influence on the expression of downstream genes and is another mechanism for methylation-induced gene repression [43,44]. We observed that methylation of the Ets-1A and Ets-1B recognition sequences in vitro decreased the binding of Ets-1 to these sites significantly.
Previous studies have indicated that Ets transcription factors are involved in gene activation in response to vascular inflammation . Ets-1 transcriptionally induces the expression of caspase 1 , which plays a prominent role in the apoptotic induction of the inflammatory response in cells . On the other hand, studies in Ets-1-null mutant mice have shown that Ets-1 acts as a transcriptional mediator for Ang II-induced vascular remodelling and the generation of reactive oxygen species in hypertension and vascular diseases [48,49]. Our recent studies have also shown that ablation of the Npr1 gene provokes an inflammatory response in Npr1-null mutant mice . Since Ets-1 is stimulated by pro-inflammatory mediators and also enhances Npr1 gene transcription, it is implied that it may have a dual function in cardiovascular disease states. On one hand, it plays a role in inflammatory responses downstream of Ang II signalling pathways, and, on the other hand, it stimulates Npr1 gene transcription, which seems to inhibit pro-inflammatory responses in hypertension and cardiovascular events.
In summary, the present study provides direct evidence that Ets-1 is essential for Npr1 gene transcription and mediates its effect by binding to its consensus sites present in the Npr1 promoter. The findings of the present study should prove important in elucidating the molecular mechanisms for the expression and regulation of members of the GC receptor family. The results of the present study will greatly enhance our understanding of the transcriptional regulation of the Npr1 gene, an important locus in the control of hypertension and cardiovascular homoeostasis.
- Ang II
atrial natriuretic peptide
activator protein 2
brain natriuretic peptide
- DAPI 4′
Dulbecco's modified Eagle's medium
electrophoretic mobility-shift assay
Ets transformation-specific sequence
- ITS insulin
transferrin and sodium selenite
mouse mesangial cell
natriuretic peptide receptor
small interfering RNA
stimulating protein 1
Tris-buffered saline and 0.1% Tween 20
transcription start site
We thank Mr Edward Au for technical assistance and Mrs Kamala Pandey for assistance during the preparation of this manuscript. We thank Dr Paul Brindle (Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, TN, U.S.A.) for the gift of expression vectors. We also thank Dr Susan L. Hamilton (Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX, U.S.A.) and Dr Bharat B. Aggarwal (Department of Experimental Therapeutics and Cytokine Research Laboratory at MD Anderson Cancer Center, University of Texas, Houston, TX, U.S.A.) for providing use of their facilities during our displacement period due to Hurricane Katrina.
This work was supported by the National Institutes of Health [grant numbers HL57531, HL62147]; and partially supported by developmental funds from the Tulane Cancer Center.