Angiotensin II represses Npr1 expression and receptor function by recruitment of transcription factors CREB and HSF-4a and activation of HDACs

The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each other. ANP acting through natriuretic peptide receptor-A (NPRA) lowers blood pressure and blood volume. We tested hypothesis that ANG II plays critical roles in the transcriptional repression of Npr1 (encoding NPRA) and receptor function. ANG II significantly decreased NPRA mRNA and protein levels and cGMP accumulation in cultured mesangial cells and attenuated ANP-mediated relaxation of aortic rings ex vivo. The transcription factors, cAMP-response element-binding protein (CREB) and heat-shock factor-4a (HSF-4a) facilitated the ANG II-mediated repressive effects on Npr1 transcription. Tyrosine kinase (TK) inhibitor, genistein and phosphatidylinositol 3-kinase (PI-3K) inhibitor, wortmannin reversed the ANG II-dependent repression of Npr1 transcription and receptor function. ANG II enhanced the activities of Class I histone deacetylases (HDACs 1/2), thereby decreased histone acetylation of H3K9/14ac and H4K8ac. The repressive effect of ANG II on Npr1 transcription and receptor signaling seems to be transduced by TK and PI-3K pathways and modulated by CREB, HSF-4a, HDACs, and modified histones. The current findings suggest that ANG II-mediated repressive mechanisms of Npr1 transcription and receptor function may provide new molecular targets for treatment and prevention of hypertension and cardiovascular diseases.


Results
AnG ii treatment dose-dependently attenuated Npr1 gene transcription and expression. To determine the presence of ANG II responsive elements in the Npr1 promoter, 5′ deletion constructs were sequentially analyzed for luciferase activity. The cells were transfected with deletion constructs and treated with 10 nM ANG II. As compared with untreated control cells, the constructs ΔA4 (−1182/+55 bp) and ΔA9 (−941/+55 bp) displayed significant decreases (70% and 50%, respectively) in luciferase activity (Fig. 1A). However, the constructs ΔA5 (−1128/+55 bp) and ΔA10 (−882/+55 bp) showed no corresponding reduction in Npr1 promoter activity in response to ANG II treatment. There was a dose-and time-dependent reduction in promoter activity in ΔA4 (−1182/+55 bp) construct, with the maximum reduction occurring at a concentration of 10 nM ANG II at 16 h after ANG II treatment (Fig. 1B,C). A schematic map of Npr1 promoter region −1982 to +55 bp containing the binding sites of various transcription factors is shown in Fig. 1D.
We next determined the activity of smaller fragments of the Npr1 promoter exhibiting responsiveness to ANG II. As shown in Fig. 2A, the ΔR1 (−1182/−1127 bp) promoter construct exhibited dose-and time-dependent repression of luciferase activity in response to ANG II, with maximal inhibition occurring with 10 nM ANG II after 16 h of incubation. Real-time qRT-PCR assay showed 58% attenuation in Npr1 mRNA levels in MMCs treated with ANG II as compared to untreated control cells (Fig. 2B). Similarly, there was a 60% reduction in NPRA protein levels in MMCs treated with increasing concentrations of ANG II as compared to untreated control cells (Fig. 2C). The treatment of MMCs with ANG II, showed a significant decrease in ANP-stimulated intracellular accumulation of cGMP; that decrease was almost 57% in comparison with results in unstimulated cells (Fig. 2D).
AnG ii repressed Npr1 gene expression via AT 1 receptor signaling. The results of Npr1 promoter activity in response to candesartan, ANG II Type 1 receptor (AT 1 R) blocker, and PD 123319, an ANG II Type 2 receptor (AT 2 R) blocker, are shown in Fig. 3. A schematic map of Npr1 promoter deletion constructs ΔR1 and ΔR5 is shown in Fig. 3A. There was a significant decrease (62%, p < 0.01) in the promoter activity of construct ΔR1 (−1182/−1127 bp) in MMCs treated with 10 nM ANG II; however, treatment with 100 nM AT 2 R blocker PD123319 had no effect on ANG II-mediated repression of Npr1 promoter activity (Fig. 3B). Nevertheless, the repressive effect of ANG II was reversed after treatment with 100 nM of the AT 1 R blocker candesartan (Fig. 3C). Similarly, cells transfected with construct ΔR5 (−984/−914 bp) exhibited a significant reduction (50%, p < 0.01) in Npr1 promoter activity after ANG II treatment. However, PD123319 did not reverse the ANG II effect (Fig. 3D). Also, similar to ΔR1 construct, treatment of MMCs with candesartan reversed the effect of ANG II on ΔR5 promoter activity (Fig. 3E).
Treatment with tyrosine kinase and phosphatidylinositol-3-kinase inhibitors reversed the repressive effects of ANG II on Npr1 promoter activity and transcription. The promoter activity of Npr1 in response to ANG II and protein kinase inhibitors, including inhibitors for protein kinase A (PKA), tyrosine kinase (TK), and phosphatidylinositol 3-kinase (PI-3K), are detailed in Fig. 4. There was a decrease of almost 55-60% in the promoter activity of ΔR1 construct after treatment with ANG II. However, the PKA inhibitor, H89 dihydrochloride, and the PI-3K inhibitor, wortmannin had no effect on the ANG II-mediated transcriptional activity of Npr1 (Fig. 4A,B). Interestingly, treatment with genistein a TK inhibitor completely reversed the repressive effect of ANG II on Npr1 ΔR1 promoter activity (Fig. 4C). Similarly, in ΔR5-transfected www.nature.com/scientificreports www.nature.com/scientificreports/ AnG ii represses Npr1 promoter activity via recruitment of HSF-4a and CREB. To elucidate the role of transcription factors HSF-4a and CREB, the consensus sequence cgttctt was mutated to ccatgat in ΔR1 construct (−1182/−1127 bp) and the sequence atgccgtca was mutated to atccggtga in ΔR5 construct (−984/−914 bp), as shown by schematic representation in Fig. 5A,D, respectively. Both the constructs were then transfected in MMCs to determine the Npr1 promoter activity. As shown in Fig. 5B, after treatment with ANG II, luciferase activity was reduced by almost 65% in the wild-type ΔR1 (−1182/−1127 bp) construct. In contrast, the mutant ΔR1 (−1182/−1127 bp) construct did not show any repressive effect of Ang II on Npr1 promoter activity. There was an almost 85% reduction in the Npr1 promoter activity of ΔR1 construct with the overexpression of transcription factor HSF-4a (Fig. 5C). A 55% reduction of promoter activity occurred in wild-type ΔR5 (−984/−914 bp) construct in the presence of ANG II. However, the mutant construct ΔR5 (−984/−914 bp) showed no repressive effect of ANG II on Npr1 promoter activity (Fig. 5E). On the other hand, in ΔR5-transfected cells, the overexpression of CREB reduced the ΔR5 promoter activity by almost 75% (Fig. 5F). The treatment of cells with ANG II only slightly increased HSF-4a protein expression, which was not significant as compared with untreated control cells, (Fig. 5G). However, ANG II significantly increased the phosphorylation of CREB (pCREB) in treated cells as compared with untreated control cells but did not increase the expression of total CREB protein (Fig. 5F). In the preliminary studies the transfection of only HSF-4a or CREB plasmids without ANG II did not significantly reduce the luciferase activity of Npr1 promoter (data not shown).
Electrophoretic mobility shift assay (EMSA) was performed with ∆R1a (−1156 to −1127 bp) and ∆R5a (−959 to −914 bp) probes, respectively, containing HSF-4a and CREB binding sites. The ∆R1a region showed a binding pattern with band I (arrow) corresponding to HSF-4a transcription factor and an additional band II (arrow) was also observed (Fig. 6A, lanes 2 and 3). The ∆R5a region showed a binding pattern with only one band AnG ii inhibits Npr1 transcription by increased HDAC expression and activity, as well as decreased histone acetylation. To delineate the epigenetic components involved in ANG II-mediated Npr1 repression, we determined the effect of ANG II on histone deacetylases (HDACs) expression and activity levels of class I HDACs (HDAC1, HDAC2, and HDAC3). Treatment with increasing concentrations of ANG II augmented total HDAC activity by more than 3-fold as compared with activity in control cells (Fig. 7A). There was a significant increase in HDAC1 and 2 protein expressions in ANG II-treated cells, but no change in HDAC3 protein expression as compared with that in untreated control cells (Fig. 7B). Treatment with mocetinostat, a www.nature.com/scientificreports www.nature.com/scientificreports/ class I-specific HDAC inhibitor (MGCD0103) of ANG II-pretreated cells, markedly induced expression of Npr1 mRNA and increased the protein levels of NPRA as quantified, respectively, by real time qRT-PCR and Western blot analyses (Fig. 7C,D). ANG II significantly (52%) attenuated global acetylation levels of histone H3 at lysine 9  MMCs were transiently transfected with wild-type ∆R1 or mutant ∆R1 constructs, treated with ANG II for 16 h, and the promoter activity was measured. (C) MMCs were cotransfected with HSF-4a expression plasmid and ∆R1 promoter construct, treated with 10 nM ANG II for 16 h, and the luciferase activity was measured. Normalized luciferase activity is shown as a percentage of the activity of untreated control groups. (D) Schematic diagram showing the sequence of the wild-type and mutated CREB binding site in the Npr1 promoter. (E) MMCs were transiently transfected with wild-type ∆R5 or mutant ∆R5 construct, treated with ANG II for 16 h, and luciferase promoter activity was measured. (F) MMCs were cotransfected with CREB expression plasmid and ∆R5 promoter construct treated with ANG II for 16 h, and the luciferase activity was measured. (G) Western blot and densitometry analysis of nuclear HSF-4a protein expression in cells treated with ANG II, for which the nuclear protein, TBP expression is shown as loading control (fulllength image: Supplementary Fig. 2). (H) Western blot and densitometry analysis of phosphorylated nuclear Repressive effect of ANG II on Npr1 expression and ANP-induced vasorelaxation in aortic rings. We confirmed the effect of ANG II on Npr1 expression by ex-vivo experiments using aortic rings from C57/BL6 male mice. There was a 45% reduction in Npr1 mRNA levels in aortic rings treated with ANG II, but not in untreated control aortic rings (Fig. 8A). Incubation of denuded aortic rings with ANG II demonstrated a 50% reduction in NPRA protein levels (Fig. 8B). Treatment with increasing concentrations of ANP (IC50 = 6 × 10 −9 M) relaxed aortic rings that had been contracted with PGF2α. However, overnight treatment of aortic rings with 100 nM ANG II significantly antagonized the ANP response curve (interaction, P = 0.024). Post-hoc analysis showed significant inhibition at 10 nM (p < 0.001) and 100 nM (p < 0.05) concentrations of ANG II (Fig. 8C). In the preliminary studies for baseline control experiments, aortic rings were treated overnight in either control medium or in ANG II-containing medium and next day the rings were exposed to increasing concentrations of ANG II in the wire myograph. Rings that had been exposed to ANG II overnight did not contract in response to ANG II, indicating that a sustained tachyphylaxis occurred with down-regulation of ANG II receptors in continuous treatment protocols as shown in the Supplementary Fig. 8.

Discussion
The results from the deletional analysis of Npr1 promoter showed that the transcriptional activity of the core promoter in the pGL3 vector was significantly reduced in response to ANG II. Our findings provide the direct evidence in signifying the role of ANG II-response elements, CREB and HSF-4a in mediating the repressive effect of ANG II on Npr1 transcription and functional responsiveness. The MMCs express both AT 1 and AT 2 receptor subtypes, which differ in their biological effects and signal transduction mechanisms 48,49 . AT 1 receptor mediates effects such as vasoconstriction, cell proliferation, and vascular remodeling 50 , while AT 2 receptor mediates its effect by lowering blood pressure, diuresis, natriuresis, and cell growth inhibition 51 . Both AT 1 and AT 2 receptors have been implicated in ANG II-dependent inhibition of ANP-stimulated GC activity of NPRA and intracellular cGMP accumulation 24,52 . However, the mechanisms involved in the mediation of the ANG II-induced effects differ for the two ANG II receptor subtypes. The stimulation of AT 1 R evokes several intracellular signals such as activation of protein kinases, including TK, PI-3K, and MAPK cascades 53,54 ; on the other hand, AT 2 R activates one or several tyrosine phosphatases and MAPK phosphatase, resulting in the inhibition of specific kinases and apoptosis 55 . We investigated the inhibitory effect of ANG II on the expression of Npr1 in the presence of receptor blockers specific to AT 1 R (candesartan) and AT 2 R (PD123319) subtypes. The AT 1 R antagonist, candesartan, but not the AT 2 R antagonist, PD123319, blocked ANG II-mediated repression of Npr1 in both the ΔR1 and ΔR5 constructs of the Npr1 promoter, suggesting the involvement of AT 1 receptor subtype in this repression. Nevertheless, at higher concentrations of AT 2 R antagonist, PD123319, there was a slight repressive effect on Npr1 promoter activity in both the ANG II responsive regions of the Npr1 gene. Earlier, we observed that Npr1 promoter showed repressive activity in the presence of ANG II 56 .
In the current work, we examined the involvement of protein kinases in the signal transduction pathway, mediating the repressive effect of ANG II on Npr1 promoter. The ANG II-mediated repression of Npr1 promoter ΔR1 construct could be blocked by genistein, a TK inhibitor, suggesting the involvement of TK in the transcriptional repression of Npr1. In the ΔR1 construct, there is a 100% match of the DNA sequence with a putative heat-shock element, which binds to heat-shock factors. It has been reported that HSF-4a represses basal transcription through interaction with transcription factor IIF (TFIIF), which occurs through inhibition of an early step in formation of the preinitiation complex 57 . Interestingly, ANG II causes an increase in heat-shock factors and that heat-shock protein 90 complex negatively regulates NP receptors 58,59 . Moreover, genistein has been shown to inhibit herbimycin A-induced over-expression of inducible heat-shock protein corresponding to 70 kDa 60 . Our UV cross-linking experiments and gel mobility shift assays demonstrated the formation of DNA-HSF-4a binding complex, which was enhanced in ANG II-treated cells. The novelty in the present study stems from the fact that HSF-4a is activated by ANG II, which negatively regulates Npr1 gene transcription and receptor function. To our knowledge, this is the first report demonstrating the role of ANG II-dependent activation of TK in regulating HSF-4a in transcriptional repression of Npr1.
In the present study, promoter activity of the ΔR5 Npr1 construct was repressed in the presence of ANG II; this repression could be blocked by genistein or wortmannin, suggesting the involvement of both TK and PI-3K in this pathway. The ΔR5 construct contains a putative cAMP-response element, TGCCGTCA (at −932 bp position), which is recognized by the transcription factor CREB, one of the few reported cis-elements through which ANG II has been shown to regulate gene expression. Interestingly, our results indicate that ANG II was able to activate the recruitment of CREB to Npr1 promoter and exerted the repressive effect on Npr1 gene transcription and function. UV cross-linking and EMSA exhibited enhanced DNA-CREB binding complex in ANG II-treated cells as compared with untreated control cells. Western blot results confirmed the enhanced phosphorylation of CREB in the presence of ANG II. It has previously been suggested that ANG II promotes the phosphorylation of CREB at Ser133 through an ERK1/2-dependent mechanism [61][62][63] . CREB activity has CREB (pCREB) and total CREB protein expression in ANG II-treated cells for which also nuclear protein, TBP expression is shown as loading control (full-length image: Supplementary Fig. 3). Densitometry analyses of pCREB and CREB protein bands were done in the samples obtained from the same experiment and gels/ blots were processed simultaneously in parallel. The results are expressed as mean + SE from 6-8 independent experiments. WB, Western blot; **p < 0.01. (2020) 10:4337 | https://doi.org/10.1038/s41598-020-61041-y www.nature.com/scientificreports www.nature.com/scientificreports/ also been shown to be regulated by PI-3K/Akt signaling in Jurkat T leukemia cells treated with tumor necrosis factor-related apoptosis-inducing ligand 64 and tyrosine kinase B/PI-3K/Akt pathway in SH-SY5Y cells treated with brain-derived neurotrophic factor 65 . Although CREB is most often described as a positive transcription factor, several reports have shown that it can also inhibit the transcriptional activity of several gene promoters such as those of c-fos and somatostatin 66,67 . It is noteworthy to mention that in our preliminary studies, the transfection of MMCs with only either HSF-4a or CREB expression plasmids alone without ANG II, did not exhibit any discernible effect on the negative repression of luciferase activity and Npr1 transcription (data not shown). www.nature.com/scientificreports www.nature.com/scientificreports/ It is implicated that the hormonal signal of ANG II is required for the activation of HSF-4a and CREB to exert a repressive effect on Npr1 promoter activity and its gene transcription.
Our present results show that ANG II treatment enhanced total HDAC activity and induced the protein expression of HDAC 1/2. Epigenetic mechanisms, including changes in histone acetylation and deacetylation have been shown to alter gene expression under various physiological and pathophysiological conditions 68,69 . Evidence suggests that ANG II treatment induces epigenetic modifications, including changes in HDACs  Supplementary Fig. 4). (C) Effect of MGCD0103 on Npr1 mRNA expression and (D) NPRA protein (135 kDa) levels in ANG II-pretreated MMCs (full-length image: Supplementary Fig. 5). (E) Western blot and densitometry analyses of acetylated histones H3-K9/14 and H4-K8 protein expression in ANG II-treated cells (full-length image: Supplementary Fig. 6). The results are expressed as mean ± SE from 6-8 independent experiments. WB, Western blot; *p < 0.05; **p < 0.01; **p < 0.001.

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(2020) 10:4337 | https://doi.org/10.1038/s41598-020-61041-y www.nature.com/scientificreports www.nature.com/scientificreports/ expression and activity, which is associated with ANG II-induced tissue hypertrophy and fibrosis 70,71 . Earlier, it was shown that ANG II treatment of intact E12.5 mouse metanephroi grown ex vivo increased HDAC1 and decreased total acetylated histone H3 protein levels 72 . Recently, it has been shown that in ApoE gene-knockout www.nature.com/scientificreports www.nature.com/scientificreports/ mice with abdominal aortic aneurysms, infusion of ANG II for 4 weeks increased expression of class I HDAC1, 2, and 3, as well as expression of class II HDAC 4 and 7; it also decreased acetylation levels of H3-K18 73 . On the other hand, selective inhibition of class I HDACs led to potent suppression of ANG II-mediated cardiac fibrosis and hypertrophy by targeting cardiac fibroblasts and bone-marrow-derived fibromyocytes 71,74 . These findings are consistent with our observation that ANG II treatment repressed acetylation of H3-K9/14 and H4-K8 and enhanced HDAC activity. Our results support the notion depicting a model that ANG II facilitated the recruitment of transcription factors, CREB and HSF-4a to Npr1 promoter via AT1R, resulting in the activation of TK and PI-3K signaling pathways, which exerted the repressive effects on Npr1 gene transcription and function (Fig. 8D). Moreover, our model also predicts that the treatment of MMCs with HDAC inhibitor attenuated the repressive effect of ANG II on Npr1 gene transcription, expression, and functional activity.
In conclusion, the present results demonstrate that ANG II mediates its repressive effects on Npr1 transcription by inducing phosphorylation of CREB protein and enhancing the expression and binding of HSF-4a and CREB to the consensus sites of Npr1 promoter. Our findings showed that the inhibitory effect of ANG II on NPRA/cGMP signaling is transduced by direct repressive effects on the Npr1 transcription and expression via AT1 receptor, TK, and PI-3K signaling. ANG II markedly increased HDAC 1/2 protein levels and HDAC activity. The cotreatment with HDAC inhibitor reversed ANG II-mediated repression of Npr1 transcription and function. These findings are noteworthy as they provide important insights and advance our understanding towards the action of ANG II in the repressive regulation of Npr1 gene transcription and the ANP/NPRA/cGMP signaling pathway, which critically mediates the pathophysiology of hypertension and cardiovascular dysfunction.

Animals.
Mice used in the present studies were C57/Bl6 wild-type and produced at Tulane Vivarium. Mice were housed under 12:12 h light-dark cycle at 25 °C and fed regular chow (Purina Laboratory and tap water ad libitum as previously described 75 . Adult mice (30 g) were euthanized by deep anesthesia with isoflurane inhalation. Thoracic part of aorta was isolated and rings were prepared as earlier reported 76 . Animals were used under the protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the Tulane University Health Sciences Center and were conducted in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animal.
Plasmid construction in pGL3-promoter. The cloning of the smaller DNA fragments from −1182 to −914 bp of Npr1 promoter region was done at the upstream of SV-40 promoter firefly luciferase gene in the pGL3-promoter vector as previously reported 25 . The ∆R1 (−1182 to −1127 bp), ∆R2 (−1128 to −1072 bp), ∆R3 (−1071 to −1028 bp), ∆R4 (−1026 to 986 bp), and ∆R5 (−984 to −914 bp) constructs were generated by PCR using the pNPRA-luc1 (−1982 to +55 bp) as a template and DNA polymerase (elongase). All the forward primers (F1, F2, F3, F4, and F5) contained a MluI restriction site; whereas the reverse primer contained a BglII restriction site at the 5′ ends. The PCR primers used are listed in Supplementary Table S2. cell culture and hormonal treatment. MMCs were cultured in Dulbecco modified Eagle's medium (DMEM) supplemented with 10% FCS and ITS as previously described 35 . Cultures were maintained at 37 °C in a 5% CO 2 /95% O 2 humidified atmosphere. For all experiments, cells were used between 4 to 12 passages. To study the effect of ANG II, cells were seeded in 24-well plates at 80% to 90% confluence. The cells were washed twice with serum-free assay medium containing 0.1% bovine serum albumin (BSA) and treated with 10 nM ANG II in fresh assay medium in the absence or presence of 100 nM dihydrochloride, 100 nM wortmannin, 100 nM genistein, or 1 µM MGCD0103. The cells were harvested at the indicated time intervals and lysed essentially as described earlier 77 . (2020) 10:4337 | https://doi.org/10.1038/s41598-020-61041-y www.nature.com/scientificreports www.nature.com/scientificreports/ transient transfection and luciferase assay. MMCs were seeded in 12-well plates at a density producing ~80% confluence. After 24 h, the cells were transfected using lipofectamine-2000 reagent according to the manufacturer's instructions, with 1 µg of test plasmid and 0.3 µg of pRL-TK carrying the renilla luciferase gene downstream of the thymidine kinase promoter, which was used as internal transfection control as earlier described 25 . The medium was changed after 24 h. The cells were harvested after 48 h by using passive lysis buffer (Promega). Luciferase activity was measured by TD 20/20 luminometer (Turner Designs, Loveland, CO) with 20 µl cell extract using a dual luciferase reporter assay system. In the transfection experiments, a pGL3-control vector containing both the SV40 promoter and enhancers was used as a positive control; the empty pGL3-basic vector was used as a negative control. The assays were performed in triplicate in 6-8 independent experiments. Results were normalized for the transfection efficiency as relative light units per renilla luciferase activity.
Preparation of whole cell lysate and nuclear extract for Western blot analysis. Whole cell lysate and nuclear extract were prepared as described earlier 77 . The protein concentration of the lysate was measured with a Bradford protein detection kit (Bio-Rad, Hercules, CA). Western blot assay was done as previously described 35,77 . The cytoplasmic fraction (50-70 µg) or nuclear extract (40-60 µg) was mixed with sample loading buffer and electrophoresed for 2 h, then transferred to a nylon membrane. The membrane was blocked with 1x Tris-buffered saline-Tween 20 (TBST; 25 mM Tris, 500 mM NaCl, and 0.05% Tween 20, pH 7.5) containing 5% fat-free milk for 1 h, then incubated overnight in TBST containing 5% fat-free milk at 4 °C with primary antibody (1:250 dilution). The membrane was treated with corresponding secondary anti-rabbit or anti-mouse horseradish-peroxidase (HRP)-conjugated antibodies. Protein bands were developed using a SuperSignal West Femto Chemiluminescent kit and visualized using a FluorChem detection system from Proteinsimple (Santa Clara, CA). The intensity of protein bands was quantified by AlphaView software (San Jose, CA). The antibodies used in Western blot assay are listed in Supplementary Table S3. cGMP assay. Twenty-four hours after plating, MMCs were made serum-free for 12 h and treated with ANG II (10 nM) for another 24 h. Cells were stimulated with ANP at 37 °C for 15 min in the presence of 0.2 mM 3-isobutyl-1-methylxanthine (IBMX), washed three times with phosphate-buffered saline (PBS), and scraped into 0.5 N HCl, as previously described 19 . The cell suspension was subjected to five cycles of freeze and thaw and then centrifuged at 10,000 × g for 15 min. The supernatant thus collected was used for cGMP assays using a direct cGMP complete Elisa kit according to the manufacturer's instructions (Enzo Life Sciences, Farmingdale, NY).
Real-time Rt-pcR analysis. Confluent MMCs were treated with or without ANG II (10 nM) in assay medium. After harvesting the cells, total RNA was extracted and 1 µg of total RNA was reverse transcribed, using a RT 2 First Strand cDNA kit from Qiagen. Primers for amplification of Npr1 and β-actin were from Qiagen. PCR amplification was done in triplicate in a 25-µl reaction volume using RT 2 Real-Time SYBR Green/ROX PCR Master Mix and PCR conditions as previously described 78 . Control experiments were done with RNA samples but without reverse transcriptase. The specific primers for ß-actin gene were included in the PCR reaction mixture as an internal control.
In vitro site-directed mutagenesis. The HSF-4a and CREB transcription factor mutants, ΔR1 mutant (−1182/−1127 bp) for HSF-4a and ΔR5 mutant (−984/−914 bp) for CREB, were constructed by using the in-vitro Site-Directed Mutagenesis kit (Stratagene). The consensus sequence of HSF-4a and CREB along with the mutant sequence is listed in Supplementary Table S4. The pGL3 plasmid with full length Npr1 gene promoter, pNPRA-Luc1 25 , was used as a template for generating mutations. The double-stranded DNA template pNPRA-Luc1 was alkaline denatured, annealed with the mutagenic oligonucleotide and selection oligonucleotide in annealing buffer, incubated at 75 °C for 5 min, and allowed to cool slowly to 37 °C. The mutant strand was synthesized with T4 DNA polymerase and T4 DNA ligase in the presence of synthesis buffer and incubated at 37 °C for 90 min. The DNA thus synthesized was transformed in XL-10 Gold super competent cells (Stratagene) and plated in LB amp agar plates containing gene editor antibiotic mix. The probable clone was confirmed by sequencing. electrophoretic mobility shift assay. The wild-type and mutant oligonucleotides corresponding to HSF-4a and CREB transcription factor binding sites were commercially synthesized and labeled at the 5′-end by phosphorylation of the 5′ hydroxyl ends with [γ 32 -P]ATP, using T4 polynucleotide kinase enzyme as previously described 79 . The radiolabeled sense oligonucleotide was annealed with the antisense unlabeled oligonucleotide in a 1:1 molar concentration by adding an equal volume of 2 x annealing buffer and incubating for 5 min in boiling water, then slowly cooling. The annealed oligonucleotides were purified on a Sephadex G-50 column. EMSA was done using ATP-labeled and annealed oligonucleotides as previously described 80 . For gel retardation, reaction mixture was prepared by adding 5-10 μg of nuclear extracts to 40,000-50,000 cpm of probe in 1x binding buffer containing 10 mM Tris (pH 7.5), 1 mM MgCl 2 , 0.05 mM EDTA, 0.05 mM DTT, 50 mM NaCl, 4% (v/v) glycerol, and 1 µg of nonspecific DNA. The reaction mixture was incubated for 30 min on ice. The DNA-protein complex was resolved from the free-labeled DNA by electrophoresis in a 4% native polyacrylamide gel at 100 V for 2 h and after drying gel was autoradiographed. For competition assays, 100-fold excess molar concentrations of unlabeled probe were added in the reaction mixture as a competitor.

UV cross-linking analysis.
To determine the molecular mass of nuclear binding proteins to the consensus sites, we did a UV cross-linking experiment. Oligonucleotide spanning the consensus site in the Npr1 promoter was 32 P-end-labeled and annealed for the EMSA reaction as described 80 . Binding reactions were set up as those for EMSA. After incubation on ice for 30 min, the reaction mixture was UV-cross-linked by pipetting onto parafilm and irradiated in a UV-cross linker. Samples were resolved by electrophoresis on 10% denaturing polyacrylamide gel and exposed to X-ray film for autoradiography.

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(2020) 10:4337 | https://doi.org/10.1038/s41598-020-61041-y www.nature.com/scientificreports www.nature.com/scientificreports/ Histone purification. Total histone was extracted from ANG II-treated and untreated MMCs, using a total histone extraction kit (Epigentek, Farmingdale, NY) as earlier reported 78 . In brief, cells were harvested and suspended in 1x pre-lysis buffer, kept on ice for 10 min, and centrifuged at 10,000 × g for 1 min at 4 °C. The supernatant was removed; the cell pellet was resuspended in 3 volumes of lysis buffer, incubated on ice for 30 min, and centrifuged at 14,000 × g for 5 min at 4 °C. Balance-dithiothreitol (DTT) buffer (0.3 volumes) was added to the supernatant, which was then stored at −80 °C. The protein concentration of the eluted histone was estimated using a Bradford protein detection kit (Bio-Rad, Hercules, CA), using BSA as a standard.
Total histone deacetylase activity assay. Total HDAC activity was measured in nuclear extracts prepared from ANG II-treated cells using a colorimetric ELISA assay kit from Active Motif (Carlsbad, CA) essentially as previously described 78 . HDAC enzyme activity was calculated by measuring the amount of HDAC-deacetylated product, which was directly proportional to HDAC enzyme activity. Absorbance was read at 450 nm. Results were calculated using a standard curve according to the manufacturer's instructions and expressed as ng/min/mg protein.
treatment of aortic rings ex vivo with ANG II for Western blot and qRT-PCR analyses. The male mice (C57/Bl6) were euthanized by deep anesthesia with isoflurane inhalation. Thoracic aorta was isolated and aortic rings were prepared using the previously described protocol with a minor modification 31,76 . Immediately after thoracotomy, the thoracic aorta was removed and placed in cold Dulbecco's PBS and cleaned by removing the surrounding fat and connective tissue. For experiments, the aorta was cut into 3 to 4 mm rings. After 2 h incubation in DMEM with 0.1% BSA, the aortic rings were treated with ANG II for 20 h. Aortic rings were then homogenized by sonication in lysis buffer, centrifuged, and supernatant was collected and stored at −80 °C for Western blot assay. For qRT-PCR, ANG II-treated and control rings were homogenized with 1.5-ml lysis buffer and RNA was extracted by an RNeasy mini-kit, following the manufacturer's protocol (Qiagen, Valencia, CA).
Aortic rings relaxation assay. Aortas were isolated and excised from C57/Bl6 male mice, cut into 2 mm rings, and incubated in DMEM containing 0.1% BSA. After 2 h, 100 nM ANG II was added to the aortic vessels. After 6 h, another dose of ANG II was added, and incubation was continued. Vessels were mounted on a wire myograph and experiments were completed as previously described 31,81 . Vessels were preconstricted with prostaglandin F2α (PGF2α) and treated with increasing concentrations of ANP. Data are expressed as percent relaxation from PGF2α contraction. For baseline control experiment, aortic rings were incubated overnight in either control media or in media containing ANG II. The next day, rings were exposed to increasing concentrations of ANG II in the wire myograph.
Statistical analysis. Statistical analysis was done using GraphPad prism software (San Diego, CA). The results are expressed as mean ± SE. Statistical significance was evaluated by one-way ANOVA and Student t test. Data was also analyzed using Two-Way ANOVA. Repeated measures by both ANG II and ANP treatments and Sidak's multiple comparisons test were used. The differences were considered significant with the probability value < 0.05.