Prerna Kumar

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.

Histone Deacetylase Inhibitors Modulate the Transcriptional Regulation of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Gene INTERACTIVE ROLES OF MODIFIED HISTONES, HISTONE ACETYLTRANSFERASE, p300, AND Sp1

Background: Natriuretic peptide receptor-A (NPRA) lowers blood pressure and blood volume. Results: Data show that histone deacetylase inhibitors (HDACi) enhance NPRA expression by blocking HDACs and interacting with Sp1, histone acetyltransferase, and acetylated histones. Conclusion: Results suggest that epigenetic mechanisms regulate NPRA gene transcription. Significance: Findings will help to identify molecular targets regulating NPRA expression and function in vivo. Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPRA) and produces the intracellular second messenger, cGMP, which regulates cardiovascular homeostasis. We sought to determine the function of histone deacetylases (HDACs) in regulating Npr1 (coding for GC-A/NPRA) gene transcription, using primary mouse mesangial cells treated with class-specific HDAC inhibitors (HDACi). Trichostatin A, a pan inhibitor, and mocetinostat (MGCD0103), a class I HDAC inhibitor, significantly enhanced Npr1 promoter activity (by 8- and 10-fold, respectively), mRNA levels (4- and 5.3-fold, respectively), and NPRA protein (2.7- and 3.5-fold, respectively). However, MC1568 (class II HDAC inhibitor) had no discernible effect. Overexpression of HDAC1 and HDAC2 significantly attenuated Npr1 promoter activity, whereas HDAC3 and HDAC8 had no effect. HDACi-treated cultured cells in vitro and intact animals in vivo showed significantly reduced binding of HDAC1 and -2 and increased accumulation of acetylated H3-K9/14 and H4-K12 at the Npr1 promoter. Deletional analyses of the Npr1 promoter along with ectopic overexpression and inhibition of Sp1 confirmed that HDACi-induced Npr1 gene transcription is accomplished by Sp1 activation. Furthermore, HDACi attenuated the interaction of Sp1 with HDAC1/2 and promoted Sp1 association with p300 and p300/cAMP-binding protein-associated factor; it also promoted the recruitment of p300 and p300/cAMP-binding protein-associated factor to the Npr1 promoter. Our results demonstrate that trichostatin A and MGCD0103 enhanced Npr1 gene expression through inhibition of HDAC1/2 and increased both acetylation of histones (H3-K9/14, H4-K12) and Sp1 by p300, and their recruitment to Npr1 promoter. Our findings define a novel epigenetic regulatory mechanism that governs Npr1 gene transcription.

All-Trans Retinoic Acid and Sodium Butyrate Enhance Natriuretic Peptide Receptor A Gene Transcription: Role of Histone Modification

The objective of the present study was to delineate the mechanisms of GC-A/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) expression in vivo. We used all-trans retinoic acid (ATRA) and histone deacetylase (HDAC) inhibitor, sodium butyrate (NaBu) to examine the expression and function of Npr1 using gene-disrupted heterozygous (1-copy; +/−), wild-type (2-copy; +/+), and gene-duplicated heterozygous (3-copy; ++/+) mice. Npr1+/− mice exhibited increased renal HDAC and reduced histone acetyltransferase (HAT) activity; on the contrary, Npr1++/+ mice showed decreased HDAC and enhanced HAT activity compared with Npr1+/+ mice. ATRA and NaBu promoted global acetylation of histones H3-K9/14 and H4-K12, reduced methylation of H3-K9 and H3-K27, and enriched accumulation of active chromatin marks at the Npr1 promoter. A combination of ATRA-NaBu promoted recruitment of activator-complex containing E26 transformation–specific 1, retinoic acid receptor α, and HATs (p300 and p300/cAMP response element–binding protein-binding protein–associated factor) at the Npr1 promoter, and significantly increased renal NPRA expression, GC activity, and cGMP levels. Untreated 1-copy mice showed significantly increased systolic blood pressure and renal expression of α-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) compared with 2- and 3-copy mice. Treatment with ATRA and NaBu synergistically attenuated the expression of α-SMA and PCNA and reduced systolic blood pressure in Npr1+/− mice. Our findings demonstrate that epigenetic upregulation of Npr1 gene transcription by ATRA and NaBu leads to attenuation of renal fibrotic markers and systolic blood pressure in mice with reduced Npr1 gene copy number, which will have important implications in prevention and treatment of hypertension-related renal pathophysiological conditions.

Regulation of natriuretic peptide receptor-A gene expression and stimulation of its guanylate cyclase activity by transcription factor Ets-1

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.

Cooperative Activation of Npr1 Gene Transcription and Expression by Interaction of Ets-1 and p300

The objective of the present study was to gain insight into the cooperative roles of Ets-1 and p300 in transcriptional regulation and expression of the Npr1 gene (coding for guanylyl cyclase-A/natriuretic peptide receptor-A). Overexpression of Ets-1 and p300 in mouse mesangial cells increased Npr1 promoter activity by 12-fold, natriuretic peptide receptor-A mRNA levels by 5-fold, and ANP-dependent intracellular accumulation of cGMP by 26-fold. Knockdown of Ets-1 and p300 expression by small interfering RNA inhibited Npr1 gene transcription by 90%. Sequential chromatin immunoprecipitation assay demonstrated a direct physical association between p300 and Ets-1 on binding to the Npr1 promoter, suggesting that a physical interaction between Ets-1 and p300 is important to enhance Npr1 gene transcription. Mutant p300 lacking histone acetyltransferase activity did not show a functional effect with Ets-1, suggesting that histone acetyltransferase activity of p300 is required for the cooperative interaction in modulating Npr1 gene transcription. Overexpression of wild-type adenovirus E1A significantly decreased the Npr1 promoter activity by 40%, whereas mutant E1A, which is incapable of binding to p300, did not show any effect. The results indicate that Npr1 gene transcription is critically controlled by histone acetyltransferase p300 and Ets-1. The present findings should yield important insights into the molecular signaling governing Npr1 gene transcription, an important regulator in the control of hypertension and cardiovascular events.

Transcriptional regulation of guanylyl cyclase/natriuretic peptide receptor-A gene.

Activation of natriuretic peptide receptor-A (NPRA) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. In the present study, we have examined the role of trans-acting factor Ets-1 in transcriptional regulation of Npr1 gene (coding for NPRA). Using deletional analysis of the Npr1 promoter, we have defined a 400 base pair (bp) region as the core promoter, which contains consensus binding sites for transcription factors including: Ets-1, Lyf-1, and GATA-1/2. Overexpression of Ets-1 in mouse mesangial cells (MMCs) enhanced Npr1 gene transcription by 12-fold. However, overexpression of GATA-1 or Lyf-1 repressed Npr1 basal promoter activity by 50% and 80%, respectively. The constructs having a mutant Ets-1 binding site or lacking this site failed to respond to Ets-1 activation of Npr1 gene transcription. Collectively, the present results demonstrate that Ets-1 greatly stimulates Npr1 gene promoter activity, implicating its critical role in the regulation and function of NPRA at the molecular level.

Inhibition of HDAC enhances STAT acetylation, blocks NF-κB, and suppresses the renal inflammation and fibrosis in Npr1 haplotype male mice

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) plays a critical role in the regulation of blood pressure and fluid volume homeostasis. Mice lacking functional Npr1 (coding for GC-A/NPRA) exhibit hypertension and congestive heart failure. However, the underlying mechanisms remain largely less clear. The objective of the present study was to determine the physiological efficacy and impact of all-trans-retinoic acid (ATRA) and sodium butyrate (NaBu) in ameliorating the renal fibrosis, inflammation, and hypertension in Npr1 gene-disrupted haplotype (1-copy; +/-) mice (50% expression levels of NPRA). Both ATRA and NaBu, either alone or in combination, decreased the elevated levels of renal proinflammatory and profibrotic cytokines and lowered blood pressure in Npr1+/- mice compared with untreated controls. The treatment with ATRA-NaBu facilitated the dissociation of histone deacetylase (HDAC) 1 and 2 from signal transducer and activator of transcription 1 (STAT1) and enhanced its acetylation in the kidneys of Npr1+/- mice. The acetylated STAT1 formed a complex with nuclear factor-κB (NF-κB) p65, thereby inhibiting its DNA-binding activity and downstream proinflammatory and profibrotic signaling cascades. The present results demonstrate that the treatment of the haplotype Npr1+/- mice with ATRA-NaBu significantly lowered blood pressure and reduced the renal inflammation and fibrosis involving the interactive roles of HDAC, NF-κB (p65), and STAT1. The current findings will help in developing the molecular therapeutic targets and new treatment strategies for hypertension and renal dysfunction in humans.

Retinoic acid and sodium butyrate suppress the cardiac expression of hypertrophic markers and proinflammatory mediators in Npr1 gene-disrupted haplotype mice

The objective of the present study was to examine the genetically determined differences in the natriuretic peptide receptor-A (NPRA) gene (Npr1) copies affecting the expression of cardiac hypertrophic markers, proinflammatory mediators, and matrix metalloproteinases (MMPs) in a gene-dose-dependent manner. We determined whether stimulation of Npr1 by all-trans retinoic acid (RA) and histone deacetylase (HDAC) inhibitor sodium butyric acid (SB) suppress the expression of cardiac disease markers. In the present study, we utilized Npr1 gene-disrupted heterozygous (Npr1(+/-), 1-copy), wild-type (Npr1(+/+), 2-copy), gene-duplicated (Npr1(++/+), 3-copy) mice, which were treated intraperitoneally with RA, SB, and a combination of RA/SB, a hybrid drug (HB) for 2 wk. Untreated 1-copy mice showed significantly increased heart weight-body weight (HW/BW) ratio, blood pressure, hypertrophic markers, including beta-myosin heavy chain (β-MHC) and proto-oncogenes (c-fos and c-jun), proinflammatory mediator nuclear factor kappa B (NF-κB), and MMPs (MMP-2, MMP-9) compared with 2-copy and 3-copy mice. The heterozygous (haplotype) 1-copy mice treated with RA, SB, or HB, exhibited significant reduction in the expression of β-MHC, c-fos, c-jun, NF-κB, MMP-2, and MMP-9. In drug-treated animals, the activity and expression levels of HDAC were significantly reduced and histone acetyltransferase activity and expression levels were increased. The drug treatments significantly increased the fractional shortening and reduced the systolic and diastolic parameters of the Npr1(+/-) mice hearts. Together, the present results demonstrate that a decreased Npr1 copy number enhanced the expression of hypertrophic markers, proinflammatory mediators, and MMPs, whereas an increased Npr1 repressed the cardiac disease markers in a gene-dose-dependent manner.

Transforming growth factor β1 antagonizes the transcription, expression and vascular signaling of guanylyl cyclase/natriuretic peptide receptor A - role of δEF1.

The objective of this study was to determine the role of transforming growth factor β1 (TGF‐β1) in transcriptional regulation and function of the guanylyl cyclase A/natriuretic peptide receptor A gene (Npr1) and whether cross‐talk exists between these two hormonal systems in target cells. After treatment of primary cultured rat thoracic aortic vascular smooth muscle cells and mouse mesangial cells with TGF‐β1, the Npr1 promoter construct containing a δ‐crystallin enhancer binding factor 1 (δEF1) site showed 85% reduction in luciferase activity in a time‐ and dose‐dependent manner. TGF‐β1 also significantly attenuated luciferase activity of the Npr1 promoter by 62%, and decreased atrial natriuretic peptide‐mediated relaxation of mouse denuded aortic rings ex vivo. Treatment of cells with TGF‐β1 increased the protein levels of δEF1 by 2.4–2.8‐fold, and also significantly enhanced the phosphorylation of Smad 2/3, but markedly reduced Npr1 mRNA and receptor protein levels. Over‐expression of δEF1 showed a reduction in Npr1 promoter activity by 75%, while deletion or site‐directed mutagenesis of δEF1 sites in the Npr1 promoter eliminated the TGF‐β1‐mediated repression of Npr1 transcription. TGF‐β1 significantly increased the expression of α‐smooth muscle actin and collagen type I α2 in rat thoracic aortic vascular smooth muscle cells, which was markedly attenuated by atrial natriuretic peptide in cells over‐expressing natriuretic peptide receptor A. Together, the present results suggest that an antagonistic cascade exists between the TGF‐β1/Smad/δEF1 pathways and Npr1 expression and receptor signaling that is relevant to renal and vascular remodeling, and may be critical in the regulation of blood pressure and cardiovascular homeostasis.