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; 1/2), wild-type (2-copy; 1/1), and gene-duplicated heterozygous (3-copy; 11/1) mice. Npr1 1/2 mice exhibited increased renal HDAC and reduced histone acetyltransferase (HAT) activity; on the contrary, Npr111/1 mice showed decreased HDAC and enhanced HAT activity compared with Npr1 1/1 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 a, 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 a-smooth muscle actin (a-SMA) and proliferating cell nuclear antigen (PCNA) compared with 2-and 3-copy mice. Treatment with ATRA and NaBu synergistically attenuated the expression of a-SMA and PCNA and reduced systolic blood pressure in Npr1 1/2 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.

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is activated by cardiac hormones atrial and brain natriuretic peptides (ANP, BNP) and produces intracellular second messenger cGMP. The physiological actions of ANP/NPRA/cGMP signaling cascade largely include diuresis, natriuresis, vasorelaxation, and organ protection, which maintains blood pressure and opposed renal, cardiac, and vascular injury and remodeling. However, the mechanisms that contribute to the activation of Npr1 (coding for GC-A/NPRA) expression is not well understood. The present study was aimed at understanding the epigenetic mechanisms of DNA methyltransferase (DNMT) inhibitor, 5-Azacytidine (5-Aza)-mediated Npr1 gene transcription. The studies were carried out in mouse mesangial cells (MMCs) and rat thoracic aortic smooth muscle (RTASM) cells, cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with 5-Aza for 24 h. Western blot analysis showed that 5-Aza enhanced NPRA protein levels by 5-fold and 7-fold in a dose-dependent manner in MMCs and RTASM cells, respectively, and greatly stimulated intracellular accumulation of cGMP in ANP-treated cells. Treatment with 5-Aza attenuated DNMT activity and repressed DNMT1 protein levels in cells. The MethPrimer search result showed the presence of three cytosine-phosphate-guanine (CpG) islands in the Npr1 full length promoter and 5'-untranslated region (UTR), namely island 1 (-886 to -752, 134 bp), island 2 (-310 to -158, 152 bp), and island 3 (-154 to +310, 464 bp) from the transcription start site. Addition of 5'-UTR encompassing +56 to +359 bp region to the Npr1 promoter constructs, (-1982/+55) and proximal promoter (-356/+55), was inhibitory to Npr1 transcriptional activity, independent of the cell type. Furthermore, treatment with 5-Aza distinctly induced Npr1 promoter activity of the construct -356/5'-UTR by 10-fold in MMCs suggesting that the repressive effect of DNA methylation on Npr1 gene transcription might be via 5'-UTR. The results demonstrate that 5-Aza acts as a potent inducer of Npr1 expression and signaling and provides new insights in the epigenetic control of Npr1, an important player in the control of hypertension and cardiorenal axis. Supported by the NIH grants (HL062147 and DK133833). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Atrial natriuretic peptide (ANP) exerts its antihypertensive effects by binding to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which generates the second messenger cGMP. The present study was aimed at understanding the epigenetic signaling governing Npr1 (coding for GC‐A/NPRA) gene transcription. The mouse mesangial cells were cultured in DMEM containing 10% FBS and ITS (insulin, transferrin, and sodium selenite), transfected using Lipofectamine‐2000, and treated with histone deacetylase (HDAC) inhibitors. Luciferase assay showed that trichostatin A (TSA, pan‐inhibitor) and mocetinostat (MGCD0103, class I inhibitor) induced Npr1 promoter activity by 8‐fold and 10‐fold, Npr1 mRNA levels by 4‐ and 5.3‐fold, and protein expression by 2.5‐ and 3‐fold, respectively. TSA and MGCD0103 inhibited HDAC activity by 25% and 50%, respectively. Furthermore, TSA and MGCD0103 enhanced acetylation and binding of H3‐K9/14, H4‐K12, and Sp1 to Npr1 promoter. Taken together, the present results demonstrate that TSA and MGCD0103 enhanced Npr1 gene expression via inhibition of HDAC1 and HDAC2 and increased histone H3, H4, and Sp1 acetylation. The present findings provide a novel regulatory mechanism for Npr1 gene transcription, an important player in the control of hypertension and cardiovascular homeostasis. This work was supported by the NIH grant (HL57531).