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 (1-4). 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 an...

Objectives The objective of this study was to investigate the efficiency of uptake of PEGylated polylactide-co-gycolide (PLGA) nanoparticles by breast cancer cells. Methods Nanoparticles of PLGA containing various amounts of polyethylene glycol (PEG, 5%–15%) were prepared using a double emulsion solvent evaporation method. The nanoparticles were loaded with coumarin-6 (C6) as a fluorescence marker. The particles were characterized for surface morphology, particle size, zeta potential, and for cellular uptake by 4T1 murine breast cancer cells. Key findings Irrespective of the amount of PEG, all formulations yielded smooth spherical particles. However, a comparison of the particle size of various formulations showed bimodal distribution of particles. Each formulation was later passed through a 1.2 μm filter to obtain target size particles (114–335 nm) with zeta potentials ranging from −2.8 mV to −26.2 mV. While PLGA-PEG di-block (15% PEG) formulation showed significantly higher 4T1 cellular uptake than all other formulations, there was no statistical difference in cellular uptake among PLGA, PLGA-PEG-PLGA tri-block (10% PEG), PLGA-PEG di-block (5% PEG) and PLGA-PEG di-block (10% PEG) nanoparticles. Conclusion These preliminary findings indicated that the nanoparticle formulation prepared with 15% PEGylated PLGA showed maximum cellular uptake due to it having the smallest particle size and lowest zeta potential.

Atrial natriuretic peptide (ANP) exerts its biological effects by binding to guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), 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 this study was to elucidate the mechanism by which Ets-1 contributes towards 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 Npr1 promoter. Overexpression of Ets-1 significantly enhanced NPRA mRNA levels, protein expression, guanylyl cyclase (GC) activity, and ANP-stimulated intracellular accumulation of cGMP levels in transfected cells. Depletion of endogenous Ets-1 by small interfering RNA (siRNA) dramatically decreased promoter activity by 80%. Moreover, methylation of the Npr1 promoter region −356 to +55 significantly reduced the promoter activity and hypermethylation around the Ets-1 binding sites directly reduced the Ets-1 binding to Npr1 promoter. Collectively, our present results demonstrate that the Npr1 gene transcription and GC activity of the receptor are critically controlled by Ets-1 in target cells.

Chitosan-modified PLGA nanoparticles showed significant uptake by neoplastic 4T1 cells, and were distributed to several major organs frequently seen as sites of cancer metastasis in mice.

Activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) by cardiac hormones atrial and brain natriuretic peptides produces the second messenger cGMP, which activates downstream signaling and biological effects of NPRA including vasorelaxation, anti‐mitogenic, and anti‐hypertrophic effects. The objective of the present study was to gain insight into the signaling mechanism of all‐trans retinoic acid (ATRA) in the regulation of Npr1 gene (coding for GC‐A/NPRA) transcription in cultured mouse mesangial cells. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin,transferrin, and sodium selenite) and were transiently transfected using Lipofectamine. The results showed that ATRA significantly increased Npr1 mRNA, NPRA protein expression, and intracellular accumulation of cGMP in a dose‐dependent manner. ATRA caused a 7‐fold increase in luciferase activity from the region −365 to +55 base pairs of the Npr1 promoter. ATRA‐dependent Npr1 gene transcription was effectively inhibited by Ro 41‐5253, a specific antagonist for RARα. ATRA enhanced in vivo binding of Ets‐1 to Npr1 promoter as confirmed by ChIP assay. Moreover, sequential ChIP assay showed simultaneous presence of RARα and Ets‐1 in the Npr1 promoter containing Ets‐1 binding sites. Collectively, our results show that retinoic acid induces Npr1 gene transcription and expression via RARα and Ets‐1 transcription factors. The findings of the present studies will have important implications in prevention of high blood pressure and cardiac remodeling.