Ligand-Dependent Downregulation of Guanylyl Cyclase/Natriuretic Peptide Receptor-A: Role of miR-128 and miR-195

Abstract

Cardiac hormones act on the regulation of blood pressure (BP) and cardiovascular homeostasis. These hormones include atrial and brain natriuretic peptides (ANP, BNP) and activate natriuretic peptide receptor-A (NPRA), which enhance natriuresis, diuresis, and vasorelaxation. In this study, we established the ANP-dependent homologous downregulation of NPRA using human embryonic kidney-293 (HEK-293) cells expressing recombinant receptor and MA-10 cells harboring native endogenous NPRA. The prolonged pretreatment of cells with ANP caused a time- and dose-dependent decrease in ¹²⁵I-ANP binding, Guanylyl cyclase (GC) activity of receptor, and intracellular accumulation of cGMP leading to downregulation of NPRA. Treatment with ANP (100 nM) for 12 h led to an 80% decrease in ¹²⁵I-ANP binding to its receptor, and BNP decreased it by 62%. Neither 100 nM c-ANF (truncated ANF) nor C-type natriuretic peptide (CNP) had any effect. ANP (100 nM) treatment also decreased GC activity by 68% and intracellular accumulation cGMP levels by 45%, while the NPRA antagonist A71915 (1 µM) almost completely blocked ANP-dependent downregulation of NPRA. Treatment with the protein kinase G (PKG) stimulator 8-(4-chlorophenylthio)-cGMP (CPT-cGMP) (1 µM) caused a significant increase in ¹²⁵I-ANP binding, whereas the PKG inhibitor KT 5823 (1 µM) potentiated the effect of ANP on the downregulation of NPRA. The transfection of miR-128 significantly reduced NPRA protein levels by threefold compared to control cells. These results suggest that ligand-dependent mechanisms play important roles in the downregulation of NPRA in target cells.

Keywords: ANP-NPRA/cGMP signaling; GC/NPRA; HEK-293 cells; MA-10 cells; internalization.

Figure 1

Effect of ANP pretreatment on ¹²⁵I-ANP binding and ANP-induced eGFP-NPRA internalization in stably expressing recombinant HEK-293 cells and endogenously expressing MA-10 cells. (A,B) Confluent HEK-293 cells and MA-10 cells were pretreated with varying concentrations of ANP for the indicated times at 37 °C. Cells were transferred to 4 °C and ¹²⁵I-ANP binding was done in assay medium containing 0.1% BSA as described under the Section 4 for 30 min at 4 °C. Cells. (C) A series of single confocal plane images were taken from HEK-293 cells fixed with 4.0% formaldehyde to visualize the internalization of NPRA after stimulation by 100 nM ANP. The images of mid-focal planes were collected from 5–6 independent experiments. The magnified images of areas have been indicated in red squares. (D) The highlighted changes in the images have been quantified and scored. Values are expressed as means ± SE of 6–7 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (untreated vs. drug-treated group).

Figure 2

Effect of BNP, c-ANF and CNP pretreatment on ¹²⁵I-ANP binding to NPRA in HEK-293 and MA-10 cells. (A) Confluent HEK-293 cells and (B) MA-10 cells were pretreated with 100 nM of BNP, c-ANF, or CNP for indicated times at 37 °C. Cells were transferred to 4 °C and receptor binding was carried out as described under the Section 4. Values represents mean ± SE of 7 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (untreated vs. drug-treated group).

Figure 3

Effect of ANP pretreatment on guanylyl cyclase activity in HEK-293 cells and MA-10 cells. (A) HEK-293 cells and (B) MA-10 cells were treated with 100 nM ANP for 0, 8, 16, and 24 h. Cells were incubated with glycine acetate buffer (pH 3.8) for 2 min, plasma membranes were prepared. GC activity was determined as described under the Section 4. Values represent mean ± SE of 8 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (untreated vs. drug-treated group).

Figure 4

Effects of A71915 and KT 5823 pretreatment on ¹²⁵I-ANP binding to NPRA in HEK-293 and MA-10 cells. Confluent HEK-293 cells and MA-10 cells were pretreated with 100 nM ANP for the indicated times at 37 °C after which cells were transferred to 4 °C. Receptor binding was carried out as indicated under the Section 4. (A) Effect of NPRA antagonist, A71915 and (B) effect of PKG inhibitor, KT-5823 on ¹²⁵I-ANP binding in HEK-293 and MA-10 cells. The bars represent the mean ± SE of 6 determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 (untreated vs. drug-treated group).

Figure 5

Effect of ANP pretreatment on the synthesis and immunofluorescence analysis of cGMP in HEK-293 and MA-10 cells. To assay the stimulation of intracellular accumulation of cGMP, (A) HEK-293 cells and (B) MA-10 cells were treated with 100 nM ANP for different times (0, 2, 4, 6 and 8 h) in the presence of IBMX and cGMP was assayed by ELISA as described in the Section 4. (C) Untreated cells stained with DyLight™405 anti-rabbit antibody without prior incubation with the first rabbit antiserum. Cells were treated with ANP for 1, 5, 10, 15 or 30 min, which showed the accumulation of cGMP (blue). (D) Bars represent the densitometric analysis of the cGMP fluorescence intensities. Values represent means ± SE of 6 independent experiments. Images of mid-focal planes were used from 6 independent experiments. ** p < 0.01, *** p < 0.001 (untreated vs. drug-treated group).

Figure 6

The effect of CPT-cGMP and okadaic acid pretreatments on ¹²⁵I-ANP binding to NPRA and guanylyl cyclase activity. (A) Confluent MA-10 cells were pretreated with 1 µM concentrations of 8-(4-chlorophenylthio)-cGMP (CPT-cGMP) for the indicated times at 37 °C and then transferred to 4 °C. Receptor binding was carried out as described under the Section 4. (B) MA-10 cells were treated with 1 mM CPT-cGMP for 24 h. The medium was aspirated. Cells were incubated with glycine acetate buffer (pH 3.8) for 2 min and plasma membranes were prepared to assay the GC activity. (C) Confluent MA-10 cells were pretreated with CPT-cGMP in the presence or absence of indicated concentrations of okadaic acid (OA) for 24 h at 37 °C. Receptor binding was carried for 30 min at 4 °C as described in the Section 4. Values are expressed as means ± SE of 6 determinations. ** p < 0.01, *** p < 0.001 (untreated vs. drug-treated group).

Figure 7

Effect of miRNA overexpression and ANP treatment on NPRA protein levels. HEK-293 cells were transfected with miR-128, miR-195, or vector control (miR control). At 48 h post-transfection, cells were treated with ANP (100 nM) for the indicated times of short-term (0, 2.5, 10, and 30 min) and long-term (0, 1, 4, 8, 12, and 24 h) ANP treatments. After short-term treatments, (A) Western blot and (B) densitometric analysis of NPRA protein was done with the control miR. β-actin level is shown as loading control, (C) Western blot and (D) densitometric analysis of NPRA protein levels after transfection with miR-128, and (E) Western blot and (F) densitometric analysis of NPRA protein levels are shown after transfection with miR-195. Similarly, after long-term treatments, (G) Western blot and (H) densitometric analysis of NPRA protein levels after transfection with the control miR. (I) Western blot and (J) densitometric analysis of NPRA protein levels after transfection with miR-128, and (K) Western blot and (L) densitometric analysis of NPRA protein levels in cells after transfection with miR-195. β-actin level is shown are loading control. Bar represents mean ± S.E. of 5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (untreated vs. drug-treated group).

Figure 8

Schematic representation of the role of miR-128 and miR-195 in the downregulation of NPRA. miR-128 significantly downregulated NPRA compared to miR-195. ANP-dependent downregulation of NPRA (ANP-NPRA complex) as compared to control cells, and downregulation of NPRA through miR affects ANP/NPRA/cGMP signaling.