Functional silencing of guanylyl cyclase/natriuretic peptide receptor-A by microRNA interference: analysis of receptor endocytosis

Abstract

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is the principal receptor for the regulatory action of atrial and brain natriuretic peptides (ANP and BNP) and an important effector molecule in controlling of extracellular fluid volume and blood pressure homeostasis. We have utilized RNA interference to silence the expression of GC-A/NPRA gene (Npr1), providing a novel system to study the internalization and trafficking of NPRA in intact cells. MicroRNA (miRNA)-mediated small interfering RNA (siRNA) elicited functional gene-knockdown of NPRA in stably transfected human embryonic kidney 293 (HEK-293) cells expressing a high density of recombinant NPRA. We artificially expressed three RNA polymerase II-driven miRNAs that specifically targeted the Npr1 gene, but shared no significant sequence homology with any other known mouse genes. Reverse transcription-PCR (RT-PCR) and Northern blot analyses identified two highly efficient Npr1 miRNA sequences to knockdown the expression of NPRA. The Npr1 miRNA in chains or clusters decreased NPRA expression more than 90% as compared with control cells. ANP-dependent stimulation of intracellular accumulation of cGMP and guanylyl cyclase activity of NPRA were significantly reduced in Npr1 miRNA-expressing cells by 90-95% as compared with control cells. Treatment with Npr1 miRNA caused a drastic reduction in the receptor density subsequently a deceased internalization of radiolabeled (125)I-ANP-NPRA ligand-receptor complexes. Only 12%-15% of receptor population was localized in the intracellular compartments of microRNA silenced cells as compared to 70%-80% in control cells.

Keywords: Atrial natriuretic peptide; gene-knockdown; guanylyl cyclase/natriuretic peptide receptor-A; human embryonic kidney cells; microRNA; receptor internalization.

Figure 1

Scheme and identification of recombinant plasmids: A. The double-stranded oligo coding for mature miRNA was cloned into the linearized pcDNA™6.2-GW/EmGFP-miR plasmid vector. B. Upon transcription, the mature Npr1 miRNA sequence and its complement of pre-miRNA have a short internal loop that is separated by a larger terminal loop. C. Chaining of two Npr1 miRNAs is derived from different vector into one miRNA expression vector.

Figure 2

Transfection of pCMV-Npr1miRNA of different oligonucleotide sequences efficiently silences Npr1 gene expression in recombinant HEK 293 cells: A. Representative RT-PCR image from three independent experiments showing the effect of pCMV-Npr1miR1595, pCMV-Npr1miR2931, and pCMV-Npr1miR2187 on Npr1 gene expression in recombinant HEK 293 cells. B. The effect of target mRNA reduction is measured by the percentage of the transcript level in miRNA transfected cells relative to the transcript levels in controlled cells. C. Specific probe for Npr1 cDNA was used to detect the mRNA level of Npr1 by Northern blot after 48 h of transfection. D. The ratio of specific band to β-actin in recombinant HEK 293 cells was standardized as 100%. and the changes in Npr1 mRNA level analyzed from Northern blots.

Figure 3

Npr1microRNA cluster decreases the NPRA mRNA level in HEK 293 cells: A. Example of a representative reverse transcription-PCR experiment evaluating the transcript level of NPRA and β-actin in HEK 293 cells after treatment with different controls and pCMV-Npr1miR-chain 2931-1595. B. Results are expressed as a percentage of untreated control of the ratio of optical densities of NPRA RT-PCR product versus β-actin. Vertical bars represent the mean ± SE from three independent experiments. P < 0.05 versus control.

Figure 4

Fluorescence-based evaluation of the silencing efficacy of Npr1 miRNA: HEK-293 cells were transfected with Npr1-RFP (panel A) or co-transfected with Npr1-RFP and pcDNA6.2-GW/GFP-miR vectors containing miR negative target (panel B) and Npr1-directed miRNA inserts (panel C). Cells were fixed after 48 h of transfection. A single field of cells is shown in each row to reveal nuclei (DAPI), EmGFP (autofluorescence), Npr1-RFP (autofluorescence), and overlay of all fields in each panel. The Npr-1 miRNA transfected cells in which EmGFP fluorescence is detectable show markedly reduced Npr1 RFP expression, indicating tight correlation between Npr1 miR EmGFP expression and Npr1-RFP knockdown. In control miR- negative transfected wells, Npr1-RFP signals in non-EmGFP and EmGFP-expressing cells are similar, indicating miR-negative on Npr1-RFP expression.

Figure 5

Saturation binding of ¹²⁵I-ANP in 293 cells stably expressing recombinant NPRA: HEK-293 cells were transfected with pCMV-Npr1miR-chain 2931-1595. The equilibrium binding of ¹²⁵I-ANP was done in 2 ml of assay medium at 4°C for 1 h in the presence or absence of the indicated concentrations of unlabeled and labeled hormones. The results shown are representative of four binding experiments performed on triplicate dishes with specific ANP binding. The inset shows the Scatchard analysis of ¹²⁵I-ANP binding data.

Figure 6

Knockdown of the Npr1 gene in recombinant HEK-293 cells reduced internalization of ¹²⁵I-ANP: Recombinant HEK-293 cells were transfected in the presence or absence of pCMV-Npr1miR-chain 2931-1595. In 6-cm² dishes, NPRA-HEK-293 cells expressing pCMV-Npr1miR-chain 2931-1595 were allowed to bind ¹²⁵I-ANP at 4°C for 1 h. Cells were washed four times, each time with 2 ml of assay medium, to remove unbound ligand, then reincubated in fresh medium at 37°C. At the indicated time points, dishes were transferred at 4°C and the media collected. Cell-surface-associated (acid-sensitive) radioactivity was eluted with glycine acidic buffer (pH 3.4) and cells were dissolved in 1 M NaOH to measure internalized (acid-resistant) radioactivity. A. Cell-surface-associated, B) internalized, and C) released ¹²⁵I-ANP radioactivity were respectively determined in the acid elute, cell extract, and culture medium, respectively.

Figure 7

Knockdown of the Npr1 gene in recombinant HEK-293 cells reduced degraded and intact ¹²⁵I-ANP release: NPRA-HEK-293 cells were transfected in the presence or absence of pCMV-Npr1miR-chain 2931-1595. In 60-cm² dishes, NPRA- HEK-293 cells expressing pCMV-Npr1miR-chain 2931-1595 were allowed to bind ¹²⁵I-ANP at 4°C for 1 h. The cells were washed four times with ice-cold assay medium to remove the unbound ligand, then reincubated in 2 ml of fresh assay medium at 37°C for 30 min. After incubation, the culture medium was collected and the intact and degraded ligand products were quantified.

Figure 8

Knockdown of the Npr1 gene in recombinant HEK-293 cells inhibited cGMP stimulated with ANP: A. NPRA-recombinant HEK293 cells were transfected in the presence or absence of pcDNA^TM6.2-GW/EmGFP-miRNpr-neg and pCMV-Npr1miR-chain 2931-1595. Confluent cells were incubated at different concentrations of ANP at 37°C in the presence of 0.2 mM IBMX. After washing the dishes with serum-free assay medium, cells were dissolved in 0.5N HCl. The intracellular accumulation of cGMP was measured in cell extracts by an immunoassay kit. B. Knockdown of the Npr1 gene in recombinant HEK293 cells inhibited guanylyl cyclase activity in response to different concentration of ANP. NPRA-recombinant HEK293 cells were transfected in the presence or absence of pcDNA^TM6.2-GW/EmGFP-miRNpr-neg and pCMV-Npr1miR-chain 2931-1595. Plasma membrane was prepared, guanylyl cyclase activity was measured, and cGMP assay was done using an immunoassay ELISA kit.