A common genetic variant in the 3'-UTR of vacuolar H+-ATPase ATP6V0A1 creates a micro-RNA motif to alter chromogranin A processing and hypertension risk

Abstract

Background: The catecholamine release-inhibitor catestatin and its precursor chromogranin A (CHGA) may constitute "intermediate phenotypes" in the analysis of genetic risk for cardiovascular disease such as hypertension. Previously, the vacuolar H(+)-ATPase subunit gene ATP6V0A1 was found within the confidence interval for linkage with catestatin secretion in a genome-wide study, and its 3'-UTR polymorphism T+3246C (rs938671) was associated with both catestatin processing from CHGA and population blood pressure. We explored the molecular mechanism of this effect by experiments with transfected chimeric photoproteins in chromaffin cells.

Methods and results: Placing the ATP6V0A1 3'-UTR downstream of a luciferase reporter, we found that the C (variant) allele decreased overall gene expression. The 3'-UTR effect was verified by coupled in vitro transcription/translation of the entire/intact human ATP6V0A1 mRNA. Chromaffin granule pH, monitored by fluorescence of CHGA/EGFP chimera during vesicular H(+)-ATPase inhibition by bafilomycin A1, was more easily perturbed during coexpression of the ATP6V0A1 3'-UTR C-allele than the T-allele. After bafilomycin A1 treatment, the ratio of CHGA precursor to its catestatin fragments in PC12 cells was substantially diminished, though the qualitative composition of such fragments was not affected (on immunoblot or matrix-assisted laser desorption ionization (MALDI) mass spectrometry). Bafilomycin A1 treatment also decreased exocytotic secretion from the regulated pathway, monitored by a CHGA chimera tagged with embryonic alkaline phosphatase. 3'-UTR T+3246C created a binding motif for micro-RNA hsa-miR-637; cotransfection of hsa-miR-637 precursor or antagomir/inhibitor oligonucleotides yielded the predicted changes in expression of luciferase reporter/ATP6V0A1-3'-UTR plasmids varying at T+3246C.

Conclusions: The results suggest a series of events whereby ATP6V0A1 3'-UTR variant T+3246C functioned: ATP6V0A1 expression probably was affected through differential micro-RNA effects, altering vacuolar pH and consequently CHGA processing and exocytotic secretion.

Figure 1

ATP6V0A1 3′-UTR variant T+3246C (rs938671) influences gene expression. In each case, results are plotted as the mean value ± SEM. A. Luciferase reporter/3′-UTR variant transfection into PC12 cells (n=4 per group at each time point). Transcription is driven by the SV40 early promoter. The structure of the reporter plasmid is given in supplemental Figure 1. B. In vitro transcription/translation of full-length human ATP6V0A1 cDNA. Full-length cDNAs for human ATP6V0A1 (including 3′-UTR in two versions: T+3246C) were subcloned into a plasmid with the prokaryotic SP6 promoter. SP6-luciferase was used as positive control, while vehicle (H₂O) was used as negative control. Using a Transcend^™ non-radioactive translation detection system (left panel), proteins synthesized in a rabbit reticulocyte lysate were labeled with biotinylated lysine, and then detected by streptavidin-HRP chemiluminescence. Using the immunoblot technique (right panel), ATP6V0A1 was detected by a rabbit polyclonal antibody (sc-28801, Santa Cruz Biotechnology, Santa Cruz, CA). Both strategies showed higher expression of ATP6V0A1 from wild-type (T-allele) than variant (C-allele) 3′-UTRs.

Figure 1

ATP6V0A1 3′-UTR variant T+3246C (rs938671) influences gene expression. In each case, results are plotted as the mean value ± SEM. A. Luciferase reporter/3′-UTR variant transfection into PC12 cells (n=4 per group at each time point). Transcription is driven by the SV40 early promoter. The structure of the reporter plasmid is given in supplemental Figure 1. B. In vitro transcription/translation of full-length human ATP6V0A1 cDNA. Full-length cDNAs for human ATP6V0A1 (including 3′-UTR in two versions: T+3246C) were subcloned into a plasmid with the prokaryotic SP6 promoter. SP6-luciferase was used as positive control, while vehicle (H₂O) was used as negative control. Using a Transcend^™ non-radioactive translation detection system (left panel), proteins synthesized in a rabbit reticulocyte lysate were labeled with biotinylated lysine, and then detected by streptavidin-HRP chemiluminescence. Using the immunoblot technique (right panel), ATP6V0A1 was detected by a rabbit polyclonal antibody (sc-28801, Santa Cruz Biotechnology, Santa Cruz, CA). Both strategies showed higher expression of ATP6V0A1 from wild-type (T-allele) than variant (C-allele) 3′-UTRs.

Figure 2

Chromaffin granule pH (pH_ves) in PC12 cells: Estimation by fluorescence intensity of a transfected/expressed human CHGA/EGFP chimera, and effects of ATP6V0A1. A. CHGA/EGFP fluorescence as a function of pH. PC12 cells expressing CHGA/EGFP were subjected to pH calibration as described in Methods. The cells display punctate fluorescence in a subplasmalemmal distribution typical of chromaffin granules, as well as a proportional log/linear relationship between fluorescence intensity and pH, over the range pH=5.0~8.0 (n=10 determinations at each calibration pH). The resting/basal intragranular pH (pH_ves) was estimated by interpolation at pH=5.8±0.3. B. ATP6V0A1 and chromaffin granule pH. CHGA/EGFP was co-transfected/co-expressed with the full-length ATP6V0A1 cDNA (3′-UTR T+3246C alleles) to monitor pH_ves using fluorescence intensity, as described in Methods and in panel 2A. Fluorescence was monitored every 30 sec over a 25-min period after exposure to bafilomycin (100 nM), with n=6 replicates per allele group.

Figure 2

Chromaffin granule pH (pH_ves) in PC12 cells: Estimation by fluorescence intensity of a transfected/expressed human CHGA/EGFP chimera, and effects of ATP6V0A1. A. CHGA/EGFP fluorescence as a function of pH. PC12 cells expressing CHGA/EGFP were subjected to pH calibration as described in Methods. The cells display punctate fluorescence in a subplasmalemmal distribution typical of chromaffin granules, as well as a proportional log/linear relationship between fluorescence intensity and pH, over the range pH=5.0~8.0 (n=10 determinations at each calibration pH). The resting/basal intragranular pH (pH_ves) was estimated by interpolation at pH=5.8±0.3. B. ATP6V0A1 and chromaffin granule pH. CHGA/EGFP was co-transfected/co-expressed with the full-length ATP6V0A1 cDNA (3′-UTR T+3246C alleles) to monitor pH_ves using fluorescence intensity, as described in Methods and in panel 2A. Fluorescence was monitored every 30 sec over a 25-min period after exposure to bafilomycin (100 nM), with n=6 replicates per allele group.

Figure 3

CHGA proteolytic processing to fragments during inhibition of the vacuolar pH gradient in chromaffin cells. A. Immunoblot after SDS-PAGE. Rat pheochromocytoma PC12 cells (n=3 replicate cultures) were treated with bafilomycin A1 (100 nM) or vehicle (DMSO) for 22 hours, then lysed, subjected to SDS-PAGE, and immunoblotted with anti-rat catestatin antibody (top and middle panels). The upper panel (Mr 25–75 kDa) was exposed for 30 sec. The middle panel (Mr 15–37 kDa) shows a darker exposure (45 min) of the lower part of the same immunoblot. A parallel blot was stained with anti-actin antibody (bottom panel), to indicate comparable protein load/lane. B. Mass spectrometry. Cell lysates from the vehicle and bafilomycin A1 treated cells were subjected to immunoprecipitation with anti-rat catestatin antibody followed by MALDI-TOF analysis. Calculated mass (M/Z) values for fragments of the parent molecule (rat Chga) are listed in the table.

Figure 4

V-ATPase inhibition by bafilomycin A1 alters trafficking of CHGA to the regulated secretory pathway in chromaffin cells. Secretion of EAP enzymatic activity of chimeric CHGA-EAP photoproteins exposed for 18 hours to bafilomycin A1 (10 nM) or vehicle (DMSO), is shown. Expression of each chimera is driven by the strong pCMV promoter. Units for basal secretion and stimulated secretion are % (of cell total stores). Sorting index is a dimensionless ratio. Values represent the mean ± SEM, for n=3 replicates per condition.

Figure 5

3′-UTR variant T+3246C influences miRNA hsa-miR-637 regulation of ATP6V0A1 gene expression. A. Computation. The calculated miRNA::mRNA interaction is shown, with the SNP highlighted. B and C. Luciferase reporter activity of luciferase/ATP6V0A1 3′-UTR plasmids co-transfected with hsa-miR-637 precursor (B), inhibitor (C), or their negative control; values represent the mean ± SEM in n=6 replicates for each condition.