Paraoxonase 2 is an ER chaperone that regulates the epithelial Na⁺ channel

Abstract

The mammalian paraoxonases (PONs) have been linked to protection against oxidative stress. However, the physiological roles of members in this family (PON1, PON2, and PON3) are still being characterized. PON2 and PON3 are expressed in the aldosterone-sensitive distal nephron of the kidney and have been shown to negatively regulate expression of the epithelial sodium channel (ENaC), a trimeric ion channel that orchestrates salt and water homeostasis. To date, the nature of this phenomenon has not been explored. Therefore, to investigate the mechanism by which PON2 regulates ENaC, we expressed PON2 along with the ENaC subunits in fisher rat thyroid (FRT) cells, a system that is amenable to biochemical analyses of ENaC assembly and trafficking. We found that PON2 primarily resides in the endoplasmic reticulum (ER) in FRT cells, and its expression reduces the abundance of each ENaC subunit, reflecting enhanced subunit turnover. In contrast, no effect on the levels of mRNAs encoding the ENaC subunits was evident. Inhibition of lysosome function with chloroquine or NH4Cl did not alter the inhibitory effect of PON2 on ENaC expression. In contrast, PON2 accelerates ENaC degradation in a proteasome-dependent manner and acts before ENaC subunit ubiquitination. As a result of enhanced ENaC subunit ubiquitination and degradation, both channel surface expression and ENaC-mediated Na+ transport in FRT cells were reduced by PON2. Together, our data suggest that PON2 functions as an ER chaperone to monitor ENaC biogenesis and redirects the channel for ER-associated degradation.

INTRODUCTION

Members of the paraoxonase (PON) family (PON1, PON2, and PON3) are known for their protective roles against oxidative stress-induced apoptosis and lipid peroxidation in several major organs and tissue types (1–5). Notably, deleting Pon1 in mice increases oxidative stress and accelerates atherosclerosis, and Pon2-deficient mice are prone to develop diet-induced atherogenesis due to elevated mitochondrial oxidative stress and inflammation (6–9). Similarly, Pon3 KO mice are more susceptible to obesity, gallstone formation, and atherosclerosis when challenged with high-fat diets (10). The mammalian PONs are evolutionarily conserved with the Caenorhabditis elegans MEC-6 and several other nematode proteins with undefined functions. MEC-6 is a type-II integral membrane protein that is essential for the gentle body touch response in nematodes (11, 12), an avoidance behavior mediated by an ion channel complex expressed in touch receptor neurons (TRNs). At the core of this mechanosensitive complex are MEC-4 and MEC-10, two members of the epithelial Na⁺ channel (ENaC)/degenerin family (11, 13). MEC-6 primarily resides in the endoplasmic reticulum (ER) and functions as a chaperone to facilitate proper folding, assembly, and surface expression of the MEC-4/MEC-10 channels (12, 14). Loss-of-function MEC-6 mutations abolish the gentle touch response in nematodes due to defects in biogenesis and surface targeting of the mechanosensitive degenerin channels (15, 16). These findings highlight the importance of the chaperone function of MEC-6 and potentially other members of this family. However, it is unknown whether mammalian PONs also exhibit chaperone-like activity. Moreover, the identities of most PON clients have not been defined.

The homology between MEC-6 and mammalian PONs, and between MEC-4/MEC-10 and ENaC, raises the possibility that PONs likewise regulate ENaC. Functional ENaC complexes consist of three homologous subunits, α (or δ), β, and γ, which form a Na⁺-selective channel in epithelial tissues (17). To date, a number of molecular chaperones have been implicated in key steps during ENaC biogenesis, trafficking, and degradation (18–27). Although Hsp70 and Erp29 facilitate the ER exit and forward trafficking of the assembled channel complex in higher cells (20, 24, 27), two Hsp40s (Scj1 and Jem1) and the Hsp70-like protein Lhs1 promote the ER-associated degradation (ERAD) of ENaC in the early secretory pathway in a yeast model (22, 25). The effects of the Lhs1 homolog, GRP170, and of an ER membrane protein Derlin-1 on ENaC ERAD have also been confirmed in higher cells (25, 28). In addition, we previously showed that both PON2 and PON3 are expressed in principal cells of the aldosterone-sensitive distal nephron, where ENaC resides (29, 30). When expressed in Xenopus oocytes, both PON2 and PON3 inhibited amiloride-sensitive whole cell Na⁺ currents by reducing ENaC surface expression (29, 30). The inhibitory effect of PON2 on ENaC activity was retained in catalytically dead PON2 mutants (29), suggesting that PON2-dependent ENaC regulation was independent of lactonase activity. Based on these data and the homology between MEC-6 and PON and MEC-4/MEC-10 and ENaC subunits, we chose to investigate the mechanism by which PON2 regulates ENaC expression. To this end, ENaC subunits were expressed in cultured fisher rat thyroid (FRT) cells, and ENaC biogenesis was monitored in the presence or absence of exogenous PON2. We found that PON2 is primarily present in the ER and reduces the abundance of ENaC subunits in the whole cell and at the cell surface by promoting their ubiquitination and proteasomal degradation. Our data suggest that PON2 functions as ER chaperone, one that is able to regulate ENaC surface and functional expression by targeting the channel for ERAD.

MATERIALS AND METHODS

Plasmids and FRT Cell Transfection

Untagged mouse Pon2 was cloned in the pCMV6 vector (OriGene, MR205558). Wild-type (WT) mouse ENaC α, β, and γ subunits were cloned in the pcDNA3 vector. In some experiments, ENaC subunits with an N-terminal hemagglutinin (HA) epitope tag and a C-terminal V5 epitope tag were used. In that case, only one ENaC subunit had an N-terminal HA tag and a C-terminal V5 epitope tag to generate trimeric channel complex of _HAα_V5βγ, α_HAβ_V5γ, or αβ_HAγ_V5, respectively. Fisher rat thyroid (FRT) cells were cultured in DMEM/F-12 medium (plus 8% FBS) and transiently transfected with plasmids (0.5 µg per construct) using Lipofectamine 3000 (Invitrogen, L3000008) according to the manufacturer’s instruction. An equal amount of a mouse PON2 cDNA or the pCMV6 vector was cotransfected with ENaC constructs to study the effect of PON2 on ENaC expression. Experiments were normally performed 24–48 h after transfection. In selected experiments, FRT cells were transfected with a WT mouse Na-Cl cotransporter (NCC) construct with an HA tag at N-terminus (31) in the presence or absence of mouse PON2. The expression of NCC in the whole cell lysates was probed using an anti-HA horseradish peroxidase (HRP) antibody (0.05 μg/mL, Sigma, 3F10) 24 h after transfection.

Immunofluorescence Staining in FRT Cells

Cells grown on the cover glass were transiently transfected with a mouse PON2 plasmid for 24 h, fixed with 4% paraformaldehyde, and permeabilized with 1% SDS. After incubation with 10% horse serum to block the nonspecific binding, cells were incubated overnight at 4°C with a monoclonal mouse anti-PON2 antibody (2 µg/mL, Thermo Fisher, MA5-17251) and antibodies against markers of different cellular organelles: rabbit anti-calnexin (5 μg/mL, Cell Signal, 2679), rabbit anti-protein disulfide-isomerase (PDI; 5 µg/mL, Cell Signal, 3501), rabbit anti-receptor-binding cancer antigen expressed on SiSo cells (RCAS1; 5 µg/mL, Cell Signal, 12290), rabbit anti-early endosome antigen 1 (EEA1; 5 µg/mL, Cell Signal, 3288), or rabbit anti-lysosomal-associated membrane protein 1 (LAMP1; 5 µg/mL, Cell Signal, 9090). Cells were subsequently labeled with fluorescent secondary antibodies, donkey anti-rabbit Alexa488 (2 µg/mL, Jackson Immuno-Research, 711-545-152) and donkey anti-mouse Cy3 (0.5 µg/mL, Jackson Immuno-Research, 705-165-150), for 2 h at room temperature. Nuclei were stained with TO-PRO-3 (1:1,000, Thermo Fisher, T3605). Slides were mounted with VECTASHIELD antifade mounting media (Vector Laboratories, H-1000-10). Slides were imaged on a Leica TCS SP5 CW-STED confocal imaging system with a ×40 glycerol immersion lens with a 1.25 numerical aperture.

Total RNA Isolation and Semiquantitative PCR Analysis

Twenty-four hours after transfection, total RNA was isolated from FRT cells expressing ENaC ± PON2 with a Direct-zol RNA MiniPrep Kit (ZymoResearch, R2051) according to the manufacturer’s instructions. cDNA was prepared with an iScript cDNA Synthesis Kit (Bio-Rad, 1708891), and semiquantitative (qPCR) was performed using the iTaq Universal SYBR Green SuperMix (Bio-Rad, 1725120) on a Bio-Rad CFX96 Touch Real-Time PCR Detection System. Primers were used for SCNN1a (Forward: 5′- CCTTCTCCTTGGATAGCCTGG-3′ and Reverse: 5′- CAGACGGCCATCTTGAGTAGC-3′), SCNN1b (Forward: 5′- GGCCCAGGCTACACCTACA-3′ and Reverse: 5′- AGCAGCGTAAGCAGGAACC-3′), SCNN1g (Forward: 5′- GCACCGACCATTAAGGACCTG-3′ and Reverse: 5′- GCGTGAACGCAATCCACAAC-3′), or the loading control GAPDH (Forward: 5′- AGGTCGGTGTGAACGGATTTG-3′ and Reverse: 5′- TGTAGACCATGTAGTTGAGGTCA-3′). Analysis of gene expression was calculated using the following equation: ΔCt = 2^{−(Ctgene-Ctreference)}.

Cycloheximide Chase Assay

FRT cells were transfected with _HAα_V5βγ ± PON2 for 24 h. To block de novo protein system, cycloheximide (CHX; Sigma-Aldrich, 239765) was added to the culture media at a final concentration of 100 µg/mL, and after 0, 2, 4, or 6 h, cells were processed for immunoblot analysis. In some experiments, cells were pretreated with 10 µM MG132 (Sigma-Aldrich, 474791) for 2 h before the CHX chase assay to block proteasomal degradation. At each time point, proteins were extracted with a detergent solution (20 mM HEPES, 100 mM NaCl, 40 mM KCl, 1 mM EDTA, 10% glycerol, 1% NP40, 0.4% deoxycholate, pH 7.4) supplemented with the protease inhibitor cocktail III (Calbiochem, 535140). Whole cell lysates were subjected to 18-well, 4%–15% gradient gels (Bio-Rad, 5671084) under reducing conditions to probe for αENaC (anti-V5, 0.2 µg/mL, Thermo Fisher, R96025), βENaC (0.4 μg/mL, StressMarq, SPC404), γENaC (0.4 µg/mL, StressMarq, SPC405), PON2 (0.5 µg/mL, Thermo Fisher, MA5-17251), actin (0.2 μg/mL Sigma, A1978), or GAPDH (0.2 µg/mL, ProteinTech, HRP-60004). Immunoblots were developed using Clarity Western ECL Blotting Substrates (Bio-Rad, 1705060) and imaged with a Bio-Rad ChemiDoc system. The band intensity for ENaC subunits at each time points was first normalized to the loading control (Actin or GAPDH) and then expressed as a relative value to the signal intensity before CHX was added to the cells at t = 0. The rate constant (k) was determined by plotting the relative protein abundance over the 6 h CHX treatment by using a one-phase exponential decay function, Y = e^−kX, where X is the CHX treatment time, and Y is the relative protein abundance at each time point. The half-life (t_1/2) of each ENaC subunit was estimated from the equation, t_1/2 = ln 2/k.

Ubiquitination Assay

FRT cells were transfected with _HAα_V5βγ ± PON2 as described earlier for 24 h. An equal amount of a human HA-ubiquitin plasmid (32) was cotransfected, and cells were pretreated with 10 µM MG132 for 4 h to enhance the levels of ubiquitinated proteins. Five percent of the whole cell lysate was saved as “input.” The remaining material was incubated overnight with 50 µL of agarose-immobilized V5 antibodies (Bethyl, S190-119) with end-over-end mixing at 4°C to pull down αENaC. Both the V5-IP and input were separated by SDS-PAGE under reducing conditions to probe for the ubiquitinated αENaC species with an anti-ubiquitin antibody (0.4 µg/mL, Santa Cruz, P4D1). Total recovered αENaC was probed with anti-V5 antibody (0.2 µg/mL, Thermo Fisher, R96025).

Surface Biotinylation Assay

To measure the surface expression of ENaC, FRT cells were cotransfected with plasmids encoding ENaC (_HAα_V5βγ) with either a mouse PON2 cDNA or the pCMV6 vector (0.5 µg per construct) till reaching confluency. Transfected FRT cells were washed four times with cold Dulbecco’s PBS with 1.0 mM CaCl₂ and 0.5 mM MgCl₂ (Thermo Fisher, 14040216) and then labeled with EZ-Link Sulfo-NHS-SS-Biotin (Thermo Fisher, 21331) for 30 min on ice. The biotin was freshly dissolved in DMSO and diluted to the final concentration of 1 mg/mL in a buffer containing 137 mM NaCl, 15 mM sodium borate (pH 9.0). Excess biotin was quenched with DMEM/F12 containing 10% FBS for 20 min on ice and then extensively washed with ice-cold PBS containing Ca²⁺ and Mg²⁺. Cells were lysed in the detergent solution mentioned earlier to extract the total protein. To isolate the biotinylated proteins, 95% of the whole cell lysates were incubated with NeutrAvidin agarose (Thermo Fisher, 29200) overnight at 4°C, as previously described (33, 34). The recovered surface fractions and 5% of the whole cell lysate were separated by SDS-PAGE and blotted for γENaC using the StressMarq antibody (0.4 µg/mL, SPC405). The blots were then stripped and reblot for PON2 (0.5 µg/mL, Thermo Fisher, MA5-17251) and GAPDH (0.3 μg/mL, Proteintech, HRP-60004).

Ussing Chamber Recording

FRT cells transfected with ENaC (HAαV5βγ) or with ENaC + PON2 were cultured on permeable Snapwell filters (Corning, 3801) until transepithelial resistance reached ≥800 MΩ, as measured via chopstick voltohmmeter. During this incubation period, cells were maintained in media containing 10 μM amiloride as previously described (35). On the day of experiment, cells were washed with PBS to remove amiloride-containing media, and the inserts were mounted on specialized sliders for use in an Ussing-style recording chamber (Physiologic Instruments, 2302). Cells were bathed bilaterally with modified Ringer’s solution containing the following (in mM): 120 NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂, and 10 glucose. Chambers were prewarmed to 37°C and the buffer was gassed continuously with 95% O₂-5% CO₂ to maintain a pH of 7.4. Cells were allowed to equilibrate until transepithelial potential stabilized (∼15 min). After equilibration, cells were clamped to 0 mV and the resulting short-circuit current (I_SC) was recorded using pClamp 10 (Molecular Devices). ENaC-mediated currents were measured as the change in I_SC induced by the application of 10 μM amiloride to the apical compartment of the chamber.

Statistical Analyses

All experiments were repeated at least three times using cells of different passages. Data were expressed as the means ± standard deviation (SD) in the main text or shown in Scatter-dot plots. Statistical analyses were performed using Prism9 (Kolmogorov–Smirnov t test for comparing two groups and one-way or two-way ANOVA followed by Sidak’s multiple comparisons for comparing three or more groups). A P value of <0.05 was considered statistically significant.

RESULTS

PON2 is Mainly Localized to the ER

MEC-6, the C. elegans ortholog of mammalian PONs, primarily resides in the ER of the nematode TRNs (14). We previously showed that PON2 negatively regulates ENaC activity when expressed in Xenopus oocytes (29). This inhibitory effect reflects a reduction in channel surface expression and did not require PON2 lactonase activity (29). Based on its residence, we hypothesized that PON-2 exhibits paraoxonase-independent chaperone activity, and one of its potential targets is ENaC.

To test these hypotheses, we first examined the subcellular localization of PON2 using indirect immunofluorescence (IF) staining in FRT cells transiently transfected with plasmids encoding mouse PON2. Cells were colabeled with an antibody against mouse PON2 and antibodies recognizing markers for several subcellular organelles. As shown in Fig. 1, the anti-PON2 antibody specifically detected mouse PON2 overexpressed in FRT cells. The specificity of this antibody was also confirmed by immunoblots shown in Figs. 2, 3, 4, 5, 6, and 7. Importantly, we found that PON2 was mainly detected in a perinuclear region in FRT cells and colocalized with both calnexin and PDI, two ER marker proteins. The staining pattern of PON2 was clearly different than that of markers for the Golgi (RCAS1), early endosome (EEA1), and lysosome (LAMP1), although the merged images indicate a small portion of PON2 might be present in these cellular compartments (Fig. 1). Overall, our data indicate that the ER is the primary location of PON2 when expressed in FRT cells.

Figure 1.

Subcellular localization of PON2 in FRT cells. FRT cells were transiently transfected with mouse PON2 and cultured on cover glasses for 24 h. Cells were coincubated with the anti-PON2 antibody and antibodies against markers of specific cellular organelles, including calnexin and protein disulfide-isomerase (PDI) for the ER, receptor-binding cancer antigen expressed on SiSo cells (RCAS1) for the Golgi apparatus, early endosome antigen 1 (EEA1) for the early sorting endosome, and lysosomal-associated membrane protein 1 (LAMP1) for the lysosome. The anti-PON2 antibody specifically recognized the overexpressed mouse PON2 but not the endogenous rat PON2 in FRT cells. Experiments were repeated three times with FRT cells of different passages. Images were taken on a Leica SP5 confocal microscope using a ×40 objective. Overlay images were shown for PON2 (red) and individual intracellular organelle marker (green). An enlarged image was shown for each group on the right, bars = 5 µm. ER, endoplasmic reticulum; FRT, fisher rat thyroid; PON2, paraoxonase 2.

Figure 2.

Coexpression of PON2 reduces the protein abundance of each ENaC subunit in FRT cells. FRT cells were transiently transfected with plasmids encoding the three ENaC subunits in the presence of mouse PON2 (+ PON2) or an equal amount of the pCMV6 vector (−PON2). In each case, only one ENaC subunit had an N-terminal HA and a C-terminal V5 tag to produce _HAα_V5βγ, α_HAβ_V5γ, or αβ_HAγ_V5, respectively. Whole cell lysates were collected 24 h after transfection. A: individual ENaC subunits were assessed using the anti-V5 antibodies. The coexpression of PON2 was confirmed. GAPDH was also probed as a loading control. Markers of molecular mass (kDa) are shown on the right. B: the abundance of each ENaC subunit (V5 signal) was normalized to GAPDH levels of the same sample and shown as values of each ENaC subunit expressed in the presence of exogenous PON2 (+PON2) relative to each ENaC subunit expressed in the absence of exogenous PON2 (−PON2). C: total mRNA was isolated from FRT cells 24 h post transfection. The message level of each ENaC subunit, analyzed using quantitative PCR using specific primers, was normalized to GAPDH levels in the same sample. Transcript levels for each ENaC subunit expressed in the presence of exogenous PON2 (+PON2) were normalized to mRNA levels for each ENaC subunit expressed in the absence of exogenous PON2 (−PON2). “n” in B and C indicates the number of times that experiments were repeated using cells of difference passages. Statistical comparisons between the PON2-expressing cells (+PON2) and control cells (−PON2) were analyzed with two-way ANOVA followed by a Sidak’s multiple comparisons test (**P < 0.01; ***P < 0.001; ns, not significant). D: FRT cells were cotransfected with HA-NCC and either mouse PON2 (+PON2) or the empty vector control (−PON2) for 24 h. Whole cell abundance of NCC was examined using anti-HA-HRP antibodies. The coexpression of PON2 was confirmed. GAPDH was also probed as a loading control. Markers of molecular mass (kDa) are shown on the left. NCC abundance in the presence of PON2 (+PON2) is shown as percentages of that in the absence of PON2. No statistically significant difference was observed when data were analyzed with a nonparametric Wilcoxon signed-rank test. ENaC, epithelial sodium channel; FRT, fisher rat thyroid; NCC, Na-Cl cotransporter; PON2, paraoxonase 2.

Figure 3.

PON2 accelerates ENaC degradation in FRT cells. A: FRT cells were transfected with plasmids encoding a tagged ENaC α subunit and the untagged β or γ subunit (_HAα_V5βγ) in the presence or absence of mouse PON2 cDNA. Twenty-four hours after transfection, cells were treated with CHX (100 µg/mL) for 0, 2, 4, or 6 h, and cell lysates were collected for immunoblotting analysis. The α subunit was probed with anti-V5 antibodies, whereas the β and γ subunits were assessed using the StressMarq ENaC antibodies. The β-antibody recognizes a nonspecific band (>100 kDa) in FRT cells. The expression of PON2 was confirmed. GAPDH was probed in the same blot as a loading control. Markers of molecular mass (kDa) are shown on the left. B: the abundance of ENaC α, β (indicated by the arrowhead), or γ subunit at each time point was normalized to GAPDH levels of the same samples and then expressed as a relative value (means ± SD) to that before the CHX treatment (T = 0). Data were fit to one phase exponential decay curves to calculate the half-life (red dashed lines) as described in the materials and methods. Experiments were repeated five times using FRT cells of different passages. Statistical comparisons between groups or different time points (+PON2 and −PON2) were analyzed with two-way ANOVA followed by a Sidak’s multiple comparisons test (*P < 0.05; ***P < 0.001). CHX, cycloheximide; ENaC, epithelial sodium channel; FRT, fisher rat thyroid; PON2, paraoxonase 2.

Figure 4.

The effect of PON2 on ENaC degradation was blunted by MG132 pretreatment. A: FRT cells expressing ENaC (_HAα_V5βγ) with or without PON2 were pretreated in the absence [control (Ctrl)] or presence of MG132 (10 µM) for 2 h before the CHX-chase assay. The blot was incubated with the following antibodies: anti-V5 for αENaC, anti-PON2, or anti-actin (loading control). The effect of MG132 treatment was confirmed by probing total ubiquitinated proteins in the whole cell lysates with anti-ubiquitin antibodies. Molecular mass (kDa) markers are shown. B–D: αENaC abundance at each time point was normalized to actin levels and then expressed as a relative value to that at T = 0 (means ± SD). Black dashed lines represent αENaC degradation in the absence of MG132 (data presented in Fig. 3A). E: mouse PON2 abundance at each time point in the presence or absence of MG132 was normalized to the loading control and then expressed as a relative value to that at T = 0 (means ± SD). Experiments were repeated five times using FRT cells of different passages. Statistical comparisons between groups or different time points (+MG132 and −MG132, or +PON2 and −PON2) were analyzed with two-way ANOVA followed by a Sidak’s multiple comparisons test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). CHX, cycloheximide; ENaC, epithelial sodium channel; FRT, fisher rat thyroid; PON2, paraoxonase 2.

Figure 5.

The inhibitory effect of PON2 on ENaC was not altered by lysosome inhibitors. A: FRT cells were transfected with ENaC (_HAα_V5βγ) or ENaC + PON2 for 24 h. To block protein degradation via the lysosomal pathway, cells were treated with 5 mM NH₄Cl or 100 µM chloroquine in the culture media for 6 h before collecting the whole cell lysates for an immunoblot assay. The blot was incubated with anti-V5 antibodies to estimate the abundance of αENaC. The expression of mouse PON2 was confirmed with an anti-PON2 antibody. The inhibitory effect of NH₄Cl or chloroquine on lysosome function was assessed by probing for the accumulation of LC3-II in whole cell lysates. GAPDH was probed as a loading control. Molecular mass markers (kDa) are shown on the left. B: summary data of PON2 effect on αENaC abundance under each treatment. The number of experimental repeats is noted in the panel as “n”. Statistical comparisons were analyzed with two-way ANOVA followed by a Sidak’s multiple comparisons test (***P < 0.001; ****P < 0.0001; ns, not significant). ENaC, epithelial sodium channel; FRT, fisher rat thyroid; PON2, paraoxonase 2.

Figure 6.

PON2 promotes αENaC ubiquitination in FRT cells. A: FRT cells were transfected with ENaC (_HAα_V5βγ) along with the PON2 plasmid or a vector control. FRT cells that did not express ENaC or PON2 were also examined to assess background levels of substrate ubiquitination. Cells were treated with MG132 for 4 h to allow cells to accumulate ubiquitinated ERAD substrates. Whole cell lysates were then prepared and incubated with anti-V5 agarose overnight at 4°C to immunoprecipitate the α subunit (V5-IPs). The ubiquitinated αENaC, total αENaC, and the expression of PON2 were probed in both the total whole cell lysates and the V5-IPs. Markers of molecular mass (kDa) are shown on the left. B: the effect of PON2 on αENaC protein abundance was quantified as the V5 signal intensity in the V5-IPs. C: the extent of αENaC ubiquitination was estimated by normalizing the ubiquitinated αENaC signal (anti-ubiquitin) to the signal corresponding to total αENaC (anti-V5) within the V5-IPs. Experiments were repeated six times using cells of different passages. Statistical comparisons between the PON2-expressing cells (+PON2) and the control cells (−PON2) were analyzed with a Kolmogorov–Smirnov t test (*P < 0.05; **P < 0.01). ENaC, epithelial sodium channel; ERAD, endoplasmic reticulum-associated degradation; FRT, fisher rat thyroid; PON2, paraoxonase 2.

Figure 7.

ENaC surface abundance was reduced by PON2. A: FRT cells were transiently transfected with ENaC (_HAα_V5βγ) and either mouse PON2 (+PON2) or an equal amount of the pCMV6 vector (−PON2). Surface proteins were labeled with biotin and recovered with NeutrAvidin beads at 4°C. The ENaC γ subunit (StressMarq γ antibody), PON2, and GAPDH were probed in both the biotinylated surface fractions and in 5% of the total volume of whole cell lysates. Molecular mass markers (kDa) are shown on the left. B: the effect of PON2 on γENaC abundance in the whole cell lysate and at the cell surface are shown in Scatter-dot plots with horizontal bars indicating the means. Experiments were repeated four times using cells of different passages. Statistical comparisons were analyzed with one-way ANOVA followed by a Sidak’s multiple comparisons test (*P < 0.05, **P < 0.01). ENaC, epithelial sodium channel; FRT, fisher rat thyroid; PON2, paraoxonase 2.

PON2 Negatively Regulates ENaC Subunits Expression by Accelerating Degradation

To determine whether PON2 negatively regulates ENaC levels in FRT cells, as was observed in Xenopus oocytes (29), we expressed murine ENaC with only one subunit containing N- and C-terminal epitope tags (_HAα_V5βγ, α_HAβ_V5γ, or αβ_HAγ_V5) in FRT cells in the presence or absence of mouse PON2. As shown in Fig. 2A, the anti-V5 antibody can recognize each of the three ENaC subunits, which has the V5-tag at the C-terminus, and the anti-PON2 antibody can specifically detect the overexpressed mouse PON2 instead of endogenous rat PON2 in FRT cells. As summarized in Fig. 2B, the presence of mouse PON2 reduced the abundance of each ENaC subunit (40 ± 38% for αENaC, 37 ± 15% for βENaC, and 39 ± 29% for γENaC) in FRT cells. To exclude the possible effects of PON2 on ENaC transcription, we performed quantitative PCR to examine the message levels of the genes encoding the three ENaC subunits. Our data suggest that the levels of SCNN1a, SCNN1b, and SCNN1g were not altered by PON2 (Fig. 2C). In contrast to its effects on ENaC subunits, PON2 did not alter the abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) in FRT cells (Fig. 2D). Moreover, we previously showed that coexpression of PON2 had no effect on the renal outer medullary potassium channel activity in oocytes (29). Together, our data suggest that the inhibitory effect of PON2 is specific to ENaC.

Based on its potential chaperone-like function, and because ENaC expression is known to be modulated by other chaperones, we asked whether the ENaC subunits are less stable in the presence of PON2. To test this hypothesis, we performed cycloheximide (CHX) chase assays in FRT cells expressing ENaC alone or ENaC with PON2. As shown in Fig. 3A, the abundance of ENaC subunits declined gradually when de novo synthesis was inhibited by CHX. This inherent instability of the ENaC subunits, even when assembled, is well established (18, 36). Consistent with our hypothesis, the degradation of all three ENaC subunits was accelerated by PON2 (Fig. 3B). Specifically, the estimated half-life of the ENaC subunits appears to be reduced from 2.4 h to 1.5 h for αENaC, from 2.3 h to 1.5 h for βENaC, and from 4.3 h to 2.6 h for γENaC. Together, our data suggest that PON2 reduces ENaC expression by accelerating subunit degradation.

PON2 Facilitates the ER-Associated Degradation of ENaC

As PON2 is primarily localized to the ER in FRT cells (Fig. 1), we hypothesized that PON2, like many other ER chaperones (37), promotes the ERAD of misfolded ENaC subunits. Since ERAD substrates are delivered to the proteasome, the effect of PON2 on ENaC degradation should be blunted when the proteasomal function is inhibited. Therefore, we pretreated cells with 10 µM MG132 for 2 h before the CHX chase assay and selectively monitored the disappearance of the α subunit (Fig. 4A). As expected, the degradation of αENaC was slowed by MG132 pretreatment in the presence or absence of PON2 (Fig. 4, B and C). More importantly, there was no difference in the rates of ENaC degradation between cells expressing ENaC alone and cells coexpressing ENaC and PON2 when proteasomal degradation was blocked by MG132 (Fig. 4D). As another control for this experiment, we showed that MG132 treatment increased the total cellular pool of ubiquitinated proteins (Fig. 4A, bottom). Combined with the results presented earlier, our data strongly suggest that PON2 delivers ENaC to the ERAD pathway. Interestingly, we also found that MG132 delayed the degradation of mouse PON2 in FRT cells (Fig. 4E), suggesting that the chaperone itself is somewhat unstable.

Some ERAD substrates escape the ER and are destroyed after transit to the lysosome (38). Therefore, we also tested whether inhibiting lysosome function, and by definition lysosomal proteases, with NH₄Cl or chloroquine would reverse the PON2 effect. As a control for this study, both NH₄Cl and chloroquine increased the levels of LC3-II (Fig. 5A), a marker that accumulates if lysosomal degradation is inhibited (39, 40). In contrast, the inhibitory effect of PON2 on ENaC expression was unchanged in cells treated with chloroquine or NH₄Cl (Fig. 5B). Taken together, our data suggest that PON2 mainly affects ENaC degradation through the proteasome pathway.

PON2 Promotes αENaC Ubiquitination

Ubiquitination is a critical modification that targets misfolded proteins for ERAD in the early biosynthetic pathway. Factors that facilitate ERAD act either up or downstream of this critical posttranslational modification (41). To better define the mechanism by which PON2 enhances ENaC turnover, we examined the extent of ENaC ubiquitination in the presence or absence of PON2. First, αENaC was efficiently precipitated using V5-conjugated agarose beads (Fig. 6A). Next, the ubiquitinated αENaC was assessed by probing the V5-IPs with anti-ubiquitin antibodies. The presence of PON2 reduced αENaC abundance by 40 ± 29% within the V5-IPs (V5, Fig. 6B). More importantly, the amount of ubiquitinated αENaC, relative to the total V5 pulldown (i.e., Ubi/V5), was significantly increased (2.0 ± 0.9-fold) in cells coexpressing PON2 (Fig. 6C). These results strongly suggest that PON2 directly enhances ENaC ubiquitination, thus providing an explanation for the mechanism of this prodegradative ER chaperone.

PON2 Reduces ENaC Surface Expression and Amiloride-Sensitive Na⁺ Transport

Our collective data indicate that PON2 facilitates the degradation of ENaC subunits through the proteosome pathway (Figs. 2, 3, 4, 5, and 6). Thus, we hypothesized that the prodegradative effect of PON2 would lead to reduced channel density at the cell surface in FRT cells. To test this hypothesis, we examined the effect of PON2 on ENaC surface expression using a biotinylation assay as previously described (30). γENaC and PON2 were detected in both the surface precipitates and the whole cell lysates. GAPDH is absent in the biotinylated samples, indicating that the surface fractions were free of contamination from intracellular proteins (Fig. 7A, bottom). As summarized in Fig. 7B, PON2 reduced the abundance of γENaC in the biotin-labeled surface fractions (43 ± 27%, P < 0.05) and the whole cell lysates (48 ± 27%, P < 0.01). Consistent with downregulation of channel surface expression, the amiloride-sensitive I_SC was also reduced by ∼20% (n = 6, P < 0.05) in cells co-expressing PON2 compared with those expressing ENaC alone (Fig. 8). This inhibitory effect of PON2 on ENaC functional expression is further consistent with our previous observation in oocytes, where ENaC-mediated amiloride-sensitive whole cell Na⁺ currents were reduced by PON2 (29).

Figure 8.

Amiloride-sensitive short circuit currents are reduced by PON2. FRT cells were transiently transfected with _HAα_V5βγ ENaC alone (−PON2) or cotransfected with mouse PON2 (+PON2). Transfected cells were cultured on permeable filters for 3 and 4 days before short-circuit current (I_SC) measurements. A: representative traces of cells expressing ENaC (black) or ENaC + PON2 (red). After currents reached steady state, 10 μM amiloride was added to the apical side of the chamber to block ENaC activity. B: the amiloride-sensitive I_SC of cells expressing ENaC + PON2 is shown as a percentage relative to the currents in cells expressing only ENaC. Statistical comparisons were made with a Kolmogorov–Smirnov t test (*P < 0.05). ENaC, epithelial sodium channel; FRT, fisher rat thyroid; PON2, paraoxonase 2.

DISCUSSION

Among the three mammalian paraoxonases, PON2 has the broadest tissue distribution and the most diverse cellular functions. PON2 plays a protective role against oxidative stress-induced apoptosis and lipid peroxidation (42–44), and as noted in the introduction, PON2-deficient mice develop diet-induced atherogenesis in association with increased mitochondrial oxidative stress and inflammation (8, 9). In the kidney, PON2 inhibits NADPH oxidase activity and prevents excessive production of reactive oxygen species within the proximal tubule (45). PON2 also has the highest catalytic activity toward N-acyl-homoserine lactones, including 3OC₁₂-HSL, and thus also plays an important role in suppressing quorum sensing and enhancing bacterial clearance (46–49). In addition to serving as an antioxidant, recent work implicated PON2 in pancreatic ductal adenocarcinoma formation and B-cell leukemogenesis by regulating glucose-transporter 1 (GLUT1; 50, 51). Specifically, the binding of PON2 to GLUT1 releases GLUT1 from an inhibitory factor, stomatin, and therefore enhances glucose uptake (51). Interestingly, stomatin is a mammalian homolog of C. elegans MEC-2, a key ancillary protein of the touch-transducing apparatus in the TRNs (52). Because MEC-2 and MEC-6 facilitate MEC-4/MEC-10 channel expression, and the loss-of-function mutations of these proteins result in the touch-insensitive phenotype in nematodes (12, 53–56), there was precedence that other MEC-6-related gene products would similarly regulate ENaC biogenesis. In accordance with this view, our previous studies showed that both MEC-6 and PON3, as well as PON2, suppress ENaC levels (29, 30). Yet, until the current study, it was unclear how PON2 gave rise to this phenomenon.

PON2 is ubiquitously expressed in many tissues and major organs, such as heart, lung, and kidney (42, 43). Among different cell types, PON2 has been found within multiple intracellular compartments, including the ER, Golgi, mitochondria, endosome, and lysosome (43, 57). Our data identify the ER as a primary site of PON2 expression in FRT cells. The importance of the ER quality control pathway, which is overseen by ER and ER-associated chaperones (38, 41, 58), in regulating ion channels has been implicated in the pathogenesis of several human diseases, including cystic fibrosis, type II Bartter syndrome, Gitelman syndrome, and congenital nephrogenic diabetes insipidus (23, 59–64). Like many other membrane-spanning proteins, the α-, β-, and γ-ENaC subunits are synthesized at the ER, where they are folded and assembled into a trimeric complex. Nevertheless, a significant portion of misfolded or orphaned ENaC subunits is degraded in the proteasome (22, 65). In the present study, we found that PON2 negatively regulates ENaC expression by accelerating the degradation of all three subunits. Using pharmacological treatments that specifically inhibit lysosomal or proteasomal functions, we established that PON2 mainly affects proteasome-dependent ENaC degradation, suggesting that PON2 is involved in the ERAD of ENaC. Indeed, PON2 enhanced the ubiquitination of ENaC, a key step during the targeting of misfolded proteins for ERAD. In addition, the prodegradative effect of PON2 led to reduced channel surface density and ENaC-mediated Na⁺ transport. Based on these features, we propose that PON2 functions as an ER chaperone to negatively regulate ENaC surface and functional expression. This inhibitory effect is somewhat specific for ENaC, as the coexpression of PON2 in FRT cells did not alter the abundance of NCC, the thiazide-sensitive Na-Cl cotransporter that has been identified as an ERAD substrate (63, 64). It will be important to determine whether PON2 functions as a chaperone in the biogenesis of other multimeric membrane proteins in future studies.

Although the physiological functions of mammalian PONs in the kidney remain elusive, there is evidence suggesting a role of PONs in maintaining normal blood pressure. Pon1 KO mice were hypotensive with a lower serum aldosterone level (66), and renal subcapsular infusion of Pon2 siRNA led to hypertension in rats (45). Although PON3 is specifically expressed in the aldosterone-sensitive distal nephron (30), PON2 appears to be more broadly expressed along the nephron segments with the highest levels of mRNA found in the long descending limb of Henle’s loop, cortical collecting ducts, and inner medullar collecting ducts (67). Additional studies need to investigate whether altered ENaC function contributes to phenotypes seen with the PON2-deficient models. Nonetheless, we propose that the mammalian PONs represent a class of novel quantity control factors that regulate ENaC expression in tissues where Na⁺ transport via ENaC is essential for salt and volume homeostasis and blood pressure control.

GRANTS

This work was financially supported, in whole or in part by National Institutes of Health Grants: DK103834 (to S.S.), DK119752 (to S.S.), DK130901 (to S.S.), DK117162 (to T.M.B), GM131732 (to J.L.B), HL147818 (to T.R.K.), DK038470 (to T.R.K.), and DK079307 (Pittsburgh Center for Kidney Research). Andrew J Nickerson is supported by a T32 training Grant (DK061296).

DISCLAIMERS

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.S., T.M.B., J.L.B., and T.R.K. conceived and designed research; S.S., T.M.B., and A.J.N. performed experiments; S.S. and A.J.N. analyzed data; S.S. and T.R.K. interpreted results of experiments; S.S. prepared figures; S.S. drafted manuscript; S.S., T.M.B., A.J.N., J.L.B., and T.R.K. edited and revised manuscript; S.S., T.M.B., A.J.N., J.L.B., and T.R.K. approved final version of manuscript.