To specifically modify ENaC, we adopted shRNA-based knockdown of ENaCα, the rate limiting subunit of ENaC (Canessa et al., 1994), in Ishikawa (ISK) cells, a commonly used human endometrial epithelial cell line (Nishida, 2002; Sun et al., 2018b). Three designs of shRNAs, shENaCα-1, -2 and -3 with predicated knockdown efficiency of 64%, 78% and 95% respectively were packaged in lentivirus and used to stably knock down ENaC in ISK cells. As a result, the mRNA level of ENaCα was reduced to 48 ± 5%, 30 ± 2%, and 27 ± 2% in cells treated with shENaCα-1, -2 and -3, respectively, as compared to the line with scrambled shRNAs as the negative control (shNC) (Figure 1A). We next used the ISK lines with shNC, shENaCα-1 and shENaCα-3 (ISK-shNC, ISK-shENaCα-1 and ISK-shENaCα-3) for subsequent experiments. Western blot results confirmed that ENaCα protein level was knocked down by about 50% and 70% in ISK-shENaCα-1 and ISK-shENaCα-3 cells, respectively, as compared to that in ISK-shNC (Figure 1B). To confirm that ENaC was functionally knocked down, we examined ENaC channel function by measuring the membrane potential (Vm) with a voltage-sensitive fluorescent dye, DiBAC4(3), which we previously published (Ruan et al., 2012). Results showed that in all these ISK lines, the addition of benzamil (10 µM) induced a decrease in DiBAC4(3) intensity indicating Vm hyperpolarization (Figure 1C), consistent with the known role of active ENaC in endometrial epithelial cells in mediating Na⁺ influx and thus Vm depolarization. Such a benzamil-induced Vm hyperpolarization (indicating ENaC activity) was found to be reduced by about 23% and 50% in ISK-shENaCα-1 and ISK-shENaCα-3 lines, respectively, as compared to that in ISK-shNC (Figure 1C).

In these ISK lines with a low (ISK-shENaCα-1, 23%) or high (ISK-shENaCα-3, 50%) degree of ENaC knockdown, we measured the expression of CFTR. Quantitative PCR (qPCR) results showed that the CFTR mRNA level in ISK-shENaCα-1 significantly increased by about 4 folds as compared to that in ISK-shNC (Figure 2A). However, in ISK-shENaCα-3 where ENaC was more knocked down, the mRNA level of CFTR did not further increase but was significantly lower than that in ISK-shENaCα-1 (Figure 2A). Consistently, only ISK-shENaCα-1 but not ISK-shENaCα-3 showed higher CFTR protein level compared to that in ISK-shNC (Figure 2B). We next examined Cl⁻ channel function of CFTR in the ISK lines by measuring intracellular Cl⁻ levels using a Cl⁻-quenched fluorescent indicator, MQAE. While fluorescent intensity of MQAE was monitored under the microscope, a selective CFTR inhibitor, CFTRinh-172 (10 μM) was added onto the cells. Upon the addition of CFTRinh-172, there was a drop of fluorescent signal indicating an increase of intracellular Cl⁻ concentration and thus suggesting CFTR function in mediating Cl⁻ efflux in the cells (Figure 2C). Comparing these ISK lines, we found that ISK-shENaCα-1 exhibited a significantly larger CFTRinh-172-sensitive MQAE change compared to ISK-shNC (Figure 2C), indicating an increased gross CFTR activity in ISK-shENaCα-1. Whereas, in the ISK-shENaCα-3 with further ENaC knockdown, the CFTRinh172-sensitive change was similar compared to that in ISK-shNC, and significantly smaller than that in ISK-shENaCα-1 (Figure 2C). These results therefore consistently suggested that the regulation of CFTR by ENaC might be biphasic, i.e., CFTR upregulation by a low degree of ENaC deficiency but downregulation by further ENaC knockdown.

We next asked how ENaC could regulate CFTR in such a biphasic manner. Since the promoter of CFTR gene is reported to contain CRE element that is activated by cAMP response element binding protein (CREB) (Cheng et al., 1991; McCarthy and Harris, 2005), we tested possible involvement of cAMP. We measured the intracellular cAMP concentration of the ISK lines, which interestingly showed that, without any treatment, ISK-shENaCα-1 had a significantly higher basal level of intracellular cAMP (2.51 ± 0.14 pmol/mg) as compared to that of ISK-shNC (1.71 ± 0.02 pmol/mg) (Figure 3A). ISK-shENaCα-3 had a significantly lower basal level of cAMP (1.69 ± 0.27 pmol/mg) than that in ISK-shENaCα-1, which was not different with that in ISK-shNC. Therefore, the effect of ENaC knockdown on basal cAMP level in ISK cells was also biphasic.

We next treated the cells with drugs that inhibit cAMP production, 5-dideoxyadenosine (DDA), a selective inhibitor of transmembrane adenylyl cyclase (tmAC), and KH-7, a selective inhibitor of soluble adenylyl cyclase (sAC). Interestingly, in the presence of KH-7 (10 µM, 24 h), the biphasic effect of ENaC on CFTR mRNA levels was found completely reversed with the CFTR mRNA increase in ISK-shENaCα-1 abolished (Figure 3B). However, although DDA (100 µM, 24 h) slightly lowered the CFTR mRNA level in all three ISK lines, the biphasic effect was not altered by DDA (Figure 3B). There results therefore suggested the involvement of sAC-mediated cAMP production in the regulation of CFTR by ENaC.

sAC activity is well noted to be modulated by intracellular Ca²⁺ level (Jaiswal and Conti, 2003). We previously observed in endometrial epithelial cells that ENaC activity affected intracellular Ca²⁺ level (Ruan et al., 2012). We therefore wondered if the regulation of CFTR by ENaC could possibly be through Ca²⁺. We measured intracellular Ca²⁺ levels ([Ca²⁺]_i) in the ISK lines by Fura-2 imaging with calibration (Figure 4A), which showed that the basal [Ca²⁺]_i was significantly lower in ISK-shENaCα-1 (64.9 ± 2.8 nM) and ISK-shENaCα-3 (50.39 ± 1.9 nM), as compared to that in ISK-shNC (73.2 ± 3.0 nM) (Figure 4B). Consistently, the inhibition of ENaC by benzamil (0.1–10 µM) induced reduction in [Ca²⁺] a dose-dependent manner, although the response to benzamil was reduced when ENaC was knocked down in ISK-shENaCα-1 and ISK-shENaCα-3 (Figure 4C). To mimic a situation of intracellular Ca²⁺ reduction, we treated the cells with BAPTA-AM, an intracellular Ca²⁺chelator, which indeed produced decreases in [Ca²⁺]_i in ISK cells (Figure 4D). Interestingly, treating the cells with a low dose of BAPTA-AM (5 µM) for 24 h increased CFTR mRNA expression as compared to the DMSO control, while at a higher dose of 50 μM, it decreased mRNA CFTR expression in ISK cells (Figure 4E), mimicking the biphasic effect of ENaC knockdown. Similarly, intracellular cAMP level in ISK cells was found to be increased by the low dose (5 µM, 1 h) of BAPTA-AM treatment, but lowered by the higher dose (50 μM, 1 h), as compared to the control (Figure 4F). Moreover, the BAPTA-AM (5 µM)-induced increase in CFTR mRNA level was reversed by treatment with KH-7 (10 µM) and attenuated by DDA (10 µM) (Figure 4G). These results therefore suggested the intracellular Ca²⁺-modulated cAMP production underlying the regulation of CFTR by ENaC.

To further understand the regulatory action of ENaC on CFTR expression, we adopted a dual luciferase reporter to indicate the initiation of CFTR transcription in cells (see method). To our surprise, the reporter activity was found significantly higher in both ISK-shENaCα-1 and ISK-shENaCα-3, as compared to that in ISK-shNC, with no difference between ISK-shENaCα-1 and ISK-shENaCα-3 (Figure 5A), suggesting that CFTR transcription was stimulated with both degrees of ENaC knockdown. We then treated ISK cells with benzamil to inhibit ENaC, and found that benzamil at all doses tested (0.1–10 μM, 24 h) enhanced the luciferase reporter activity in a dose-dependent manner (Figure 5B). However, we also measured CFTR mRNA in ISK cells by qPCR after the treatment with benzamil (0.1–10 μM, 24 h), which showed that at 0.1 µM benzamil slightly increased CFTR mRNA level, although at 10 μM, it significantly decreased CFTR mRNA (Figure 5C). These results may suggest that inhibition of ENaC may at one hand stimulate CFTR transcription, while at the other hand affect CFTR mRNA maturation or stability.

Having observed the relationship between CFTR and ENaC, we wondered whether this phenomenon was specific to ISK cells. We established ENaC knockdown model in a human bronchial epithelial cell line (HBE). Stable transfection of shENaC-1, -2 and -3 resulted in ENaCα expression reduction in HBE cells by about 44%, 34% and 65% respectively (Figure 6A). Only with the lowest degree (34%) of ENaC knockdown (shENaCα-2) among the three, the mRNA expression of CFTR was found to be significantly increased compared to that in shNC-treated cells (Figure 6B). As ENaC was further knocked down (44%) by shENaCα-1, the mRNA level of CFTR started to drop, which significantly decreased when ENaC was knocked down to a high degree (65%) by shENaCα-3 (Figure 6B). Therefore, a similar biphasic regulation of CFTR by ENaC was also seen in HBE cells.

Human endometrial epithelial cell line Ishikawa (ISK) was purchased from ATCC (Virginia, United States) and cultured in RPMI-1640 (Thermo Fisher, 31800022) supplemented with 10% fetal bovine serum (v/v) and 1% penicillin–streptomycin (v/v) in 5% CO₂ incubators at 37°C. The cell line was authenticated by STR profiling at the Department of Anatomical and Cellular Pathology, Faculty of Medicine, The Chinese University of Hong Kong. Human bronchial epithelial cell line (16HBE14o-, ATCC) was a gift from Prof Zhou Wenliang at Sun Yat-sen University and cultured in Minimal Essential Medium (Thermo Fisher, 41500034) supplemented with 10% fetal bovine serum (v/v) and 1% penicillin–streptomycin (v/v) in 5% CO₂ incubators at 37°C.

Three designs of shRNAs targeting ENaCα and the scrambled non-coding shRNAs (shNC) were purchased from (Sigma-Aldrich) (Table 1). To package the shRNAs into the lentivirus, envelope vector pMD2.G (Addgene, 12259), packaging vector psPAX2 (Addgene, 12260) and shRNAs together with lipofectamine 2000 (ThermoFisher Scientific) were transfected into 293FT cells. Packaged lentivirus was harvested 72 h after transfection and next transfected into ISK or HBE cells for 24 h in the presence of polybrene (6 μg/ml). The cells were then cultured in the presence of puromycin (1 μg/ml) for 14 days to achieve stable knockdown.

Cells were lysed in TRIzol (Thermo Fisher Scientific, 15596026) for RNA extraction according to the manufacturer’s instructions. 1 μg RNA was then reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, 4368814) following the manufacturer’s instructions. Afterward, targeting genes (Table 2, all sequences were listed from 5′ to 3′) were amplified and detected by Touch Real-Time PCR Detection System (Bio-Rad, CFX96) using STBR Green Master mix (TAKARA, RR420A). GAPDH was used as a housekeeping gene for normalization. The data were analyzed using ΔΔCT method.

Cells were seeded on coverslips and grown for 24 to 48 h before the experiment. Cells on the coverslip were washed off the growth medium with Margo-Ringer buffer (NaCl 130 mM, KCl 5 mM, MgCl₂ 1 mM, CaCl₂ 2.5 mM, Hepes 20 mM and glucose 10 mM, pH 7.4), mounted to a chamber containing 1 ml Margo-Ringer buffer with the presence of a membrane potential-sensitive fluorescence dye, DiBAC4(3) (1 μM, Thermo Fisher Scientific, B438) and then placed under a fluorescence microscope (Nikon, ECLIPSE Ti2). After 15 min stabilization, the DiBAC4(3) was excited at 488 nm with emission lights collected at 535 nm and pictures taken every 3–5 sec. The data were collected and analyzed through NIS-Elements software.

Cells were lysed in pre-cooled RIPA lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% DOC and 0.1% SDS) with protease inhibitor cocktail (Thermo Fisher Scientific, 78443) for 30 min on ice. The supernatant was collected after centrifugation at 13,000 rpm for 30 min at 4°C. The protein concentration of each sample was measured with a BCA protein assay kit (Beyotime, P0011). Proteins were denatured in LDS sample buffer (Thermo Fisher Scientific, NP0007) plus 4% β-mercaptoethanol (Acros, 125472500) with heating at 70°C for 10 min to detect ENaCα, or incubated at room temperature for 30 min to detect CFTR. After denaturation, proteins were separated by SDS-polyacrylamide gel electrophoresis and subsequently transferred to equilibrated nitrocellulose membrane. The membrane was blocked by 5% non-fat milk in TBS for 30 min and subsequently incubated with the primary antibody in 5% non-fat milk in TBS at 4°C overnight. Primary antibodies against CFTR (1:500, Alomone Labs, ACL-006), ENaCα (1:500, StressMarq, SPC403), and actin (1:2000, Sigma Aldrich, MAB1501R) were used. After three washes with 0.05% Tween 20 in TBS, the membrane was incubated with HRP-conjugated antibodies at room temperature for 1 h and visualized by ECL substrates (Bio-Rad, 170-5060) and ChemiDoc MP Imaging System (Bio-Rad). HRP-conjugated secondary antibodies were goat anti-rabbit IgG (1:2,000, Bio-Rad, 12004159) and goat anti-mouse IgG (1:2,000, Bio-Rad, 12004159). Image J software was used for the densitometry of western blots.

Cells were seeded on coverslips and grown for 24 to 48 h before the experiment. Prior to imaging, cells on the coverslip were incubated with a Cl^- sensitive fluorescent dye, MQAE (10 mM, MedChemExpress, HY-D0090) in Margo-Ringer buffer for 30 min at 37°C followed by washing and stabilizing for another 10 min. The coverslip was then mounted to a chamber containing 1 ml Margo-Ringer buffer under a fluorescence microscope (Nikon, ECLIPSE Ti2). MQAE was excited at 340 nm with emission at 460 nm every 3–5 sec. The data were collected and analyzed through NIS-Elements software.

Intracellular cAMP concentration was measured with Direct cAMP ELISA Kit (Enzo Life Sciences, Cat. No. ADI-900-066). Cells were lysed with 0.1M HCl at room temperature for 10 min. The lysates were centrifuged at 1000 g for 5 min, and the supernatants were used for subsequent analysis. Part of the supernatants were used to determine protein concentration in each sample with BCA Protein Assay Kit (Beyotime, Cat No. P0011). Another part of the supernatants was used for cAMP measurement, added with acetylating reagent (10 μl for every 200 μl sample) to increase assay sensitivity. Sample wells, cAMP standard wells and other reference wells were loaded and treated according to assay layout sheet and product manual. Optical density (OD) of each well was read at 405 nm with Ledetect 96 Absorbance Plate Reader (Labexim Products), subtracting mean OD of substrate blank from all measurements. Concentration of cAMP of each sample was calculated with a four-parameter logistic curve fitting program.

Cells seeded on coverslips were incubated with Fura-2 (2 μM Thermo Fisher Scientific, F1221) for 30 min at 37°C before mounted onto a fluorescence microscope (Eclipse Ti, Nikon, Tokyo, and Japan). Fura-2 fluorescence was alternately excited at 340 and 380nm, and the emission signals were recorded at 510 nm. Ca²⁺ calibration was done according to a previous protocol (Williams and Fay, 1990). Briefly, cells were washed with Ca²⁺-free bath solution containing (in mM): NaCl 130, KCl 5, Hepes 20, EGTA 10 and glucose 10 (pH 7.2) before loaded with Fura-2 (2 μM) in the bath solution at 37°C for 30 min. The coverslip was then transferred to a mini chamber containing 1 ml Ca²⁺-free bath solution supplemented with nigericin (5 μM) and ionomycin (2 μM), and mounted on to the fluorescence microscope. To calibrate the change in Fura-2 fluorescence intensity into Ca²⁺ concentrations, a series of volumes of a high Ca²⁺ buffer containing (in mM): NaCl 130, KCl 5, CaCl₂ 10, Hepes 20, EGTA 10 and glucose 10 (pH 7.2) supplemented with nigericin (5 μM) and ionomycin (2 μM) was added into the Ca²⁺-free bath to achieve CaEGTA concentrations at 0, 1, 2, 4, 6, 8, and 10 mM. The concentration of free Ca²⁺ was estimated using the equation: [Ca²⁺]_free = K_d-EGTA x (C_CaEGTA/C_K2EGTA), where K_d-EGTA is a constant of 150.5 at 20°C with a pH of 7.2. The calibration curve was obtained by fitting Fura-2 240/380 ratios with [Ca²⁺]_free.

The proximal 5′region of human CFTR (- 1930 to +70, where +1 represents the transcriptional start site of CFTR) was amplified and cloned into pGL3-basic luciferase reporter by KpnI and NheI sites to make the pGL3/hCFTR construct (MiaoLingPlasmid Company). ISK cells were seeded into 96-well culture plates at a density of 1 × 10⁴ cells/well and co-transfected with luciferase reporter plasmid (pGL3/hCFTR, 1,000 ng/well) and internal control plasmid (Renilla-Luc, 10 ng/well) using Lipofectamine 2000 (Invitrogen). Firefly and renilla luciferase activities were assessed using the Dual-Glo Luciferase Assay System (Promega, E2920) according to the manufacturer’s instructions. The relative luciferase activity was defined as the ratio of readout for firefly luciferase to that for renilla luciferase with that of control group set as 1.0. Triplicate wells were used for measurement at each group.

One-way ANOVA was used for comparisons among more than 2 groups. Two-way ANOVA was used when there were two different categorical independent variables. A p value smaller than 0.05 was regarded as statistically significant. All graphs were generated and all statistical analyses were done with GraphPad Prism 9.