NHERF1 and NHERF2 are necessary for multiple but usually separate aspects of basal and acute regulation of NHE3 activity

Abstract

Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border (BB) of intestinal epithelial cells and accounts for the majority of neutral NaCl absorption. It has been shown that the Na(+)/H(+) exchanger regulatory factor (NHERF) family members of multi-PDZ domain-containing scaffold proteins bind to the NHE3 COOH terminus and play necessary roles in NHE3 regulation in intestinal epithelial cells. Most studies of NHE3 regulation have been in cell models in which NHERF1 and/or NHERF2 were overexpressed. We have now developed an intestinal Na(+) absorptive cell model in Caco-2/bbe cells by expressing hemagglutinin (HA)-tagged NHE3 with an adenoviral infection system. Roles of NHERF1 and NHERF2 in NHE3 regulation were determined, including inhibition by cAMP, cGMP, and Ca(2+) and stimulation by EGF, with knockdown (KD) approaches with lentivirus (Lenti)-short hairpin RNA (shRNA) and/or adenovirus (Adeno)-small interfering RNA (siRNA). Stable infection of Caco-2/bbe cells by NHERF1 or NHERF2 Lenti-shRNA significantly and specifically reduced NHERF protein expression by >80%. NHERF1 KD reduced basal NHE3 activity, while NHERF2 KD stimulated NHE3 activity. siRNA-mediated (transient) and Lenti-shRNA-mediated (stable) gene silencing of NHERF2 (but not of NHERF1) abolished cGMP- and Ca(2+)-dependent inhibition of NHE3. KD of NHERF1 or NHERF2 alone had no effect on cAMP inhibition of NHE3, but KD of both simultaneously abolished the effect of cAMP. The stimulatory effect of EGF on NHE3 was eliminated in NHERF1-KD but occurred normally in NHERF2-KD cells. These findings show that both NHERF2 and NHERF1 are involved in setting NHE3 activity. NHERF2 is necessary for cGMP-dependent protein kinase (cGK) II- and Ca(2+)-dependent inhibition of NHE3. cAMP-dependent inhibition of NHE3 activity requires either NHERF1 or NHERF2. Stimulation of NHE3 activity by EGF is NHERF1 dependent.

Fig. 1.

Adenoviral expression of hemagglutinin (HA)-tagged Na⁺/H⁺ exchanger 3 (NHE3) in Caco-2/bbe cells. Caco-2/bbe cells were infected with purified adenovirus (Adeno)-HA-NHE3 at 0.5 × 10¹⁰, 1.0 × 10¹⁰, and 2.0 × 10¹⁰ particles/ml medium for 6–7 h (1 ml apical + 1 ml basal). Approximately 40 h after infection, cells were placed in serum-free medium for 4 h and collected for Western blot analysis, NHE3 activity assay, and immunostaining. A: Western blot analysis was performed on lysates of Caco-2/bbe cells infected with different amounts of Adeno-HA-NHE3; 40 μg of protein was used for Western blot analysis, and NHE3 expression was detected with mouse anti-HA monoclonal antibody. NHE3 expression linearly correlated with number of viral particles exposed. B: confocal microscopic x-y section (left), x-z section (top), and y-z section (right) of Caco-2/bbe monolayer infected with 2.0 × 10¹⁰ Adeno-HA-NHE3 particles/ml and immunostained with anti-HA antibody and nuclear marker (Hoechst). NHE3 expression was mostly at/near the apical surface. C: 50 μg of total cell lysate protein from Caco-2/bbe, Caco-2/bbe/Adeno-NHE3, and mouse jejunum were immunoblotted (IB) with polyclonal antibodies against NHE3 (top), monoclonal HA antibody (middle), and monoclonal β-actin (bottom). NHE3 expression was quantitated and normalized with β-actin from the same samples. Adeno-HA-NHE3 expression in Caco-2 cells (Adeno) was similar to endogenous mouse jejunum NHE3 expression, but endogenous NHE3 expression in Caco-2 cells (Endo) was negligible. AU, arbitrary units. D: cells grown on Transwell filters were infected with Adeno-HA-NHE3 and immunostained under permeabilized conditions with mouse monoclonal anti-HA antibodies and Hoechst nuclear staining. The number of NHE3-positive and -negative cells was counted with a Zeiss LSM510 confocal fluorescence microscope. The calculated rate of infection (NHE3-positive cells/total no. of cells) linearly correlated with viral particle number, with a maximum infection rate of ∼75%. Results are means ± SE from 3 experiments. E: Caco-2/bbe cells were grown on “filterslips” and infected with Adeno-HA-NHE3. Approximately 48 h later, basal NHE3 activity was measured with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)/fluorometry. Activity of NHE3 was dependent on the number of viral particles used for infection. Results are means ± SE from 3 similar experiments.

Fig. 2.

Apical domain location of endogenous NHE regulatory factor (NHERF)1 and NHERF2 in Caco-2/bbe cells. z-Sections of Caco-2/bbe monolayers infected with 2.0 × 10¹⁰ particles/ml were immunostained with anti-HA monoclonal antibodies and NHERF1 or 2 polyclonal antibodies. NHERF1 and NHERF2 were localized to the apical pole, with NHERF1 demonstrating more overlap with NHE3 than NHERF2. The same settings (laser power, gain) were used for images in A–C. In D, pairs of images are shown, with images on the left representing staining using the same gain settings as A–C and images on the right shown with increased gain in order to demonstrate the different location of NHERF2 compared with NHERF1 and NHE3. Scale bars, 10 μm (A, C) and 2.5 μm (B, D). z-Series images were captured with a ×63 water immersion objective, and images were reconstructed with MetaMorph software; x–z, y–z, and x–y sections are shown in A and C and x–z sections in B and D.

Fig. 3.

Stable knockdown of NHERF1 or NHERF2 in Caco-2/bbe cells using lentivirus-short hairpin RNA (shRNA) to study NHE3 regulation. A: Caco-2/bbe cells were transduced with lentiviral particles prepared from 3 different shRNA constructs specific for either NHERF1 or NHERF2 genes. After selection with puromycin (10 μg) for at least 2 passages, cell lysates were prepared; 40 μg of cell lysate was used to immunoblot with anti-NHERF1 and -NHERF2 antibodies. Cells infected with lentivirus prepared from shRNA specific for green fluorescent protein (GFP) was used as a negative control (Cont; human cells do not endogenously express GFP). NHERF1 shRNA construct 1-1 knocked down >90% NHERF1 expression, and NHERF2 shRNA construct 2-3 knocked down NHERF2 expression by >85%. Intensities of protein expression were calculated with Odyssey software. Results shown are from a single experiment that was repeated 4 times with similar results. B: Caco-2/bbe cells grown on filterslips were infected with Adeno-NHE3, and NHE3 basal activity was measured with BCECF/fluorometry. Cells with NHERF1 knocked down (shRNA construct 1-1) had significantly reduced NHE3 activity (30–40%) compared with wild-type (WT) or GFP-knockdown (KD) controls. Cells with NHERF2 knocked down (shRNA construct 2-3) had significantly increased NHE3 activity (6). Results represent means ± SE from n independent experiments. Control was GFP-shRNA (6). shRNA for GFP did not alter basal NHE3 activity compared with empty vector (data not shown). P values are comparison with control (ANOVA). C–E: apical cell membrane biotinylation was performed in Caco-2/bbe cells transduced with lentivirus-shRNA-GFP, shRNA-NHERF1, and shRNA-NHERF2. Total and apical surface amounts of NHE3 expression were determined by Western blot analysis. Two different volumes of each sample (10 μl and 20 μl) were loaded for Western blot analysis. Total and surface NHE3 expression were normalized to total β-actin. Calculated total and surface NHE3 used as a control lentivirus-shRNA-GFP set to 100% for each experiment. Results are means ± SE of 4 independent experiments. P values are in comparison to control (unpaired t-tests). NS, not significant.

Fig. 4.

NHERF1 or NHERF2 knockdown individually in Caco-2/bbe cells did not change the cAMP inhibition of NHE3, but the cAMP effect was abolished when both NHERF1 and NHERF2 were knocked down. A: Caco-2/bbe cells stably expressing lentivirus-shRNA for GFP (control cell), NHERF1 (construct 1-1) and NHERF2 (construct 2-3) were grown on filterslips and infected with Adeno-HA-NHE3. NHE3 activity was measured in these cells with or without 20 μM forskolin (FSK) treatment for 15 min. FSK inhibited NHE3 activity (30–40%) in control cells, and similar inhibition occurred in NHERF1-KD and NHERF2-KD cells. Results are means ± SE from 3 independent experiments; P values represent effect of FSK (paired t-tests). B: Caco-2/bbe cells with NHERF2 knocked down (shRNA construct 2-3) were transduced with additional lentivirus prepared from shRNA for NHERF1 (construct 1-1), and cells were selected with a higher concentration of puromycin (20 μg/ml). After 2 passages, cell lysates were prepared and 40 μg of protein was used for Western blot analysis for NHERF1 and NHERF2 expression. In these double-knockdown cells, both NHERF1 and NHERF2 were effectively knocked down (>80%) compared with lentivirus-empty vector + shRNA GFP knockdown cells (control). C: NHE3 activity was measured in cells in which both NHERF1 and NHERF2 were knocked down with lentiviruses, with or without 20 μM FSK treatment for 15 min. FSK inhibited NHE3 activity in control cells (lentivirus empty virus + shRNA GFP), but the FSK effect was abolished when NHERF1 + NHERF2 were both knocked down. Results are means ± SE of 3 separate experiments. P values compare effects of FSK and control, with control being a combination of lentivirus-GFP-shRNA and empty vector (paired t-tests).

Fig. 5.

NHERF2 knockdown abolishes carbachol inhibition of NHE3 activity in Caco-2/bbe cells. NHE3 activity was measured in cells grown on filterslips and infected with Adeno-HA-NHE3. NHERF1 and NHERF2 knock used lentivirus-shRNA and control cells used lentivirus-shRNA GFP. Control cells and NHERF1-KD cells showed 30–40% inhibition of NHE3 activity when treated with 50 μM carbachol for 5 min. The effect of carbachol was abolished in cells with NHERF2 but not NHERF1 knockdown (n = 3). P values are carbachol effect compared with matched untreated controls (paired t-test).

Fig. 6.

8-p-chlorophenylthio-cGMP (8-pCPT-cGMP) inhibits NHE3 activity in Caco-2/bbe cells expressing cGMP-dependent protein kinase (cGK) II. A: NHE3 activity was unchanged in Caco-2/bbe cells infected with control adenovirus [empty vector (EV)] after treatment with 8-pCPT-cGMP (EV+cGMP) compared with untreated cells (EV); n = 4. B: Caco-2/bbe cells showed no detectable levels of endogenously expressed cGK II (not infected) (right lane) and were therefore infected with recombinant adenoviral vector expressing cGK II [cGK II (AV)] (left lane). HIS-cGK II represents purified His₆-tagged cGK II fusion protein as a standard (middle lane). GAPDH was used as a loading control. Results are representative of 3 independent experiments. C: Caco-2/bbe cells expressing cGK II displayed significant inhibition of NHE3 after treatment with 8-pCPT-cGMP (cGMP; 100 μM) compared with untreated control cells (P < 0.05, paired t-tests; n = 4).

Fig. 7.

cGMP-dependent inhibition of NHE3 activity in Caco-2/bbe cells expressing cGK II is NHERF2 dependent. A: infection of cGK II-expressing Caco-2/bbe cells with small interfering RNA (siRNA) constructs directed against NHERF2 dramatically reduced NHERF2 protein expression. Infection with siRNA construct 1 or construct 2 strongly reduced NHERF2 expression (75%), and the coinfection of both these constructs produced a knockdown of >85%. GAPDH was used as a loading control. B: in cells with NHERF-2 knocked down (Adeno-NHERF2, siRNA constructs 1 + 2, infected at same time as Adeno-HA-NHE3), there was no longer 8-pCPT-cGMP-induced inhibition of NHE3 activity, while in simultaneously studied scrambled siRNA control cells 8-pCPT-cGMP inhibited NHE3 activity (paired t-tests; n = 3 for each). C: NHE3 activities (initial rates) were measured in Caco-2/bbe cells in which NHERF1 or NHERF2 was knocked down with lentivirus-shRNA and lentivirus-GFP-shRNA was used as a negative control. NHERF1-KD cells expressing cGK II showed reduced NHE3 activity when treated with 100 μM 8-pCPT-cGMP, but NHERF2-KD cells expressing cGK II had no effect when treated with cGMP. P values indicate effect of cGMP (paired t-tests; n = 3).

Fig. 8.

Epidermal growth factor (EGF) stimulation of NHE3 activity is NHERF1 dependent in Caco-2/bbe cells. NHE3 activities were measured in Caco-2/bbe cells with or without exposure to EGF (200 ng/ml) for 30 min. EGF stimulated NHE3 activity (30–40%) in control cells (Cont; lentivirus-empty vector cells) and NHERF2-KD cells (NF2-KD) but failed to stimulate NHE3 activity in NHERF1 lentivirus knockdown cells (NF1-KD). Results are means ± SE. P values are effects of EGF (paired t-tests; n = 3).