NHE3 mobility in brush borders increases upon NHERF2-dependent stimulation by lyophosphatidic acid

Abstract

The epithelial brush border (BB) Na(+)/H(+) exchanger NHE3 is associated with the actin cytoskeleton by binding both directly and indirectly to ezrin; indirect binding is via attachment to NHERF family proteins. NHE3 mobility in polarized epithelial cell BBs is restricted by the actin cytoskeleton and NHERF binding such that only approximately 30% of NHE3 in the apical domain of an OK cell line stably expressing NHERF2 is mobile, as judged by FRAP analysis. Given that levels of NHE3 are partially regulated by changes in trafficking, we investigated whether the cytoskeleton association of NHE3 was dynamic and changed as part of acute regulation to allow NHE3 trafficking. The agonist studied was lysophosphatidic acid (LPA), an inflammatory mediator, which acutely stimulates NHE3 activity by increasing the amount of NHE3 on the BBs by stimulated exocytosis. LPA acutely stimulated NHE3 activity in OK cells stably expressing NHERF2. Two conditions that totally prevented LPA stimulation of NHE3 activity only partially prevented stimulation of NHE3 mobility: the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, and the NHE3F1 double mutant which has minimal direct binding of NHE3 to ezrin. These results show that LPA stimulation of NHE3 mobility occurs in two parts: (1) PI3K-dependent exocytic trafficking to the BB and (2) an increase in surface mobility of NHE3 in BBs under basal conditions. Moreover, the LPA stimulatory effect on NHE3 mobility required NHERF2. Although NHE3 and NHERF2 co-precipitated under basal conditions, they failed to co-precipitate 30 minutes after addition of LPA, whereas the physical association was re-established by 50-60 minutes. This dynamic interaction between NHERF2 and NHE3 was confirmed by acceptor photobleaching Förster Resonance energy Transfer (FRET). The restricted mobility of NHE3 in BBs under basal conditions as a result of cytoskeleton association is therefore dynamic and is reversed as part of acute LPA stimulation of NHE3. We suggest that this acute but transient increase in NHE3 mobility induced by LPA occurs via two processes: addition of NHE3 to the BB by exocytosis, a process which precedes binding of NHE3 to the actin cytoskeleton via NHERF2-ezrin, and by release of NHERF2 from the NHE3 already localized in the apical membrane, enabling NHE3 to distribute throughout the microvilli. These fractions of NHE3 make up a newly identified pool of NHE3 called the 'transit pool'. Moreover, our results show that there are two aspects of LPA signaling involved in stimulation of NHE3 activity: PI3K-dependent stimulated NHE3 exocytosis and the newly described, PI3K-independent dissociation of microvillar NHE3 from NHERF2.

Fig. 1.

LPA transiently stimulates NHE3 lateral mobility in polarized OK cells in the presence of NHERF2. (A) FRAP was performed before and every 10 minutes after LPA (100 μM) addition (at time zero, as indicated by arrow). The mobile fraction was obtained by calculating the FRAP data with the following equation at t=400 seconds: M_f (t=400)=[(F₄₀₀–F₀)/(100–F₀)]× 100(%) (F_i was set to 100 as shown in B). A single experiment on a single coverslip is shown with multiple bleach FRAP experiments studied over time on cells in different parts of the coverslip. The same approach is used in the time course shown in subsequent figures (Figs 2, 3, 4, 5, and 6). Similar results were obtained in three different experiments. (B) Representative FRAP studied after 30 minutes of LPA treatment and untreated control, with initial fluorescence set at 100. (C) Mobile fractions (at t=400 seconds) were measured at 30 minutes after LPA treatment. Means ± s.e.m. were calculated from n=5 experiments. P values compare untreated control with LPA-treated samples (paired Student's t-test).

Fig. 2.

Water-soluble surface cross-linker BS³ only partially blocks the LPA stimulatory effect on NHE3 mobility. (A) NHE3 transport activity was measured for untreated control cells and for cells treated with BS³, LPA and BS³+LPA. (B) Percentage of total NHE3 on the surface was measured for untreated control cells and for cells treated with BS³, LPA and BS³+LPA. A representative experiment of three is shown. (C) FRAP was performed before and every 10 minutes after LPA (100 μM) treatment and pre-incubation with BS³ (5 mM; 30 minutes). LPA (100 μM) was added at the time zero as indicated with arrow. A representative experiment is shown. (D) Mobile fractions (at t=400 seconds) measured in control, BS³, BS³+LPA and LPA, at 30 minutes after addition of LPA. Means ± s.e.m. were calculated from n=5 experiments. *P<0.05 and **P<0.01.

Fig. 3.

Stimulatory effect of LPA on NHE3 mobility is only partially inhibited by inhibition of the PI3K pathway. (A) FRAP studies of NHE3 treated with LPA were performed in OK NHERF2 cells treated with the PI3K inhibitor LY294002 (30 μM) for 30 minutes and LPA (100 μM) was added at the time zero as indicated by arrow. (B) Mobile fractions (at t=400 seconds) were measured at 30 minutes after LPA treatment and 60 minutes after LY294002 pretreatment. Means ± s.e.m. were calculated from n=5 experiments.

Fig. 4.

LPA stimulation of NHE3 mobility was significantly decreased in the ezrin-binding mutant NHE3F1D. (A) NHE3-WT and NHE3F1D were transiently transfected in OK NHERF2 cells. NHE3 transport activity (ΔpHi/minute) was measured with or without LPA (100 μM). Means ± s.e.m. were calculated from n=3 experiments. P values compare effect of LPA with untreated controls. (B) Protein expression of VSV-G tagged NHE3-WT and NHE3F1D detected using anti-VSV-G antibody. 20 μg and 30 μg of total cell lysate was loaded (upper panel). Similar amounts of NHERF2 are expressed in these cells (lower panel). (C) FRAP studies of NHE3F1D with LPA (100 μM) performed on OK NHERF2 cells. LPA was added at the time zero (arrow). A representative experiment is shown. (D) NHE3F1D mobile fraction with or without LPA (100 μM) measured at 30 minutes after addition of LPA. n=5. P represents comparison of LPA effect in NHE3F1D cells.

Fig. 5.

LPA stimulation of NHE3-EGFP mobility requires NHERF2 in OK cells. (A) FRAP studies of NHE3 with LPA performed on OK cells lacking NHERF2 but with endogenous NHERF1. (B) FRAP studies with LPA performed on OK cells stably expressing NHERF2 and NHE3585-EGFP, the latter of which lack all NHERF family PDZ-binding domains. LPA (100 μM) was added at the time zero as marked by arrow. (C,D) Graphs showing means ± s.e.m. of three experiments, representative examples of which are shown in A and B, respectively.

Fig. 6.

LPA stimulation of NHE3 mobility does not occur in OK NHERF2Δ30 cells. (A) NHERF2 and NHERF2Δ30 were stably expressed in OK cells and detected with anti-NHERF2 antibody (2570). (B) NHE3 activity (ΔpHi/minute) measured in OK NHERF2 NHE3 and OK NHERF2Δ30 NHE3 cells with and without LPA (100 μM, 30 minutes). Means ± s.e.m. were calculated from n=3 experiments. P values compare untreated control and NHE3 NHERF2Δ30 with NHE3 NHERF2 cells. (C) Basal mobile fraction of OK cells expressing NHE3, NHE3 NHERF2 and NHE3 NHERF2Δ30 cells. The low basal NHE3 mobility related to NHERF2 was abolished in NHERF2Δ30 mutant. Means ± s.e.m. were calculated from n=7 experiments. (D) Fluorescence recovery of NHE3 after photobleaching measured with LPA (100 μM) treatment for 30 minutes in OK NHERF2Δ30 cells. Unlike the FRAP wild-type cells under control conditions, an oscillating pattern occurred after LPA treatment, but without an overall increase in the mobile fraction. Results from a single cell are shown with this pattern of mobility was seen in the majority of cells studied 10, 20, 30 and 40 minutes after LPA with return to a normal response by 50 minutes.

Fig. 7.

NHERF2 co-precipitates NHE3 in OK cell membranes in a LPA-related time-dependent manner. LPA (100 μM) was added to OK NHERF2 HA-NHE3 cells for 0, 30 and 60 minutes. Total membrane preparations of OK cells were prepared for immunoprecipitation of NHERF1 (A) or NHERF2 (B). Co-precipitation of HA-NHE3 was detected by immunoblotting. NHERF2 but not NHERF1 failed to co-precipitate NHE3 at 30 minutes. Anti-NHERF2 antibody (2570) and anti-NHERF1 antibody (5199) were used for the detection of NHERF2 and NHERF1, respectively. Anti-HA antibody was used to detect NHE3. Representative results of three experiments are shown.

Fig. 8.

NHE3 transiently dissociates from NHERF2 after LPA exposure, demonstrated by disappearance of FRET. (A) FRET images in OK cells expressing NHE3-YFP and CFP-NHERF2 acquired before (top panel) and after (bottom panel) photobleaching under basal conditions. Top panel (before photobleaching) shows: CFP-NHERF2 (pseudocolored red). NHE3-YFP (pseudocolored green) and overlay image. Bottom panel (after photobleaching) shows: CFP-NHERF2 (pseudocolored red) and NHE3-YFP (pseudocolored green), demonstrating nearly complete bleaching. (B) As in A, except cells were exposed to LPA for 30 minutes before acquisition of FRET images, before (top panel) and after (bottom panel) the acceptor photobleaching. Top, CFP-NHERF2 (pseudocolored red), NHE3-YFP (pseudocolored green) and overlay image. Bottom (after photobleaching), CFP-NHERF2 (pseudocolored red), NHE3-YFP (pseudocolored green). NHERF2 and NHE3 are highly co-localized on the microvillar region of OK cells, but not in the juxtanuclear region. MetaMorph percentage localization program showed that ~50% of NHE3 co-localized with NHEF2, whereas ~90% of NHERF2 co-localized with NHE3. (C) FRET images in OK cells expressing YFP-GL-GPI and CFP-NERF2 acquired before (top panel) and after (bottom panel) the photobleaching under basal conditions. Top panel shows FRET images before photobleaching and bottom panel after photobleaching. (D) FRET images as in 8C, except 30 minutes after LPA exposure. (E) FRET efficiencies in the OK cell apical domain calculated with MetaMorph software for NHE3-YFP and CFP-NHERF2 (left pair), and YFP-GL-GPI and CFP-NHERF2 (right pair) with or without LPA (100 μM, 30 minutes). Ten ROIs from the microvillar area were analyzed for each cell and at least seven cells were quantified from three different experiments. Results are means ± s.e.m.

Fig. 9.

Schematic model for LPA stimulation of NHE3 mobile fraction. The LPA-induced increase of NHE3 mobile fraction consists of two components: NHE3 newly delivered to the apical domain by a PI3K-dependent process, which requires direct ezrin binding to NHE3 and which occurs prior to fixing of NHE3 to the microvillar cytoskeleton. This consists of functionally active NHE3 and NHE3 that is newly dissociated from NHERF2 in the intervillus clefts and in the microvillus, which allows the NHE3 to transit over the microvillus prior to cytoskeletal fixation. Definition of the ‘transit pool’ of NHE3 includes NHE3 in the intervillus clefts and lower part of the microvillus, which receives NHE3 from the recycling system and dispenses NHE3 to the microvillus as part of exocytosis. This pool of NHE3 binds NHERF2 dynamically, changing with LPA signaling and associates with the cytoskeleton by ezrin binding to NHERF2. NHERF1 distribution is shown in red and NHERF2 distribution in blue and their locations in the BB are assumed to be static.