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J. Biol. Chem., Vol. 275, Issue 37, 28599-28606, September 15, 2000

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RhoA and Rho Kinase Regulate the Epithelial Na⁺/H⁺ Exchanger NHE3

ROLE OF MYOSIN LIGHT CHAIN PHOSPHORYLATION^*

Katalin Szászi^§¶, Kazuyoshi Kurashima^§, András Kapus, Anders Paulsen, Kozo Kaibuchi^**, Sergio Grinstein, and John Orlowski^§§¶¶

From the  Cell Biology Programme, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, the  Department of Surgery, The Toronto Hospital and University of Toronto, Toronto, Ontario M5G 1L7, Canada, the ^** Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan, and the ^§§ Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada

Received for publication, February 13, 2000, and in revised form, June 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The activity of the Na⁺/H⁺ exchanger NHE3 isoform, which is found primarily in epithelial cells, is sensitive to the state of actin polymerization. Actin assembly, in turn, is controlled by members of the small GTPase Rho family, namely Rac1, Cdc42, and RhoA. We therefore investigated the possible role of these GTPases in modulating NHE3 activity. Cells stably expressing NHE3 were transiently transfected with inhibitory forms of Rac1, Cdc42, or RhoA and transport activity was assessed using microfluorimetry. NHE3 activity was not adversely affected by either dominant-negative Rac1 or Cdc42. By contrast, the inhibitory form of RhoA greatly depressed NHE3 activity, without noticeably altering its subcellular distribution. NHE3 activity was equally reduced by inhibiting p160 Rho-associated kinase I (ROK), a downstream effector of RhoA, with the selective antagonist Y-27632 and a dominant-negative form of ROK. Furthermore, inhibition of ROK reduced the phosphorylation of myosin light chain. A comparable net dephosphorylation was achieved by the myosin light chain kinase inhibitor ML9, which similarly inhibited NHE3. These data suggest that optimal NHE3 activity requires a functional RhoA-ROK signaling pathway which acts, at least partly, by controlling the phosphorylation of myosin light chain and, ultimately, the organization of the actin cytoskeleton.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Members of the Na⁺/H⁺ exchanger (NHE)¹ family mediate the electroneutral countertransport of H⁺ for Na⁺ across biological membranes (for review, see Refs. 1 and 2). To date, six mammalian NHE isoforms have been described. Some, like NHE1 and NHE6, are expressed ubiquitously and are thought to be involved in housekeeping functions such as the regulation of the cytosolic pH and cellular volume, and the maintenance of mitochondrial ion homeostasis, respectively (1, 3). Other isoforms, in contrast, have a more restricted tissue distribution and are believed to mediate specialized functions. These include NHE3, which is the predominant isoform found on the apical membranes of epithelial cells in the kidney and gastrointestinal tract (4, 5). In these tissues, NHE3 catalyzes the entry of luminal Na⁺ into the cells, which in turn drives the transport of salt and osmotically obliged water across the epithelium. The fine regulation of systemic fluid and electrolyte homeostasis requires exquisite regulation of NHE3 activity, which has been shown to be modulated by a variety of hormones and second messengers (6, 7), as well as by physical parameters including osmolarity (8, 9).

Despite the central role of NHE3 in renal and intestinal physiology, relatively little is known about the exact molecular mechanisms underlying its regulation. Electron microscopy studies of epithelial cells revealed that, in addition to being present at the apical membrane, NHE3 is also found in an intracellular vesicular pool (10). This bimodal subcellular distribution is recapitulated when NHE3 is heterologously expressed in Chinese hamster ovary cells (11). Changes in the fraction of transporters residing on the cell surface could therefore modify the rate of transport. Indeed, reduced trafficking of intracellular exchangers to the plasmalemma was found to account for the inhibitory effects of wortmannin, an antagonist of phosphatidylinositol 3-kinase, on NHE3 activity in transfected Chinese hamster ovary cells (12). Likewise, the acute inhibition of NHE3 activity upon activation of protein kinase C in colonic epithelial cells was attributed, at least in part, to internalization of exchanger molecules from the brush border into a subapical cytoplasmic compartment (13).

The activity of NHE3 is also acutely sensitive to the state of assembly of the actin cytoskeleton (14). Specifically, depolymerization of actin filaments by scavenging monomeric actin with latrunculin B, or by capping their barbed ends with cytochalasins, led to a profound inhibition of NHE3 activity. Unlike the effects of wortmannin, however, the inhibition induced by cytoskeletal disassembly was not associated with a decrease in the amount of NHE3 at the plasma membrane (14). Therefore, the activity of NHE3 is likely to be modulated by multiple regulatory mechanisms. The structural and functional basis of the interaction between NHE3 and the actin cytoskeleton remains obscure and is the subject of the present studies.

The state of actin polymerization is controlled by members of the Rho family of small GTP-binding proteins, particularly Rac1, Cdc42, and RhoA (15). Interestingly, an interaction between members of this family and NHE1, the ubiquitous isoform of the exchanger, has been postulated. Specifically, Barber and colleagues (16) have suggested that the presence of NHE1 is required for the successful induction of stress fibers by RhoA. In addition, activation of small GTPases of the Rho and Ras families is associated with stimulation of NHE1 (17, 18). However, disruption of the actin cytoskeleton with cytochalasin B did not significantly alter basal NHE1 activity (14), unlike the detrimental effects observed for NHE3. Thus, the NHE isoforms are differentially responsive to the state of actin polymerization.

The objective of the present studies was to define whether a functional interaction exists between NHE3 and Rac1, Cdc42, or RhoA. To this end, cells were transfected with dominant-negative or constitutively active forms of these small GTP-binding proteins and the activity and subcellular distribution of NHE3 were assessed by single-cell imaging. Because NHE3 is natively expressed in epithelial cells that are less amenable to transfection, we used instead NHE-deficient AP-1 cells, which are derived from Chinese hamster ovary cells (19) and readily transfected. Our studies suggest that functional RhoA, but not Rac1 or Cdc42, are essential for optimal NHE3 function and that the effects of RhoA are mediated, at least in part, by the p160 Rho-associated kinase I (ROK), which controls the state of phosphorylation of myosin light chain.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Solutions-- Nigericin, the acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), Oregon green- and rhodamine-phalloidin were from Molecular Probes, Inc. (Eugene, OR). ML-9 was from Calbiochem. All other chemicals were purchased from BDH Inc. (St. Laurent, Quebec, Canada). Mouse anti-HA antibodies were from BabCo (Berkeley, CA). Peroxidase-conjugated donkey anti-mouse IgM and Cy-3-conjugated donkey and goat anti-mouse IgG were from Jackson ImmunoResearch, Inc. (West Grove, PA). Mouse monoclonal antibody to myosin light chain and the OPD reagent were from Sigma. ¹²⁵I-Labeled anti-mouse goat IgG was from ICN (Costa Mesa, CA). Rabbit polyclonal anti-myc antibody was from Santa Cruz. FuGENE^TM 6 was from Roche Molecular Biochemicals. The enhanced chemiluminescence system (ECL) was from Amersham Pharmacia Biotech. -Minimal essential medium, fetal bovine serum, penicillin/streptomycin, and trypsin-EDTA were purchased from Life Technologies (Burlington, Ontario). Y-27632 was a generous gift from Yoshitomi Pharmaceutical Industries, Ltd., Japan.

Isotonic Na⁺ medium contained (in mM): 130 NaCl, 3 KCl, 1 MgCl₂, 1 CaCl₂, 20 Hepes, pH 7.4. Isotonic K⁺ medium (Na⁺ free medium) contained: 130 KCl, 1 MgCl₂, 1 CaCl₂, 20 Hepes, pH 7.4.

Cells-- AP-1 cells, a Chinese hamster ovary cell line that was functionally selected for its lack of endogenous NHE activity following chemical mutagenesis (20), was transfected with either wild type NHE1 or NHE3 previously engineered to contain several unique restriction endonuclease sites (renamed NHE1' and NHE3') in order to create a series of conveniently sized DNA cassettes for subsequent mutagenesis procedures (21). To allow for immunological detection of the NHE3 protein, three tandem copies of the influenza virus hemagglutinin (HA) epitope, YPYDVPDYAS, were inserted into the first extracellular loop of NHE3 between amino acids Arg³⁸ and Phe³⁹, as described previously (11). The resulting construct is called NHE3'_38HA3 hereafter. The functional properties of the HA-tagged NHE3' construct were indistinguishable from those of untagged NHE3 (12).

NHE cDNA constructs were transfected into AP-1 cells by the calcium-phosphate-DNA co-precipitation technique of Chen and Okayama (22) and stable clones were selected for survival by imposing acute NH₄Cl-induced acid loads (19). Cells were maintained in -minimal essential medium with 10% fetal bovine serum and streptomycin/penicillin in an atmosphere containing 5% CO₂.

DNA Constructs and Transient Transfection-- The plasmids encoding the epitope-tagged dominant-negative forms of RhoA (RhoA(TN19)_myc), Rac1 (Rac1(T17N)_myc), and Cdc42 (Cdc42(T17N)_myc), as well as the constitutively active RhoA (RhoA(Q63L)_myc) were kindly provided by Dr. G. Bokoch (Scripps Research Institute, La Jolla, CA) (23). Expression vectors containing cDNAs encoding the constitutively active p160 Rho-associated kinase I, ROK-CAT_myc, and the dominant-negative p160 Rho-associated kinase I, ROK-RB/PH_myc, were described previously (24). The expression vector containing the cDNA encoding the enhanced green fluorescent protein (EGFP) was from CLONTECH. For transient transfection, DNA was introduced into cells plated on coverslips by transfection using FuGENE^TM 6 as recommended by the manufacturer, using 0.5 µg of the construct of interest together with 0.1 µg of EGFP cDNA. Using this ratio, 80% of the EGFP-positive cells also expressed the vector of interest, as determined in separate experiments by immunostaining the epitope tag. Cells were analyzed 48 h after transfection.

Measurement of Na⁺/H⁺ Exchanger Activity-- NHE activity was assessed as the rate of Na⁺-induced recovery of cytosolic pH (pH_i) following an acid load, imposed by pre-pulsing with NH₄Cl, as described previously (8). Briefly, cells grown on coverslips to 70% confluency were incubated with 2 µg/ml, the acetoxymethyl ester precursor of BCECF plus 50 mM NH₄Cl at 37 °C. After 10 min, the cells were placed into Leiden CoverSlip holders and washed with isotonic Na⁺-free solution to remove excess dye and extracellular NH₄Cl. Na⁺/H⁺ exchange was then initiated by re-introduction of extracellular Na⁺ and activity was estimated from the rate of recovery of pH_i.

Two methods were used to measure the fluorescence of BCECF. Population measurements were performed using a Nikon Diaphot TMD inverted microscope coupled to the M Series Dual Wavelength Illumination and Recording System from Photon Technologies, Inc. (South Brunswick, NJ) in a dual excitation-single emission configuration, as detailed earlier (8). The fluorescence of transiently transfected single cells was measured using the ratio imaging system described in Ref. 25 controlled by the Metafluor software (Universal Imaging, West Chester, PA). Transfected cells were identified by detecting EGFP fluorescence prior to loading with BCECF. A neutral density filter was then interposed in the excitation pathway, to decrease the signal emanating from EGFP, and the cells were loaded with BCECF and ammonium while on the microscope stage. The fluorescence of BCECF clearly exceeded that of EGFP and was readily visible in the presence of the neutral density filter. In a typical experiment, the EGFP fluorescence intensity was 150-250 units prior to insertion of the filter, was reduced to 20-40 units by the neutral density filter, and increased to 100-150 units with BCECF loading. The excitation wavelengths were 440 and 490 nm and the emission wavelength 510 nm for both the population and single-cell measurements. In both instances pH_i was calibrated by equilibrating the cells with K⁺-rich media titrated to known pH values and containing 10 µg/ml nigericin, as described previously (8).

Immunofluorescence-- AP-1 cells stably expressing NHE3'_38HA3 were plated onto glass coverslips and grown to ~70% confluence. To label only surface-exposed NHE3'_38HA3, intact cells were incubated with monoclonal anti-HA antibody (1:1000 dilution) for 1 h at 4 °C. After washing 6 times to remove unbound antibody, the cells were fixed for 15 min at room temperature using 4% paraformaldehyde in PBS and blocked with 5% donkey serum in PBS. The cells were then washed and incubated with Cy3-conjugated donkey anti-mouse antibody (1:1000 dilution) for 1 h. To visualize total cellular NHE3'_38HA3, the cells were fixed as above and were then permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature, blocked with 5% donkey serum in PBS for 1 h, and incubated with mouse anti-HA antibody for 1 h. The coverslips were next washed 4-5 times with PBS and incubated with Cy3-conjugated anti-mouse IgG for 1 h. After incubation with the secondary antibody, the cells were washed 3-5 times over 15 min with PBS and mounted onto glass slides with DAKO Fluorescence mounting medium (DAKO Corp., Carpinteria, CA). Where specified, F-actin was labeled by incubating fixed and permeabilized cells with fluoresceinated phalloidin (1:500) for 1 h at room temperature.

Cells were visualized using the ×100 objective of a Leica DM1RB fluorescence microscope (Heidelberg, Germany) equipped with a Micromax cooled CCD camera (Princeton Instruments, Trenton, NJ), operated from a Dell computer using Winview^TM software (Princeton Instruments). Where indicated, cells were visualized using a Zeiss LSM 510 confocal microscope. Serial optical slices (0.5 µm thick) were acquired and a stacked image was composed using the LSM 510 software. To quantify the surface staining, the fluorescence intensity was measured digitally using Metafluor^TM software. Five regions of defined size were chosen randomly in each transfected cell and in surrounding non-transfected cells within the same field, and the brightness of the regions was averaged. The mean brightness of transfected cells was expressed as percent of the non-transfected cells (control = 100%) for each field.

Quantitation of Surface NHE3-- Two different methods were used to quantify surface NHE3 expression: (i) a modified enzyme-linked immunosorbent assay (ELISA) described earlier (12) and (ii) a radioisotopic method. AP-1 cells stably expressing NHE3'_38HA3 were plated onto 12-well plates and grown to ~70% confluence. For ELISA, cells were incubated with anti-HA antibody (1:1000 dilution) for 1 h at 4 °C to prevent endocytosis. After washing the cells 6 times with PBS/-minimal essential medium (9:1, v/v) to remove excess unbound antibody, they were fixed for 10 min at room temperature using 4% paraformaldehyde in PBS. Following fixation, the cells were washed 3-4 times with PBS and incubated with 100 mM glycine in PBS for 15 min. Cells were next blocked with 5% donkey serum for 20 min, then incubated with a peroxidase-conjugated donkey anti-mouse antibody (1:1000 dilution) for 1 h followed by washing 6 more times with PBS/-minimal essential medium and incubation with 1 ml of OPD reagent for 15 min at room temperature. The reaction was stopped by adding 250 µl of 3 M HCl. The supernatant was collected and absorbance measured at 492 nm using a U-2000 spectrophotometer (Hitachi, Tokyo, Japan). In the range studied, the absorbance varied linearly with the amount of peroxidase bound.

For isotopic determinations, the cells were treated on the plates as specified in the text and then placed on ice and incubated in PBS containing 2 mM EDTA for 20 min to detach them from the surface. All subsequent steps were carried out at 4 °C. Cells were gently scraped into ice-cold medium containing 10% goat serum and counted using a Coulter counter. Equal numbers of cells (0.5-5 × 10⁵) were suspended in 1 ml of medium and incubated with anti-HA antibody (3 µl/sample) for 1 h. The cells were then sedimented and washed 3 times with PBS. Next, they were incubated with ¹²⁵I-labeled goat anti-mouse IgG (0.4 µCi/sample) in medium with 10% goat serum for 45 min. At the end of the incubation cells were sedimented and washed 3 more times with PBS to remove unbound radiolabel. Radioactivity was counted using a 1282 Compugamma LKB counter. The radioactivity bound to cells exposed only to ¹²⁵I-IgG without prior incubation with anti-HA antibody was also determined and subtracted from all determinations. Data are expressed as % of surface labeling of untreated control cells (100%) and are the mean ± S.E. of the number of experiments indicated, each performed in duplicate or triplicate

Assessment of Myosin Phosphorylation-- Cells were grown to 70% confluency in 10-cm² dishes and treated as specified in the text. After rinsing with PBS, cellular proteins were precipitated with ice-cold 10% trichloroacetic acid in acetone containing 2 mM dithiothreitol. Precipitates were scraped from the plate and washed once with ice-cold acetone. Pellets were solubilized in sample buffer containing 9 M urea, 2 mM dithiothreitol, 22 mM glycine, and 20 mM Tris, pH 8.8. Aliquots of the lysates were fractionated by glycerol-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The blots were probed with a monoclonal anti-myosin light chain antibody (clone MY-21, 1:200 dilution) followed by a peroxidase-coupled anti-IgM antibody. The blots were visualized using enhanced chemiluminescence.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Transfection of AP-1 Cells with Rac1, Cdc42, and RhoA-- Transient transfection of dominant-negative forms of myc-tagged Rac1, Cdc42, and RhoA was used to study their possible role in the control of NHE3. Effective expression of the GTP-binding proteins was verified by immunostaining, using antibodies to the myc epitope tag. As illustrated in Fig. 1, RhoA(T19N)_myc, Rac1(T17N)_myc, and Cdc42(T17N)_myc were readily detectable in AP-1 cells that stably express an HA-tagged version of NHE3 (i.e. NHE3'_38HA3). Co-transfection with EGFP was used to identify the transfectants by non-invasive means. The vast majority of the cells that displayed EGFP fluorescence were also myc-positive (e.g. Fig. 1, A and B), implying that the fluorescent protein is an adequate marker of the expression of the GTP-binding proteins. The functional effectiveness of the dominant-negative proteins was manifested by the altered F-actin distribution and aberrant morphology of the transfected cells. As shown in Fig. 1, A-D, unlike untreated cells that are generally polygonal (inset of Fig. 1B), those transfected with dominant-negative RhoA(T19N)_myc acquired a fusiform or stellate morphology and were largely devoid of stress fibers, consistent with observations in other cell types (26-28). When transfected with inactive Rac1(T17N)_myc, the cells were less spread and their F-actin content decreased (Fig. 1, E and F). A similar phenotype was observed in cells transfected with dominant-negative Cdc42(T17N)_myc (Fig. 1, G and H), as described for other cell types (29). These results imply that the activity of GTP-binding proteins in NHE3-expressing cells can be effectively manipulated by transfection of inhibitory constructs and that the transfected cells can be conveniently identified by co-expression of EGFP, enabling functional measurements in live cells.

View larger version (68K): [in this window] [in a new window]   Fig. 1.   Expression of Rho family GTPases in AP-1 cells. AP-1 cells expressing NHE3'38HA3 grown on coverslips were transiently transfected with RhoA(T19N)myc (A-D), Rac1(T17N)myc (E and F), or Cdc42(T17N)myc (G and H). After 48 h, the cells were fixed, permeabilized, and stained with anti-myc antibodies (A, C, E, and G) and with FITC-phalloidin to detect F-actin (D, F, and H). In panels A and B, the cells were co-transfected with EGFP, which was visualized directly using fluorescein optics (panel B). The inset in B shows a control cell transfected with EGFP only. The arrows point to cells transiently transfected with the myc-tagged constructs and/or EGFP. Images are representative of at least three experiments of each type.

Effect of Dominant-negative Rac1 and Cdc42 on NHE3-- The effect of inhibitory forms of the GTP-binding proteins on NHE3 activity was assessed by measuring the rate of Na⁺-induced H⁺ (equivalent) extrusion from acid-loaded cells. To measure pH_i, transfected cells were initially identified by their expression of EGFP (Fig. 2A). The contribution of EGFP to the fluorescence measurements was next minimized by inserting a neutral-density filter (Fig. 2B) which greatly reduced the signal intensity. Next, the cells were loaded with the pH-sensitive dye BCECF, reaching levels of fluorescence that clearly exceeded (>250%; see "Experimental Procedures") those contributed by EGFP (Fig. 2C). Under these conditions, and using internal calibrations after each individual experiment we were able to monitor the activity of NHE3 in small GTPase-transfected and untransfected (control) cells simultaneously using ratiometric fluorescence imaging microscopy.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Cytosolic pH (pHi) measurements in control and Cdc42(T17N)myc-transfected cells. A-C, method used for measurement of pHi in transfected cells. Cells expressing EGFP were located (A) and a neutral density filter was then placed in the excitation pathway to curtail the EGFP signal (B). The cells were then loaded for 10 min with BCECF while on the microscope stage, reaching fluorescence intensities that were clearly visible using the neutral density filter (C). D, effect of Cdc42(T17N)myc on NHE3 activity. Cells stained with BCECF as in A-C, were acid-loaded by an ammonium pre-pulse, as described under "Experimental Procedures." Recording of pHi was initiated upon re-introduction of Na+ to the medium. Open circles, control cells transfected with EGFP only. Solid circles, cells transfected with Cdc42(T17N)myc and EGFP (cDNA ratio, 5:1). Data are mean ± S.E. of 26 determinations from three separate experiments.

The effects of transient transfection with dominant-negative Cdc42(T17N)_myc are illustrated in Fig. 2D. As reported previously (12), AP-1 cells that stably express rat NHE3 recover from an acid load within minutes of addition of extracellular Na⁺. This recovery was not prevented by expression of Cdc42(T17N)_myc. In fact, the recovery was marginally faster in the transfected cells, although the difference in the rates did not attain statistical significance in the limited number of experiments performed. A similar modest activation was recorded in cells transfected with Rac1(T17N)_myc (data not illustrated).

Effect of Dominant-negative RhoA on NHE3-- In contrast to Rac1(T17N)_myc and Cdc42(T17N)_myc, dominant-negative Rho A(T19N)_myc induced a pronounced inhibition of NHE3 activity (Fig. 3A), resembling the effects of latrunculin B and cytochalasin (14). The depression of NHE3 activity is not likely the result of a generalized detrimental effect of RhoA(T19N)_myc, since the transfectants retained BCECF and were effectively acid-loaded by the ammonium pre-pulse. More importantly, cells transfected with NHE1, which is virtually insensitive to cytoskeletal disruption (14), were similarly unaffected by expression of dominant-negative RhoA(T19N)_myc (Fig. 3B).

View larger version (58K): [in this window] [in a new window]   Fig. 3.   Effect of RhoA(T19N)myc on the activity and subcellular distribution of NHE3 and NHE1. A and B, AP-1 cells stably expressing NHE3 (A) or NHE1 (B) were transiently transfected with either RhoA(T19N)myc plus EGFP (solid circles), or with EGFP alone (open circles). After 48 h, the cells were stained with BCECF, acid-loaded, and used for measurement of pHi as in Fig. 2. Data are mean ± S.E. of 15 determinations from three separate experiments. C-F, AP-1 cells stably expressing NHE3'38HA3 were transfected with RhoA(T19N)myc. In C and D, the cells were fixed, permeabilized, and stained with antibodies to myc (C) and to HA, the epitope on NHE3'38HA3 (D). In E and F, the cells were incubated with anti-HA antibodies (1:1000 dilution) for 1 h at 4 °C prior to fixation and permeabilization, to visualize only surface-exposed epitopes. Antibodies to myc and fluorophore-conjugated secondary antibodies were added after fixation and permeabilization. Panels E and F are composite stacks of serial confocal sections. The arrows point to cells transiently transfected with RhoA(T19N)myc. Images are representative of at least three experiments of each kind. G, quantitation of surface NHE3 in control and RhoA(T19N)myc-transfected cells. Surface labeling was quantified digitally in experiments like that in E and F using Metafluor, as described under "Experimental Procedures." Data are mean ± S.E. of >20 cells of each type in three separate experiments.

The mechanism underlying the inhibition of NHE3 induced by RhoA(T19N)_myc was investigated next. This isoform of the exchanger is expressed in two distinct cellular pools: on the plasmalemma, where it catalyzes the extrusion of H⁺, and in sorting and recycling endosomes (12, 30). Inhibition of Na⁺/H⁺ exchange can therefore be envisioned to result from redistribution of plasmalemmal exchangers to the endomembrane compartment. This possibility was analyzed by immunofluorescence detection of an NHE3 construct that bears an extracellular triple HA-epitope (NHE3'_38HA3). The functional properties of the HA-tagged NHE3 construct have been shown previously to be indistinguishable from those of untagged NHE3 (12). The overall cellular distribution of NHE3'_38HA3 in permeabilized cells was not visibly affected by the expression of RhoA(T19N)_myc (cf. myc-positive and -negative cells in Fig. 3, C and D). To specifically measure plasmalemmal NHE3'_38HA3, the fluorescence emitted by surface-stained (non-permeabilized) cells was quantified digitally in images that were reconstructed by stacking serial optical slices obtained by confocal microscopy. The whole cell reconstructed images are presented in Fig. 3, E and F, and show that NHE3'_38HA3 is clearly detectable at the surface of intact (non-permeabilized) myc-positive and -negative cells. Moreover, quantitation of the fluorescence signal indicates that expression of the dominant-negative form of RhoA does not alter the number of transporters present on the surface (Fig. 3G). Thus, inhibition of transport most likely reflects changes in the intrinsic activity of the individual exchangers.

Effect of Constitutively Active RhoA on NHE3-- Having established the inhibitory effect of inactive RhoA, we next tested whether activation of RhoA has the opposite effect on NHE3 function. To this end, AP-1 cells expressing NHE3'_38HA3 were transiently transfected with a mutant form of RhoA containing a Q63L substitution that prevents hydrolysis of GTP and therefore remains constitutively active (23). The expression of RhoA(Q63L)_myc, which was verified by detection of the myc epitope tag (Fig. 4A), was accompanied by a marked increase in the number of stress fibers (Fig. 4B), as reported for other cells (31, 32, 15). Despite the changes in F-actin structure, no obvious differences in the subcellular distribution of NHE3'_38HA3 were observed (Fig. 4, C and D), nor was the surface density of NHE3'_38HA3 visibly altered (Fig. 4, E and F). More importantly, the exchange activity of NHE3'_38HA3, assessed as the rate of recovery from an acid load, was also unaffected by expression of RhoA(Q63L)_myc (Fig. 4G). In this regard, NHE3 differs from NHE1, which was found to activate upon expression of active RhoA (18).

View larger version (54K): [in this window] [in a new window]   Fig. 4.   Effect of RhoA(Q63L)myc on the activity and subcellular distribution of NHE3. A-F, AP-1 cells expressing NHE3'38HA3 were transfected with RhoA(Q63L)myc. In A-D, the cells were fixed, permeabilized, and stained with antibodies to myc (A and C) and to HA, the epitope on NHE3'38HA3 (D). In B, the cells were stained with fluorescein isothiocyanate-phalloidin to visualize F-actin. In E and F, the cells were incubated with anti-HA antibodies for 1 h at 4 °C prior to fixation and permeabilization, to visualize only surface-exposed epitopes. Antibodies to myc and fluorophore-conjugated secondary antibodies were added after fixation and permeabilization. The arrows point to cells transiently transfected with RhoA(Q63L)myc. Images are representative of at least three experiments of each kind. G, AP-1 cells stably expressing NHE3 were transiently transfected with either RhoA(Q63L)myc plus EGFP (solid circles) or with EGFP alone (open circles). After 48 h, the cells were stained with BCECF, acid-loaded, and used for measurement of pHi as in Fig. 2. Data are mean ± S.E. of 15 determinations from three separate experiments.

Role of ROK in NHE3 Regulation-- The preceding results imply that basal levels of RhoA activity are required for optimal NHE3 function, but do not identify the pathway linking these events. Among the known effectors of this small GTP-binding protein is the serine/threonine kinase, ROK. The possible involvement of ROK in the regulation of NHE3 was tested using compound Y-27632, a potent and seemingly selective inhibitor of this kinase (33). The effectiveness of this compound was initially verified by visualizing the distribution of F-actin using phalloidin. As illustrated in Fig. 5, A and B, treatment with 2 µg/ml Y-27632 for 30 min altered cellular morphology in a manner that resembled the effects of RhoA(T19N)_myc, i.e. the normally polygonal AP-1 cells became elongated in appearance. In accordance with the role of ROK in the stabilization of stress fibers (32, 34), the cells treated with Y-27632 were largely devoid of such fibers. These effects were accompanied by, and likely due to, dephosphorylation of myosin light chain (MLC; Fig. 5F), which is necessary for the stabilization of stress fibers and cell shape (32). It is well established that the phosphorylation state of MLC is controlled by ROK (35, 36).

View larger version (56K): [in this window] [in a new window]   Fig. 5.   Effect of Y-27632 on actin morphology, myosin phosphorylation, and the activity and distribution of NHE3 and NHE1. A-D, AP-1 cells expressing NHE3'38HA3 were incubated without (A and C) or with 2 µg/ml Y-27632 for 30 min at 37 °C (B and D). In A and B, the cells were stained with FITC-phalloidin to visualize F-actin. In C and D, the cells were fixed, permeabilized, and stained with antibodies to HA, the epitope on NHE3'38HA3. E, quantification of surface NHE3 using 125I-IgG. Cells were either untreated (control) or incubated with Y-27632 as above. Data are means of five determinations, each in duplicate. F, phosphorylation of myosin light chain. Control (untreated) or Y-27632-treated cells were extracted with trichloroacetic acid in acetone and samples of the precipitate were subjected to urea-PAGE, then blotting onto nitrocellulose. The blot was probed with anti-MLC antibodies (1:200) followed by horseradish peroxidase-conjugated anti-mouse IgM and visualized by ECL. The location of MLC and of its phosphorylated form (MLC-P) is indicated. G and H, AP-1 cells expressing NHE3 (G) or NHE1 (H) were treated transiently without (open circles) or with Y-27632 (solid circles) as above. After 30 min, the cells were stained with BCECF, acid-loaded, and used for measurement of pHi as described in the legend to Fig. 2. Data are mean ± S.E. of 15 determinations from three separate experiments.

Despite the pronounced effects of Y-27632 on cell morphology, the subcellular distribution of NHE3'_38HA3 was not affected. The exchangers were still observed on the plasma membrane, as well as in a punctate endomembrane compartment that concentrated in the juxtanuclear region (Fig. 5, C and D). This impression was confirmed by quantifying the number of plasmalemmal NHE3'_38HA3 molecules by two separate methods: a modified ELISA and a radiolabel binding assay. In both cases, an antibody to the exofacial HA epitope tag is added to intact cells to detect exclusively those exchangers exposed at the surface (see "Experimental Procedures"). In three separate ELISA experiments, the number of exchangers exposed at the cell surface was essentially identical before and after treatment with Y-27632 (98.6 ± 8.0% of the untreated control). Similar results were obtained using ¹²⁵I-labeled IgG to detect immunolabeled NHE3'_38HA3 (Fig. 5E).

While the distribution of NHE3'_38HA3 was unaffected, its activity was nevertheless markedly depressed when ROK was inactivated by Y-27632 (Fig. 5G). The initial rate of exchange, computed in the first minute after addition of Na⁺, was inhibited by 92%. By contrast, NHE1 was only modestly, albeit significantly (p < 0.05), affected by Y-27632 (12% inhibition of the initial rate; Fig. 5H).

Because the selectivity of Y-27632 may be imperfect, we also assessed the role of ROK by transfection of a mutated inactive form of this kinase. A point mutation in the RB domain of the protein encoded by this construct, named ROK-RB/PH_myc, prevents it from binding to RhoA (24). It is noteworthy that while this mutant exerts a dominant negative effect on ROK, it does not scavenge RhoA, and therefore does not interfere with other RhoA-activated signal transduction pathways (24). As shown in Fig. 6, expression of ROK-RB/PH_myc depleted the cells of stress fibers and changed their morphology, rendering them stellate in appearance (Fig. 6, A and B). In parallel, the activity of NHE3'_38HA3 was depressed (Fig. 6H), even though its subcellular distribution (Fig. 6D) and surface exposure (Fig. 6, E-G) were seemingly unaffected. These results are similar to those obtained with Y-27632, supporting the notion that RhoA modulates the activity of NHE3 via ROK.

View larger version (56K): [in this window] [in a new window]   Fig. 6.   Effect of ROK-RB/PH(myc) on the activity and subcellular distribution of NHE3. A-F, AP-1 cells expressing NHE3'38HA3 were transfected with ROK-RB/PHmyc. In A-D, the cells were fixed, permeabilized, and stained with antibodies to myc (A and C) and to HA, the epitope on NHE3'38HA3 (D). In B, the cells were stained with fluorescein isothiocyanate-phalloidin. In E and F, the cells were incubated with anti-HA antibodies for 1 h at 4 °C prior to fixation and permeabilization, to visualize only surface-exposed epitopes. Antibodies to myc and fluorophore-conjugated secondary antibodies were added after fixation and permeabilization. The sample was analyzed by confocal microscopy and the image shown was reconstructed by stacking optical slices. Images are representative of at least three experiments of each kind. The arrows point to cells transiently transfected with ROK-RB/PHmyc. G, quantification of surface NHE3 in control and ROK-RB/PHmyc-transfected cells. Cells were stained and analyzed as in E and F. The fluorescence intensity of equivalent areas within the reconstructed images of the cells was quantified using Metafluor software. H, AP-1 cells expressing NHE3 were transiently transfected with either ROK-RB/PHmyc plus EGFP (solid circles) or with EGFP alone (open circles). After 48 h, the cells were stained with BCECF, acid-loaded, and used for measurement of pHi as in Fig. 2. Data are mean ± S.E. of 15 determinations from three separate experiments.

We also tested whether the basal activity of NHE3 can be enhanced by stimulation of ROK. For this purpose, cells expressing NHE3'_38HA3 were transiently transfected with an epitope-tagged version of the catalytic domain of ROK, designated ROK-CAT_myc. The isolated catalytic domain can actively phosphorylate its substrates without requirement for RhoA activation (24). Transfection of ROK-CAT_myc induced the formation of thick bundles of stress fibers in AP-1 cells (Fig. 7, A and B). In addition, F-actin condensed into bright patches at or near adhesion sites, as reported for Swiss 3T3 and HeLa cells (32, 37). Even though effectors of ROK were demonstrably activated, neither the distribution of NHE3'_38HA3 (Fig. 7, C-F), nor its activity (Fig. 7G) were noticeably affected. These observations suggest that the extent of activation of ROK in the steady state is sufficient for optimal NHE3 function. This conclusion is in good agreement with the results obtained using the constitutively active RhoA(Q63L)_myc.

View larger version (60K): [in this window] [in a new window]   Fig. 7.   Effect of ROK-CAT(myc) on the activity and subcellular distribution of NHE3. A-F, AP-1 cells expressing NHE3'38HA3 were transfected with ROK-CATmyc. In A-D, the cells were fixed, permeabilized, and stained with antibodies to myc (A and C) and to HA, the epitope on NHE3'38HA3 (D). In B, the cells were stained with FITC-phalloidin. In E and F, the cells were incubated with anti-HA antibodies for 1 h at 4 °C prior to fixation and permeabilization, to visualize only surface-exposed epitopes. Antibodies to myc and fluorophore-conjugated secondary antibodies were added after fixation and permeabilization. Images are representative of at least three experiments of each kind. The arrows point to cells transiently transfected with ROK-CATmyc. G, AP-1 cells stably expressing NHE3 were transiently transfected with either ROK-CATmyc plus EGFP (solid circles), or with EGFP alone (open circles). After 48 h, the cells were stained with BCECF, acid-loaded, and used for measurement of pHi as described in the legend to Fig. 2. Data are mean ± S.E. of 15 determinations from three separate experiments.

Role of Myosin Phosphorylation in NHE3 Regulation-- ROK is known to promote the phosphorylation of myosin not only by inhibiting myosin phosphatase (36), but also by directly phosphorylating its light chain (35). This raises the possibility that the effects of RhoA and ROK on NHE3 are a consequence of changes in the state of phosphorylation of myosin, thereby altering its interaction with actin. This possibility was tested by interfering with the phosphorylation of myosin, using a pharmacological inhibitor of myosin light chain kinase (MLCK). Treatment of NHE3'_38HA3-expressing AP-1 cells with the MLCK inhibitor ML9 (50 µM for 30 min) effectively reduced the phosphorylation of the myosin light chain (Fig. 8F), indirectly via the activity of constitutive phosphatases. In parallel, ML9 induced morphological changes similar to those caused by dominant-negative RhoA and ROK constructs; namely, the cells became elongated, with multiple protrusions (Fig. 8, A and B). Stress fibers were largely absent from ML9-treated cells (Fig. 8B). The effect of the MLCK inhibitor on the activity of NHE3'_38HA3 was also tested. The Na⁺-induced recovery from an acid load was profoundly inhibited in NHE3'_38HA3-expressing cells treated with ML9 (Fig. 8G). This effect was isoform-specific, inasmuch as NHE1-expressing cells were not inhibited by ML9, but were instead marginally stimulated (Fig. 8H). The inhibition of NHE3'_38HA3 was not associated with changes in its subcellular distribution. This was established both morphologically (Fig. 8, C and D) as well as using radiolabeled IgG (Fig. 8E). Similar results were obtained using the ELISA method (ML9-treated cells were 98.8 ± 1.7% of control; n = 3). Hence, inhibition is most likely due to a change in the intrinsic activity of the plasmalemmal NHE3. Jointly, these findings suggest that RhoA modulates the activity of NHE3, at least in part by dictating the state of myosin phosphorylation through ROK.

View larger version (52K): [in this window] [in a new window]   Fig. 8.   Effect of ML9 on NHE3 and NHE1. A-D, AP-1 cells expressing NHE3'38HA3 were incubated without (A and C) or with (B and D) 50 µM ML9 for 30 min at 37 °C. In A and B, the cells were stained with fluorescein isothiocyanate-phalloidin to visualize F-actin. In C and D, the cells were fixed, permeabilized, and stained with antibodies to HA, the epitope on NHE3'38HA3. E, quantification of surface NHE3 using 125I-IgG. Cells were either untreated (Con) or incubated with ML9 as above. Data are means of five determinations, each in duplicate. F, phosphorylation of myosin light chain in control (untreated) or ML9-treated cells was analyzed as described in the legend to Fig. 5. The location of MLC and of its phosphorylated form (MLC-P) is indicated. G and H, AP-1 cells expressing NHE3 (G) or NHE1 (H) were transiently treated without (open circles) or with ML9 (solid circles) as above. After 20 min, the cells were stained with BCECF, acid-loaded, and pHi measured as described in the legend to Fig. 2. Data are mean ± S.E. of 15 determinations from three separate experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

NHE3 had been shown earlier to depend on intact F-actin structures for optimal function (14). In the present study we assessed the possible role of small GTP-binding proteins in this process. We found no evidence that either Rac1 or Cdc42 participate in the regulation of NHE3. By contrast, functional RhoA was found to be essential for effective Na⁺/H⁺ exchange by NHE3. A role of small GTPases in the regulation of the housekeeping isoform NHE1 had been described earlier by Barber and colleagues (17, 18). However, the control of NHE1 and NHE3 by GTPases differs in several respects. First, the ubiquitous NHE1 is activated not only by RhoA, but also by Cdc42 and Rac1 (17). These effects, however, are seemingly independent of F-actin, inasmuch as cytochalasin D has little effect on the activity of this isoform (14). This contrasts with the pronounced dependence of NHE3 on normal F-actin cytoarchitecture (14). Second, the basal activity of NHE3 requires the steady state function of RhoA, but stimulation of the GTPase is without further effect. Conversely, only the stimulation, but not the basal activity of Rac1, Cdc42, and RhoA was shown to be important in the case of NHE1, which is activated by a variety of receptor-initiated pathways (17).

The present studies also indicate that ROK is an important effector in the modulation of NHE3 by RhoA. Inhibition of this kinase with Y-27632 or by transfection of a dominant-negative form of ROK mimicked the effects of inhibitory RhoA (Figs. 5 and 6). ROK was also suggested to mediate the effects of RhoA on NHE1, but, once again, the mechanisms controlling the two isoforms are distinctly different. ROK is believed to stimulate NHE1 by direct phosphorylation of its cytosolic C-terminal domain (18). In contrast, our findings suggest that ROK acts on NHE3, at least in part, by controlling the state of phosphorylation of myosin light chain, which in turn dictates the functional state of the antiporter. The latter was concluded from the observation that a comparable dephosphorylation of myosin light chain by inhibition of its kinase, using ML9, closely mimicked the effects of Y-27632 and of ROK-RB/PH_myc on NHE3 activity. However, it is becoming apparent that RhoA and its effector, ROK, engage multiple pathways to control cytoskeletal structure. In addition to dictating the state of myosin phosphorylation, ROK also activates LIM kinase, which in turn phosphorylates cofilin, an inducer of actin depolymerization. Because phosphorylated cofilin is no longer capable of causing depolymerization, activation of LIM kinase by ROK leads to increased actin assembly (38). Whether this or other pathways contribute to the regulation of NHE3 by RhoA and ROK remains to be defined.

The mechanism whereby the cytoskeleton modulates the activity of NHE3 remains obscure. Quantitation of the number of transporters at the surface of cells treated with Y-27632 or ML-9 (Figs. 5 and 8) as well as in cells transfected with RhoA(T19N)_myc or ROK-RB/PH_myc (Figs. 3 and 6, respectively) indicates that their subcellular distribution remains unaffected by cytoskeletal disruption. The inhibition of transport is therefore more simply attributed to reduced intrinsic activity of a constant number of plasmalemmal exchangers. It is possible that optimal activity of NHE3 requires association with an ancillary protein that is part of, or maintained in position by, the cytoskeleton. In this regard, two related proteins, NHERF-1 (also called EBP50) and -2 (also called TKA-1 or E3KARP) have been proposed to associate with NHE3 and to modulate its activity (39, 40). Although these proteins have been invoked in mediating the inhibition of NHE3 by cAMP, they could conceivably also influence the basal rate of transport. Alternatively or conjointly, proteins of the ezrin/radixin/moesin (ERM) family may be part of the regulatory complex. ERM proteins are attractive candidates because they are expressed abundantly at the brush border where NHE3 is present and because their conformational state is controlled by RhoA and ROK (41). In addition, polycationic sequences like those shown to mediate the attachment of several transmembrane proteins to ERM are also found in the C-terminal tail of NHE3. The possibility that ERM proteins directly bind and influence transport through NHE3 is currently under investigation.

The participation of RhoA in the regulation of NHE3 in its native environment, i.e. the epithelial brush border, remains to be documented. However, it is noteworthy that RhoA is abundant in the brush border (42), where it preferentially regulates apical and junctional actin (43). In Madin-Darby canine kidney cells, RhoA regulates the association of ERM family members with the plasma membrane (44). Perhaps more relevant to NHE3 regulation, in renal cells, apical Rho-GTPases were suggested to be involved in the regulation of phosphate transport by parathyroid hormone, which utilizes cAMP as a second messenger (45). Inhibition of transport was associated with phosphorylation of small GTPases and was mimicked by the clostridial C3 exotoxin, which specifically ADP-ribosylates and inactivates Rho. These observations raise the possibility that a similar signaling mechanism underlies the inhibition of NHE3 by cAMP. Indeed, preliminary experiments indicate that the effects of cAMP and RhoA(T19N)_myc are not additive and that the constitutively active RhoA(Q63L)_myc precludes the inhibitory effect of forskolin, an adenylate cyclase agonist.² If confirmed and extended, these results may provide a framework for the understanding of basal NHE3 activity and its regulation by hormones and other modulators.

	FOOTNOTES

^* This work was supported in part by the Medical Research Council of Canada and the Kidney Foundation of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Contributed equally to the results of this study.

^¶ Supported by a Medical Research Council of Canada fellowship.

International Scholar of the Howard Hughes Medical Institute and the current holder of the Pitblado Chair in Cell Biology at The Hospital for Sick Children. Cross-appointed to the Department of Biochemistry, University of Toronto.

^¶¶ Medical Research Council of Canada Scientist. To whom correspondence should be addressed: Dept. of Physiology, McGill University, McIntyre Medical Science Bldg., 3655 Promenade Sir-William-Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-8335; Fax: 514-398-7452; E-mail: orlowski@med.mcgill.ca.

Published, JBC Papers in Press, July 11, 2000, DOI 10.1074/jbc.M001193200

² K. Szászi, K. Kurashima, S. Grinstein, and J. Orlowski, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: NHE, Na⁺/H⁺ exchanger; BCECF, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; ERM, ezrin/radixin/moesin; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; MLC, myosin light chain; NHERF, NHE regulatory factor; PBS, phosphate-buffered saline; ROK, p160 Rho-associated kinase I; ELISA, enzyme-linked immunosorbent assay; MLCK, myosin light chain kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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