INTRODUCTION

The members of the Rho family of low molecular weight GTP-binding proteins are key control elements of the organization of the actin cytoskeleton (1). RhoA regulates the formation of stress fibers (2, 3), Rac controls the dynamics of lamellipodia (2, 3, 4), and Cdc42 controls filopodia (3). Recently, it has been shown that these GTPases also regulate a wide range of other cell functions, including cell-cell and cell-substrate adhesion, cell proliferation, and lipid metabolism (1). The signaling pathways which control these various functions are still largely uncharted.

RhoA has been shown to regulate the formation of stress fibers via at least two independent pathways. One pathway controls the establishment of focal complexes and is mediated by a staurosporine-inhibitable kinase. Another pathway regulates the polymerization of actin (3). Multiple RhoA effector proteins have recently been identified, including several kinases which could be involved in the regulation of the diverse aspects of stress fiber formation. These include the serine/threonine kinases protein kinase N (5, 6), RhoA-activated kinase (6, 7, 8), and PI 5-kinase¹ (9).

We recently determined that RhoA also mediates activation of the Na⁺-H⁺ exchanger isoform NHE1 by the GTPase G13 (4). Expression of a constitutively active RhoAV14 stimulates NHE1 activity, and activation of the exchanger by constitutively active 13Q226L is inhibited by coexpression of a dominant interfering RhoAN19. NHE1 is ubiquitously expressed and plays a central role in intracellular pH (pH_i) homeostasis and cell volume regulation. Increases in NHE1 activity are correlated with increased cell proliferation (10), differentiation (11, 12), and neoplastic transformation (13, 14).

The ability of RhoA to function as an important control point in both the activation of NHE1 and the formation of stress fibers prompted us to investigate the role of NHE1 in the regulation of stress fibers. Using a selective inhibitor of NHE1 and a cell line which is NHE-deficient, we determined that stress fiber formation induced by lysophosphatidic acid (LPA) is dependent on NHE1 activity. Moreover, induction of stress fibers by expression of the constitutively active mutant RhoAV14 is abolished in the NHE1-deficient cell line, but restored after the stable expression of NHE1 in these cells. These results indicate that stimulation of NHE1 activity is an essential component of the signal transduction pathway involved in the regulation of stress fibers by RhoA.

EXPERIMENTAL PROCEDURES

CCL39 cells, a hamster lung fibroblast line which expresses only the NHE1 isoform; PS120 cells, an NHE-deficient clone derived from CCL39 cells (15); and PS120 cells stably expressing NHE1 (PS120-NHE1) were maintained in high glucose (4.5 g/liter) Dulbecco's modified Eagle's medium (DME), supplemented with 5% heat-inactivated fetal bovine serum (Life Technologies, Inc.), streptomycin (100 µg/ml), and penicillin (100 units/ml). PS120-NHE1 cells were obtained by stable transfection of PS120 cells with the complete coding region of the rat NHE1 isoform (provided by J. Orlowski, McGill University) using the LipofectAMINE reagent according to the manufacturer's specifications (Life Technologies, Inc.). After transfection, cells were maintained in DME supplemented with 5% fetal bovine serum for 2-3 days. Cells expressing NHE1 were selected by a proton suicide technique developed by Pouyssegur and colleagues (15). Briefly, cells were washed twice with a HEPES buffer and incubated for 60 min in a modified HEPES buffer supplemented with 50 mM NH₄Cl at 37 °C in a CO₂-free atmosphere. After washing, the cells were incubated for an additional 2 h in a HEPES buffer (without NH₄Cl) at 37 °C in a CO₂-free atmosphere, washed, and maintained in DME as described above. This acid selection was repeated 3 times over a 2-week period. Expression of NHE1 was confirmed by immunoprecipitating NHE1 from ³⁵S-labeled PS120-NHE1 cells and by measuring pH_i recovery from an acid load in a HEPES buffer (4).

For transient expressions, cells were plated on glass coverslips at 0.5 × 10⁶ cells per 60-mm dish 18 h prior to transfection. Cells were transfected using LipofectAMINE and 3 µg of mutationally activated Myc-RhoAV14 or Myc-Rac1V12. After 24 h, the cells were transferred to serum-free DME containing 2 g/liter NaHCO₃ and maintained for an additional 24 h before experiments. pEXV-MycRac1V12 (16) and pEXV-MycRhoAV14 (17) were described previously.

NHE1 activity and pH_i were determined using the fluorescent pH-sensitive dye BCECF (1 µM) (18). Cells plated on glass coverslips were loaded with 1 µM BCECF for 10 min at 37 °C, mounted in a cuvette, and then placed in the thermostatically controlled holder of a Shimadzu RF5000 spectrofluorometer. The coverslips were perfused continuously at 2 ml/min with solutions maintained at 37 °C. Fluorescence ratios were determined by sequentially exciting the dye at 440 nm (H⁺-independent) and 500 nm (H⁺-dependent) and measuring emission at 530 nm. Fluorescence ratios were calibrated using 10 µM nigericin in 105 mM KCl, 25 mM HEPES, and 1 mM MgCl₂ (19). Measurements were performed either in a nominally HCO₃-free HEPES buffer containing 140 mM NaCl, 5 mM KCl, 10 mM glucose, 25 mM HEPES, 1 mM MgSO₄, 1 mM KHPO₄, and 2 mM CaCl₂, pH 7.4, or in a HCO₃ buffer that was similar except that 25 mM HEPES and 10 mM NaCl were replaced with 25 mM NaHCO₃. The HCO₃ buffer was gassed with 95% O₂, 5% CO₂. NHE1 activity was determined in a HEPES buffer as the rate of pH_i recovery following an acid load induced by a 10-min prepulse with 20 mM NH₄Cl (replacing 20 mM NaCl) (20). Recovery rates (dpH_i/dt) were measured by evaluating the derivative of the slope of the pH_i tracing at pH_i intervals of 0.05 unit.

Prior to experiments, cells were plated on glass coverslips at 0.05 × 10⁶ cells per 35-mm dish, maintained for 24 h, and then transferred to serum-free DME supplemented with 2 g/liter NaHCO₃ for 18-24 h. LPA (500 nM; Sigma) and HOE694 (10 µM; a gift from Dr. H. J. Lang, Hoechst AG), a selective inhibitor of NHE1 (21), were added to this medium for 10 min. When indicated, HOE694 was also added 10 min prior to LPA treatment. Incubations were terminated by washing the cells with phosphate-buffered saline (PBS) and fixing in 4% paraformaldehyde in PBS for 10 min. After permeabilization for 5 min in 0.5% Triton X-100 in PBS, the cells were either stained directly for polymerized actin using rhodamine-conjugated phalloidin (0.15 × 10⁷ M, 30 min; Sigma) (4) or were incubated with anti-Myc antibodies (1:100; Santa Cruz Biotechnology). Myc staining was visualized with fluorescein isothiocyanate-conjugated AffinityPure goat anti-rabbit IgG (H+L) (1:100; Jackson Immunology) simultaneous to staining with rhodamine-conjugated phalloidin.

RESULTS AND DISCUSSION

Our previous finding that activation of RhoA stimulates NHE1 activity (4) led us to investigate the role of NHE1 in stress fiber formation mediated by RhoA. In quiescent CCL39 cells serum-starved for 24 h, thin and randomly oriented actin filaments were located at the cell periphery (Fig. 1A). Addition of LPA (500 nM; 10 min) resulted in the assembly of new stress fibers, consisting of long bundles of filaments that traversed the cell (Fig. 1B). To determine whether NHE1 activity was important for this LPA effect, CCL39 cells were pretreated with HOE694 (10 µM; 10 min), a selective inhibitor of NHE1 (21). Although HOE694 alone appeared to cause a slight increase in stress fiber formation compared with quiescent CCL39 cells (data not shown), stress fiber formation induced by LPA was greatly reduced in the presence of HOE694 compared to the absence of this inhibitor. This finding indicates that pharmacological inhibition of NHE1 attenuates LPA-induced stress fiber formation.

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We next confirmed that LPA and HOE694 were regulating NHE1 activity. This was determined by measuring the rate of pH_i recovery from an acute acid load induced by 20 mM NH₄Cl (4). NHE1 activity was studied in a HEPES buffer to isolate effects on NHE1 and eliminate changes in pH_i induced by Cl-HCO₃ exchangers. The rate of pH_i recovery in quiescent CCL39 cells was significantly increased in the presence of LPA (Fig. 1D; p < 0.01), indicating that LPA stimulated NHE1 activity. Pretreatment of CCL39 cells with HOE694 completely eliminated the pH_i recovery, indicating that in a HEPES buffer, H⁺ efflux after an acid load was mainly due to the activity of NHE1. Addition of LPA was unable to rescue the pH_i recovery following HOE694 pretreatment (data not shown). In a HEPES buffer, LPA also induced an increase in steady-state pH_i compared to unstimulated cells (Fig. 1E). Addition of HOE694 to quiescent cells decreased the steady-state pH_i and prevented the LPA-induced increase in pH_i (Fig. 1E). To confirm that LPA stimulated NHE1 activity under the conditions used for studying stress fiber formation, we also determined the steady-state pH_i of CCL39 cells in a HCO₃-containing buffer. In the presence of HCO₃, acute changes in pH_i are due to the activities of the Na⁺-H⁺ exchanger as well as anion exchangers. The quiescent steady-state pH_i was higher in a HCO₃-containing buffer than in a HEPES buffer (Fig. 1E). Addition of LPA resulted in a further increase in pH_i of approximately 0.2 unit, which was similar to the pH_i induced by LPA in a HEPES buffer (Fig. 1E). HOE694 alone caused a marked decrease in pH_i, indicating the contribution of NHE1 activity to steady-state pH_i relative to the activities of Cl-HCO₃ exchangers. Addition of LPA to HOE694-treated cells caused an increase in steady-state pH_i to a value similar to that of quiescent, untreated cells. This suggested that LPA might stimulate activity of an acid extruder, such as the Na⁺-dependent Cl-HCO₃ exchanger, or inhibit activity of an acid loader, such as the Cl-HCO₃ exchanger, independently of its effect on NHE1. The LPA-induced increase in pH_i in the presence of HOE694 (pH_i 0.08), however, was significantly less than that observed in its absence (p < 0.01). These findings indicate that in a HCO₃-containing buffer, LPA induces an increase in steady-state pH_i that is primarily due to the stimulation of NHE1 and to a lesser extent to the modulation of additional H⁺-regulating mechanisms.

To further examine the role of NHE1 in stress fiber formation, we used PS120 fibroblasts, an NHE-deficient cell line derived from CCL39 cells (15). The morphology of PS120 cells differed from that of parental CCL39 cells in that their shape was more fusiform and their cytoplasm contained only diffusely distributed actin filaments. In quiescent PS120 cells, no stress fibers were apparent (Fig. 2A). In contrast to CCL39 cells, LPA failed to induce stress fiber formation in PS120 cells (Fig. 2B). Stable expression of NHE1 (PS120-NHE1), however, rescued the ability of LPA to induce stress fibers (Fig. 2D), indicating a requirement for the exchanger in this process. PS120-NHE1 cells also had a cell shape that was similar to that of parental CCL39 cells. Western analysis with anti-actin antibodies indicated that the abundance of actin was similar in CCL39, PS120, and PS120-NHE1 cells (data not shown), suggesting that the lack of stress fiber formation in PS120 cells was not due to a down-regulation of actin. Although PS120 cells do not express Na⁺-H⁺ exchangers, their quiescent steady-state pH_i in a HCO₃-containing buffer was higher than that of HOE694-treated CCL39 cells, perhaps due to a compensatory regulation of Cl-HCO₃ exchangers. Addition of LPA resulted in only a small increase in steady-state pH_i (less than 0.06 unit). In contrast, in PS120-NHE1 cells, LPA induced an increase in steady-state pH_i similar to that of CCL39 cells (compare Fig. 1E and Fig. 2D).

To determine whether RhoA-induced stress fiber formation depends on activation of NHE1, we transiently expressed a Myc-tagged, constitutively active RhoAV14 allele in CCL39, PS120, and PS120-NHE1 cells. In CCL39 cells, RhoAV14-expressing cells demonstrated a strong increase in stress fibers and the cells appeared to be contracted (Fig. 3, A and B). In PS120 cells, however, RhoAV14 induction of stress fibers was markedly inhibited (Fig. 3, C and D). In contrast, expression of RhoAV14 in PS120-NHE1 cells induced stress fiber formation to a similar extent as in CCL39 cells (Fig. 3, E and F). Together, these experiments indicate that NHE1 is critical for RhoA-mediated formation of stress fibers.

In order to determine whether NHE1-dependent cytoskeletal changes were specific to those induced by RhoA, we also examined the role of NHE1 in lamellipodia formation induced by a constitutively active Rac1V12. We previously demonstrated that although expression of Rac1V12 stimulates NHE1 activity, it acts through a mitogen-activated protein kinase kinase kinase 1 (MEKK1)-dependent mechanism that is independent of RhoA (4). Expression of Rac1V12 in PS120 cells caused extensive lamellipodia formation and membrane ruffling (Fig. 3, G and H), similar to the Rac1-induced phenotype previously reported in CCL39 cells (4) and other cell types (3, 22). This suggested that lamellipodia formation by Rac1 is independent of NHE1 activity.

We have presented two lines of evidence suggesting that NHE1 activity is critical for RhoA-mediated assembly of stress fibers. First, pharmacological inhibition of the exchanger with HOE694 inhibited LPA-induced stimulation of NHE1 activity and stress fiber formation. Second, although the cytoskeletal effects of LPA and RhoAV14 were absent in NHE1-deficient PS120 cells, they were restored by the stable expression of NHE1. Together, these findings strongly suggest that stimulation of NHE1 acts upstream of RhoA-induced stress fiber formation.

An increase in NHE1 activity or pH_i is necessary, but probably not sufficient, for inducing stress fiber formation. In the absence of either LPA or RhoAV14, we were unable to induce formation of stress fibers by merely altering pH_i or regulating NHE1 activity using NH₄Cl (data not shown). Additionally, a number of GTPases, including G_q (18, 23, 24), Ras (4, 13, 14, 20), Rac (4), and Cdc42 (4), stimulate NHE1 activity without stimulating an increase in stress fiber formation. This is consistent with the notion that RhoA, in addition to activating NHE1, stimulates other pathways which have to act in concert in order to cause stress fiber formation and that NHE1 may cooperate with an undetermined RhoA target to induce the formation of stress fibers. Indeed, the establishment of stress fibers is likely to be a complex process involving at least two independent activities: actin polymerization and the assembly of focal adhesions (3). Whether NHE1 plays a role in actin polymerization or focal adhesion assembly remains to be determined. It is interesting to note in this respect that adhesion of anchorage-dependent cells to fibronectin activates Na⁺-H⁺ exchange and increases pH_i in a HCO₃ buffer (25), and NHE1 has been found to be predominantly localized at focal complexes (26).

The exact nature of the involvement of NHE1 in stress fiber formation is still unclear. In Dictyostelium, changes in pH_i have been shown to regulate the organization of the actin cytoskeleton by altering the bundling activity of EF1 (27). Increases in pH_i, however, are associated with a decrease in actin bundling by EF1. Additionally, other actin-binding proteins, including hisactophilin (28), -actinin (29), and talin (30), show reduced actin binding with increasing pH_i. It seems unlikely, therefore, that a direct effect of pH_i on the properties of actin-binding proteins could account for the role of NHE1 in stress fiber formation. Alternatively, there may be events other than pH_i that are regulated by NHE1 activity which are critical for stress fiber assembly, such as the concentration of intracellular Na⁺ or an as yet unidentified signal produced by NHE1.