Department of Surgery, The Toronto General Hospital and University Health Network, Toronto, Ontario, Canada
Submitted 28 September 2004 ; accepted in final form 23 April 2005
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ABSTRACT |
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membrane potential; Na+-alanine cotransport; epithelium; phosphatidylinositol 3-kinase; LLC-PK1 cells
In addition to electromechanical coupling, the other major mode to turn on the myosin-based contractile apparatus is pharmacomechanical coupling (52). During this process, the binding of various neurotransmitters or other mediators to their cell surface receptors provokes voltage-independent MLC phosphorylation. Many of these agonists elicit Ca2+ signals; however, there is strong evidence that they also stimulate the Rho-ROK pathway through partially or entirely Ca2+-independent mechanisms. For example, engagement of certain serpentine receptors activates the -subunits of distinct trimeric G proteins (G
12/13, G
q), which in turn stimulate Rho-specific guanine nucleotide exchange factors (GEFs) (9, 10, 20). Moreover, a recent study has shown that in fibroblasts lacking MLCK, thrombin or lysophosphatidic acid induces ROK-mediated and Ca2+-independent cell contraction (12).
Cell contractility is thought to play a key role in epithelial physiology as a major regulator of epithelial tissue remodeling (13) and paracellular transport (4, 21, 37, 54, 56). Despite its recognized importance, the regulation of MLC phosphorylation in epithelial cells is poorly characterized. Specifically, the role of membrane depolarization, the Ca2+ dependence of the process, and the relative involvement of MLCK and/or the Rho-ROK pathway have not been elucidated. A possible membrane potential-dependent regulation of MLC in this so-called nonexcitable tissue context may be of particular interest, because epithelia contain a number of electrogenic transporters such as the Na+-amino acid cotransporters (5). Furthermore, it is well known that transmembrane transport processes regulate paracellular transport, but the underlying mechanism remains elusive (55). We considered that depolarization-regulated MLC phosphorylation might serve as a link between electrogenic transmembrane and paracellular transport.
In our previous studies, we found that hyperosmotic stress induces Rho-ROK-mediated MLC phosphorylation in kidney proximal tubular (LLC-PK1) cells (11). Curiously, during the course of these experiments, we noted that certain maneuvers that were expected to depolarize the plasma membrane also led to MLC phosphorylation. This initial finding prompted us to characterize this effect and explore the underlying mechanisms. Our present studies reveal a novel mode of Rho activation and Rho-ROK-dependent MLC phosphorylation in epithelial cells, which is depolarization induced but Ca2+ signal independent. Moreover, we provide evidence that the membrane potential-mediated regulation of cell contractility appears to be relevant physiologically in kidney cells, because the activation of the electrogenic Na+-alanine cotransport leads to Rho activation and MLC phosphorylation.
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MATERIALS AND METHODS |
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Cells. LLC-PK1, a kidney proximal tubule epithelial cell line, and Madin-Darby canine kidney (MDCK) cells, a canine distal tubular epithelial cell line, were obtained from the American Type Culture Collection (Manassas, VA). Both cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and 1% antibiotic suspension (penicillin and streptomycin; Sigma-Aldrich) in an atmosphere containing 5% CO2. Confluent cells were serum depleted for 3 h in HEPES-buffered RPMI medium before the experiments.
Media. HCO3-free RPMI 1640 was buffered with 25 mM HEPES to pH 7.4 (HPMI). The Na+-based medium (referred to as Na+ medium) consisted of (in mM) 130 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 5 glucose, and 20 Na+-HEPES (pH 7.4). The K+-based medium (referred to as K+ medium) contained (in mM) 130 KCl, 1 MgCl2, 1 CaCl2, and 20 HEPES, pH 7.4. The Ca2+-free Na+ and K+ media were made by omitting CaCl2 from the corresponding solutions and supplementing them with 1 mM EGTA. For Na+-free choline medium, NaCl was replaced with choline chloride.
Detection of myosin phosphorylation by urea-glycerol polyacrylamide gel electrophoresis. Non-, mono-, and diphosphorylated forms of MLC were separated using nondenaturating urea-glycerol polyacrylamide gel electrophoresis (PAGE) as described in our previous studies (11, 38). Briefly, confluent LLC-PK1 or MDCK cells grown in six-well plates were serum depleted for 3 h and treated as indicated in the figure legends. Subsequently, they were lysed in ice-cold acetone containing 10% TCA and 10 mM DTT, followed by centrifugation for 10 min at 12,500 rpm (4°C). The resulting pellet was washed with pure acetone, allowed to air dry, and dissolved in 60 µl of sample buffer containing 8.02 M urea, 234 mM sucrose, 23 mM glycine, 10.4 mM DTT, 20 mM Tris (pH 8.6), and 0.01% bromphenol blue. Samples were separated on a 12% urea-glycerol gel and blotted onto nitrocellulose membrane. The membrane was blocked with 2% BSA (1 h) and incubated with an anti-MLC primary antibody (1:500 dilution; 1 h). After being washed, the membrane was incubated with a peroxidase-coupled anti-goat antibody (1:3,000 dilution). The blot was washed and developed with the enhanced chemiluminescence kit from Amersham Pharmacia Biotech. Total MLC (i.e., the sum of all 6 bands) was determined by performing densitometry in each lane, and the amount of non-, mono- and diphosphorylated forms was expressed as a percentage of total MLC in the same lane. This normalization allows direct comparison among the samples and prevents any potential errors that might arise from differences in loading. In some experiments, the blots were stripped and reprobed with anti-ppMLC antibody to verify specificity of the antibody.
Detection of myosin phosphorylation using a modified ELISA method. LLC-PK1 or MDCK cells were grown to confluence in 24-well plates. The cells were serum depleted for 3 h and treated as indicated in the figure legends. Subsequently, the cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 20 min, washed three times with PBS, and permeabilized for 20 min with 0.1% Triton X-100 in the presence of 100 mM glycine to quench PFA. After being washed, cells were blocked with 250 µl of PBS containing 2% BSA for 45 min. Next, the cells were incubated with anti-diphospho-MLC (ppMLC) antibody (1:1,000 dilution) in PBS containing 0.2% BSA for 1 h. The cells were then washed five times for 15 min each with PBS and incubated with peroxidase-coupled secondary antibody (1:10,000 dilution) in PBS containing 0.2% BSA. After extensive washing of the cells, binding of the antibodies was detected using the Fast OPD kit. Each well was incubated with 750 µl 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 its absorbance was measured at 492 nm using a Beckman Coulter DU 640 spectrophotometer. Each determination was performed in duplicate or in triplicate. The background (absorbance of samples without primary antibody) for each condition was determined on the same plate and subtracted. The signal obtained in control cells was typically about threefold higher than the background. The results are expressed as the relative increase compared with the control. Similar results were obtained with another ppMLC antibody (Cell Signaling Technology; data not shown).
Rho activity assay. The amount of active Rho was determined using an affinity precipitation assay as described previously (11). Briefly, confluent LLC-PK1 cells grown in 10-cm dishes were treated as indicated in the respective figure legends. Cells were lysed in 800 µl of ice-cold lysis buffer containing 100 mM NaCl, 50 mM Tris base (pH 7.6), 20 mM NaF, 10 mM MgCl2, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 20 µl/ml protease inhibitor cocktail (BD Pharmingen), 1 mM Na3VO4, and 1 mM PMSF. The lysates were clarified by centrifugation at 12,000 rpm for 1 min at 4°C. After removing 20 µl of sample from each supernatant for determination of total Rho, we incubated the rest of the supernatant at 4°C for 45 min with 1015 µg of glutathione-Sepharose beads covered with glutathione S-transferase-Rho-binding domain (GST-RBD), followed by extensive washing. Samples for total Rho and the pelleted beads were diluted in Laemmli sample buffer and boiled for 5 min. The proteins were separated using SDS-PAGE (15% gel) and transferred onto nitrocellulose membrane. Blots were blocked with 2% BSA for 1 h, followed by incubation with anti-Rho antibody (1:500 dilution; 1 h). Binding of the antibody was visualized using peroxidase-coupled anti-mouse antibody (1:3,000 dilution) and enhanced chemiluminescence. The amount of captured Rho was quantified by performing densitometry.
Immunofluorescence microscopy. Confluent cells grown on coverslips were serum depleted for 3 h and treated as indicated in the figure legends. Next, the cells were fixed with 4% PFA, washed with PBS, and permeabilized for 20 min with 0.1% Triton X-100 in the presence of 100 mM glycine. This was followed by washing and blocking the cells with 2% BSA for 1 h. The coverslips were then incubated with the primary antibody diluted in PBS containing 0.2% BSA. The primary antibody dilution for all antibodies was 1:100. Bound antibody was detected using the corresponding Cy3-conjugated secondary antibodies (1:1,000 dilution). Staining was visualized using a Nikon Eclipse TE200 microscope (x100 magnification) coupled to a Hamamatsu cooled charge-coupled device camera operated using Simple PCI software.
Vectors and transient transfection. The vector-encoding C3 transferase that we used, a kind gift from Dr. Keith Burridge, was described previously (2). The vector-encoding enhanced green fluorescent protein (EGFP), pEGFP, was purchased from Clontech Laboratories. LLC-PK1 cells were plated on coverslips, and the next day they were cotransfected with cDNA encoding for C3 transferase (1 µg) and GFP (0.2 µg) using FuGENE6 (Roche Molecular Biochemicals) according to the manufacturers instructions. Transfection with GFP, which by itself had no effect on ppMLC staining (Fig. 7D; see also our previous study, Ref. 38), was used to visualize transfected cells.
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Measurement of intracellular Ca2+. LLC-PK1 cells grown on coverslips were loaded with fura-2 AM for 30 min in serum-free HPMI at 37°C. The cells were washed and kept in HPMI at 37°C for an additional 15 min to allow complete hydrolysis of the dye. After the coverslips were washed again, ratiometric microfluorometry was performed on a small population of cells using excitation wavelengths of 340 ± 5 nm and 380 ± 5 nm and an emission wavelength of 510 ± 5 nm.
Densitometry. Densitometric analysis of blots was performed using a model GS-690 imaging densitometer and Molecular Analyst software (version 1.5) obtained from Bio-Rad Laboratories (Hercules, CA) as described previously (11).
Statistical analysis. Data are presented as means ± SE of the number of experiments indicated (n) or as representative immunoblots or images of at least three similar experiments. When the data were normalized to the control level, a paired Students t-test was performed. In other cases, one-way ANOVA for multiple comparisons or an unpaired t-test was performed, with P < 0.05 considered statistically significant.
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RESULTS |
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To test whether the high-K+-provoked MLC response is unique to LLC-PK1 cells or whether it is also present in other kidney epithelial cells, we performed the above experiments on MDCK cells, a canine distal tubular cell line. As shown in Fig. 2, MDCK cells showed both qualitatively and quantitatively similar responses to LLC-PK1 cells. Stimulation with high K+ increased MLC phosphorylation as detected using urea-glycerol PAGE and resulted in enhanced ppMLC staining along intracellular fibers, at the periphery, and in the nucleus.
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Recently, Emmert et al. (12) described Ca2+-independent MLC phosphorylation in MLCK-deficient fibroblasts. To discern the relative contribution that Ca2+ may play in the regulation of MLC phosphorylation in intact kidney epithelial cells, we examined the ability of increased [Ca2+]i to induce MLC phosphorylation. Addition of ionomycin to cells incubated in Ca2+-containing Na+ medium induced an immediate, sharp rise in [Ca2+]i, which remained elevated for the rest of the measurement period (Fig. 5C). Despite the elevated Ca2+ levels, ionomycin only marginally increased MLC phosphorylation as determined by performing ppMLC ELISA (Fig. 5E). Similarly, addition of 100 nM thapsigargin (TG), which caused a smaller but similarly long-lasting increase in [Ca2+]i, also failed to significantly elevate ppMLC (Fig. 5, D and E). Jointly, these data suggest that an increase in [Ca2+]i in LLC-PK1 cells by itself is neither necessary nor sufficient to induce ppMLC.
Depolarization-induced MLC phosphorylation is mediated by the Rho kinase pathway in kidney epithelial cells. We investigated the kinase pathways involved in mediating depolarization-induced MLC phosphorylation. To determine the role of the major myosin-phosphorylating enzyme MLCK, we used ML-7, a potent MLCK inhibitor. Even in the presence of a high (50 µM) concentration of ML-7, K+ was able to induce a sizable increase in ppMLC staining: Immunofluorescence analysis revealed that the peripheral accumulation, the nuclear staining, and the fiberlike structures were all manifest in ML-7-treated cells, similar to controls that were treated with K+ medium only (Fig. 6, A and B). This conclusion was verified by the other two methods as well. When assessed using urea-glycerol PAGE, neither the basal phosphorylation pattern nor its changes upon depolarization were significantly different between control and ML-7-treated cells (Fig. 6D). Indeed, quantitation using ELISA showed no reduction in the depolarization-induced ppMLC content in ML-7-treated cells (Fig. 6E).
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Depolarization activates Rho, which is necessary for MLC phosphorylation. The ability of Y-27632 to interfere with the depolarization-induced MLC phosphorylation suggests that changes in the membrane potential activate the Rho-Rho kinase pathway. Because ROK can be activated both by Rho-dependent and Rho-independent mechanisms (17), we wished to discern whether depolarization directly affects Rho activation. Toward this end, we performed Rho precipitation assays to assess the amount of active Rho in cell lysates from control and depolarized cells. This approach relies on a fusion protein composed of GST and the RBD of rhotekin, a downstream effector of Rho, which binds the active (GTP-bound) form but fails to react with the inactive (GDP-bound) form of Rho. As shown in Fig. 7A, K+ medium induced substantial Rho activation. Densitometric quantitation of these pull-down assays showed that the K+ medium provoked a 1.82 ± 0.24-fold increase in active Rho compared with the Na+ medium. To visualize the intracellular distribution of Rho, another indicator of its activation, cells were immunostained with an anti-Rho antibody. While in control cells Rho exhibited predominantly cytosolic distribution with occasional membrane localization, after K+ treatment, it showed a marked peripheral staining suggestive of membrane translocation (Fig. 7B). Together, these results imply that in tubular epithelial cells, depolarization activates Rho and induces its redistribution to the cell periphery.
To assess whether depolarization-induced Rho activation is indeed necessary for MLC phosphorylation, we interfered with Rho activation by various means. First, we used C. difficile toxin B, an enzyme that catalyzes the glycosylation of a threonine residue on Rho family GTPases and thereby inactivates them (28). The advantage of this toxin is that it is cell permeable; therefore, it can be introduced into the entire cell population. Cells were incubated with toxin B and exposed to K+ medium. Toxin-treated cells showed typical morphological changes as described in our earlier work (11) but remained firmly attached to the plates. As shown in Fig. 7C, while K+ medium induced a more than twofold increase in ppMLC, as demonstrated earlier, the increase was completely blocked in toxin B-treated cells, suggesting that depolarization requires functional Rho to exert its effect on myosin.
Although toxin B offers the advantage of allowing treatment of the whole cell population, it is not specific for Rho but inhibits all Rho family GTPases. To interfere specifically with endogenous Rho activity, we used Clostridium botulinum C3 transferase, a toxin that selectively ADP ribosylates and thereby inactivates Rho. Because this protein is not cell permeable, we cotransfected the cells with a vector-encoding C3 toxin, together with GFP, to identify the successfully transfected (green) cells. Subsequently, we challenged the monolayer with K+ medium and stained it for ppMLC. Figure 7D shows that in cells expressing GFP only, depolarization induced marked ppMLC accumulation (Fig. 7D, top). In contrast, in cells expressing C3 transferase, the depolarization-induced ppMLC accumulation was completely abolished (Fig. 7D, bottom).
The mechanism whereby the Rho-ROK pathway is activated upon depolarization remains elusive. Because previous studies have shown that phosphatidylinositol 3-kinase (PI3-kinase) can be activated by depolarization (40, 59) and has been implicated as an upstream regulator of the Rho-ROK pathway (39), we wondered whether PI3-kinase might be involved in the depolarization-induced MLC response. To address this question, we used LY-294002, a specific PI3-kinase inhibitor. Figure 6, G and H, shows that LY-294002 suppressed the basal MLC phosphorylation and significantly mitigated the depolarization-provoked rise in ppMLC. This finding is consistent with a contributory role of PI3-kinase as one of the upstream mediators of the potential-sensitive MLC phosphorylation.
Electrogenic L-alanine-Na+ cotransport induces myosin phosphorylation. Proximal tubule epithelial cells contain a large number of apical transporters, many of which are electrogenic (31, 62). Accordingly, Na+-driven cotransport processes, such as Na+-coupled glucose or alanine uptake, have been shown to depolarize the cells (25, 44). Therefore, we next investigated whether physiological membrane potential changes associated with the operation of the Na+-alanine cotransporter were able to induce MLC phosphorylation in LLC-PK1 cells.
Figure 8A shows that the addition of 20 mM L-alanine to the Na+ medium induced a drop in the membrane potential and a concomitant increase in the general ppMLC staining (Fig. 8B). The accumulation of ppMLC was detected, both in the cytoplasm and at the cell periphery. Moreover, we often observed ppMLC-positive thin stress fibers near the basal surface of alanine-treated cells (data not shown). Thus the pattern of ppMLC distribution resembled that observed in the high-K+ medium, albeit that the overall effect appeared weaker. Importantly, similarly to the K+-induced response, the L-alanine-triggered increase in MLC phosphorylation was also entirely prevented by Y-27632, suggesting that ROK is a key mediator of this process as well (Fig. 8B, right). In agreement with the qualitative observations, 10-min exposure of the cells to L-alanine caused a 1.26 ± 0.08-fold increase in ppMLC as measured using the ELISA technique (Fig. 8C, first two columns). Similar MLC phosphorylation was detected in MDCK cells upon the addition of L-alanine (data not shown). We thought that if the effect of L-alanine was indeed due to its electrogenic transport, it should depend on the presence of external Na+. To test this prediction, we examined whether replacement of Na+ with choline would alter the effect of alanine on MLC phosphorylation. Figure 8, A and C, shows that in Na+-free (choline chloride) medium, the addition of alanine did not depolarize the membrane and failed to induce MLC phosphorylation. This observation gives strong support to the notion that the effect of alanine was indeed due to its Na+-dependent uptake into the cells.
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DISCUSSION |
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In smooth muscle cells, the depolarization-induced activation of MLC phosphorylation is entirely dependent on Ca2+ influx (41, 46). On the other hand, Ca2+-independent Rho-ROK-mediated MLC phosphorylation has been observed in fibroblasts (12); however, these effects were provoked by chemical agonists and not by depolarization. Thus the depolarization-induced, Rho-ROK-mediated, but Ca2+ signal-independent MLC phosphorylation represents a new mode of regulation of the contractile apparatus present in kidney epithelial cells.
Several lines of evidence suggest that the triggering factor for MLC phosphorylation is indeed depolarization. Dissipation of the membrane potential by each of three distinct mechanisms (high K+, which eliminates the major driving force for the resting potential; nystatin, which permeabilizes the membrane for monovalent ions; and the lipophilic cation TPP+, which accumulates excessively and at higher concentrations may induce an inside positive diffusion potential) provoked similar changes in ppMLC content and distribution. While these maneuvers might have changed other parameters (e.g., intracellular ion concentrations) as well, and although the participation of these cannot be excluded, the most straightforward interpretation of the data implicates the membrane potential as the critical component. Moreover, we found that a physiological stimulus, L-alanine, that caused depolarization by its electrogenic uptake through Na+-coupled cotransport (25, 31, 44) also induced MLC phosphorylation in the presence but not in the absence of Na+. We observed that the alanine-induced rise in total ppMLC as determined with our newly developed ELISA was less than that observed with the other depolarizing stimuli. This is expected to be so because alanine induced only a partial drop in the membrane potential, in agreement with patch-clamp data (25, 44).
Our findings imply that the membrane potential may directly regulate the cytoskeleton in epithelial cells. Consistent with this notion, in bovine corneal endothelial cells that are functionally equivalent to secretory epithelial cells, depolarization alters the actin cytoskeleton and causes retraction of the cells from each other (8). Although in this study the phosphorylation state of MLC and the underlying signaling mechanisms were not investigated, our present findings are consistent with increased epithelial contractility upon depolarization. Our results suggest a plausible molecular mechanism for these depolarization-provoked structural changes.
We found that depolarization resulted in the accumulation of ppMLC at the cell periphery, in fine fibers at the base of the cells, and in the nucleus. While increased contractility in the central areas and at the membrane may affect a variety of functions, including paracellular permeability (see below), the importance of nuclear ppMLC remains elusive. Nonetheless, the nuclear signal appears to be specific because it was eliminated by a specific blocking peptide and was effectively suppressed by the Rho kinase inhibitor Y-27632. In accordance with our findings, Ueda et al. (57) described specific nuclear staining by a ppMLC antibody in HELA cells that was eliminated by preadsorbing the antibody with its specific antigen.
Our results suggest that the depolarization-provoked MLC phosphorylation is mediated by the Rho-ROK pathway. This conclusion is supported by the findings that 1) depolarization activated Rho and induced its translocation to the periphery, 2) inhibition of Rho by either C. difficile toxin B or the more specific C3 transferase eliminated the depolarization-triggered MLC phosphorylation, and 3) the ROK inhibitor Y-27632 largely prevented the MLC response. While the mechanism of depolarization-induced Rho activation and peripheral translocation remains unknown, it appears to be tissue specific because it was found to be Ca2+ dependent in smooth muscle cells (46) but not in epithelia. Interestingly, Rac, another member of the Rho family, was also shown to be translocated to the membrane upon depolarization in endothelial cells (49). Because depolarization activates PI3-kinase in certain cells (40, 59) and has been suggested to act upstream of the Rho-ROK pathway (39), we investigated whether PI3-kinase might participate in the MLC response. Our findings that LY-294002 reduced the basal and depolarization-stimulated MLC phosphorylation are consistent with such a role. Conceivably, PI3-kinase may facilitate small GTPases by stimulating their GEFs. Indeed, several of the identified GEFs harbor lipid-binding PH domains (13) that could interact with and are activated by the products of PI3-kinase. Alternatively, changes in the membrane potential might modulate the exposed charges or the composition of the lipid bilayer, directly influencing the binding and/or activity of GEFs. Further work is needed to validate these possibilities. It is noteworthy that we and others have shown that Rac, Cdc42, and Rho are also responsive to changes in cell volume and intracellular ionic strength (6, 11, 36). Taken together, these data imply that the Rho family small GTPases are not only regulated by biochemical changes but also sensitive to physical parameters.
While Rho is the major activator of ROK, the kinase can be stimulated by Rho-independent pathways as well, such as by caspase-mediated cleavage (48). However, we found no evidence for the involvement of this mechanism, because ROK was not cleaved in depolarized cells, and caspase inhibitors did not interfere with MLC phosphorylation (data not shown).
The relative contribution of various kinases in MLC phosphorylation appears to differ significantly between tissues. In smooth muscle cells, MLC is phosphorylated predominantly by the Ca2+-dependent MLCK, while the major target of ROK is thought to be myosin phosphatase (51). Accordingly, MLC phosphorylation is Ca2+ dependent and is inhibited by MLCK blockers (41, 64). In contrast, in epithelial cells, we found no change upon depolarization in intracellular Ca2+, and ML-7 had only a marginal effect on the MLC response. The applied dose of ML-7 likely inhibited MLCK because it abolished spreading, an MLCK-dependent process (19, 47) (C Di Ciano-Oliveira and A Kapus, unpublished observations). Furthermore, [Ca2+]i elevation failed to increase MLC phosphorylation, arguing against the role of MLCK as the centrally regulated element of the pathway. Similar findings were reported in chicken embryonic cells, in which [Ca2+]i rise induced only a small, transient MLC phosphorylation (33). These observations, together with the strong inhibitory effect of Y-27632, suggest that ROK acts not only by the inhibition of MLC phosphatase but also by directly phosphorylating MLC. This conclusion is consistent with previous reports showing that Rho kinase can directly diphosphorylate MLC in nonmuscle cells and can mediate effective MLC phosphorylation even in the absence of MLCK (12, 57). A novel and intriguing regulator of myosin activity is the protein kinase C-mediated inhibitor CPI-17. Both ROK and PKC have been shown to phosphorylate CPI-17, which in turn enhances myosin phosphorylation by inhibiting myosin phosphatase (23). Future studies are warranted to assess the role of CPI-17 in the depolarization-induced increase in contractility.
Interestingly, removal of extracellular Ca2+ itself promoted myosin phosphorylation. This finding is consistent with earlier reports showing that removal of extracellular Ca2+ causes an immediate contraction of MDCK cells (15, 35). Because we found that Ca2+ removal-triggered MLC phosphorylation was also inhibited by Y-27632, we suggest that Rho-ROK activation is the underlying mechanism. Rho activation under these conditions might be related to the disassembly of intercellular contacts. Accordingly, the inverse process, i.e., the formation of cadherin-based cell-cell adhesions, was reported to strongly downregulate Rho activity (42). Importantly, the Rho-ROK-MLC system remains sensitive to the membrane potential and was further stimulated by depolarization even after Ca2+ removal.
Finally, we consider the physiological importance of the depolarization-induced MLC phosphorylation in epithelia. Cell contractility, governed by the phosphorylation state of MLC, is a key modulator of many epithelial functions, including the regulation of apical ion transporters (53) and the permeability of tight junctions (TJs) (21, 56) that are crucial determinants of the paracellular transport pathway. While basal Rho activity and contractility are required to maintain the integrity of intercellular contacts (16, 24), increased MLC phosphorylation is usually associated with loosening of the TJ and decreased transepithelial resistance (21, 54, 56). It has long been known that certain Na+-coupled membrane transport processes (e.g., Na+-glucose or Na+-amino acid cotransport) induce substantial increases in paracellular permeability in intestinal and renal epithelia (4, 55, 61). Remarkably, the same transport processes have been shown to induce increased myosin phosphorylation and the formation of a perijunctional contractile actomyosin ring at the apical pole (4, 37, 55). These structural changes were suggested to underlie the increase in TJ permeability (54). However, the mechanism whereby Na+-coupled membrane transport leads to enhanced MLC phosphorylation has not been elucidated. On the basis of our results that alanine activates Rho and provokes Y-27632-sensitive MLC phosphorylation, we propose the following mechanism. The initiation of the Na+-coupled electrogenic transport depolarizes the apical membrane, which leads to Rho activation and subsequent ROK-mediated myosin phosphorylation, results in increased TJ permeability. This intriguing mechanism would explain how transmembrane and paracellular transport processes are coupled and coordinated. Consistent with this proposition, recently a novel TJ-associated Rho-GEF was described, the overexpression of which resulted in augmented paracellular permeability (3). Future studies are warranted to explore the mechanism of depolarization-induced Rho activation and to validate the proposed membrane potential-dependent regulation of paracellular permeability.
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FOOTNOTES |
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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.
* K. Szászi and G. Sirokmány contributed equally to this work.
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