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Am J Physiol Cell Physiol 292: C1476-C1484, 2007; doi:10.1152/ajpcell.00375.2006
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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Hypotonicity causes actin reorganization and recruitment of the actin-binding ERM protein moesin in membrane protrusions in collecting duct principal cells

Grazia Tamma, Giuseppe Procino, Maria Svelto, and Giovanna Valenti

Department of General and Environmental Physiology, University of Bari, Italy

Submitted 10 July 2006 ; accepted in final form 4 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypotonicity-induced cell swelling is characterized by a modification in cell architecture associated with actin cytoskeleton remodeling. The ezrin/radixin/moesin (ERM) family proteins are important signal transducers during actin reorganization regulated by the monomeric G proteins of the Rho family. We report here that in collecting duct CD8 cells hypotonicity-induced cell swelling resulted in deep actin reorganization, consisting of loss of stress fibers and formation of F-actin patches in membrane protrusions where the ERM protein moesin was recruited. Cell swelling increased the interaction between actin and moesin and induced the transition of moesin from an oligomeric to a monomeric functional conformation, characterized by both the COOH- and NH2-terminal domains being exposed. In this conformation, which is stabilized by phosphorylation of a conserved threonine in the COOH-terminal domain by PKC or Rho kinase, moesin can bind interacting proteins. Interestingly, hypotonic stress increased the amount of threonine-phosphorylated moesin, which was prevented by the PKC-{alpha} inhibitor Gö-6976 (50 nM). In contrast, the Rho kinase inhibitor Y-27632 (1 µM) did not affect the hypotonicity-induced increase in phosphorylated moesin. The present data represent the first evidence that hypotonicity-induced actin remodeling is associated with phosphorylated moesin recruitment at the cell border and interaction with actin.

ezrin/radixin/moesin; protein kinase C; Rho

AN IMPORTANT FEATURE of animal cells is the ability to regulate their volume in response to alterations in extracellular tonicity. On exposure to hypotonic media, cells restore their volume through a mechanism known as regulatory volume decrease (RVD), which includes the activation of K+ and Cl– channels. In this context, the cytoskeleton is part of the volume-sensing system involved in the mechanochemical transduction of cell volume changes through intracellular signals regulating both ion transport (20, 23, 37) and F-actin reorganization (11, 35).

Proteins of the Rho family are key regulators of the actin network and can therefore play a pivotal role during cell swelling (15). Incubation of human intestine 407 cells with C3 exoenzyme, affecting RhoA activity, causes a significant decrease in osmosensitive anion efflux (44) but does not prevent the formation of membrane protrusions and actin reorganization induced by hypotonicity in Rat-1 fibroblasts (9). In contrast, preincubation with toxin B, which inhibits RhoA, Rac, and Cdc42, abolishes hypotonicity-induced actin remodeling (9).

Important regulators of Rho family members, the ezrin/radixin/moesin (ERM) proteins, cross-link actin filaments with the plasma membrane (13). ERM proteins are able to recruit Rho regulators including Rho guanine dissociation inhibitors and the Rho GDP/GTP exchange protein Dbl (7). The observation that ERM proteins function both upstream and downstream of Rho GTPases implies that there could be a feedback loop for Rho pathway autoregulation (18, 27, 41). The COOH-terminal domain of ERM family proteins binds F-actin with high affinity (47), whereas the NH2-terminal domain interacts with integral membrane proteins like ICAM (3, 46). ERM proteins were found at the cell adhesion sites (40), suggesting that they might be part of the machinery regulating the structural integrity of the cells. ERM proteins might exist in a closed and inactive monomeric conformation characterized by an interaction between the NH2-terminal and COOH-terminal domains or in head-to-tail oligomers. A specific phosphorylation at a threonine residue in the COOH-terminal domain of the ERM family proteins promotes the transition from oligomers to active monomers (14). In the phosphorylated conformation, ERM proteins unmask the binding sites for actin and plasma membrane and regulate the formation of membrane protrusions and apical microvilli (22). Several protein kinases are known to phosphorylate the ERM family proteins at a threonine residue in the COOH-terminal domain. In gastric parietal cells, activation of acid secretion promoting tubulovesicle apical targeting is associated with ezrin phosphorylation at the conserved threonine in position 567 (16, 49). Rho kinase-induced phosphorylation of moesin causes the formation of microvilli-like structure in COS7 cells (33) and modulates the head-to-tail association in Swiss-3T3 cells (29). Thrombin-dependent activation of human platelets is accompanied by phosphorylation of moesin at threonine 558 at the actin binding site (31), promoting filopodia formation (31). A threonine residue in the COOH-terminal domain of the ERM family proteins might also be phosphorylated by phosphatidylinositol 4-phosphate 5-kinase, myotonic dystrophy kinase-related Cdc42 binding kinase, or PKC-{alpha} and -{theta} (26, 29, 31, 36). In MCF7 cells, the COOH-terminal phosphorylation of ezrin is dependent on PKC-{alpha}, which was found to colocalize with beta1-integrin and ezrin in vivo (26).

We report here that in collecting duct CD8 cells hypotonicity-induced cell swelling resulted in deep actin reorganization, consisting in loss of stress fibers and the formation of F-actin patches in membrane protrusions, where moesin was recruited in an active monomeric and phosphorylated form. These findings point to a functional involvement of the ERM protein moesin in the modification of cell architecture associated with actin cytoskeleton remodeling during hypotonic shock. These observations are of particular physiological relevance, since variations of extracellular osmolarity occur in the kidney medulla during transition from antidiuretic to diuretic conditions (4).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies. Moesin and PKC-{alpha} and -{gamma} antibodies were purchased from BD Transduction Laboratories (Milan, Italy). Phospho-Thr-558 moesin (phospho-moesin) antibody was from Santa Cruz Biotechnology (Segrate Milano, Italy).

Solutions. Cells were perfused with a phosphate buffer (pH 7.4, 310 mosmol/kgH2O) containing (in mM) 137 NaCl, 1 CaCl2, 0.5 MgCl2, and 2.7 KCl. Hypotonic NaCl solution had the same composition except that the NaCl concentration was reduced to 45.6 mM. When appropriate, cells were exposed to the hypotonic solution for 10 min and then processed for the indicated experiments.

Immunofluorescence. CD8 cells grown on glass coverslips were fixed either with 4% paraformaldehyde in PBS for 20 min or with cold methanol. After blocking in 0.1% gelatin in PBS for 20 min, cells were either incubated with monoclonal anti-moesin (1:100) or with monoclonal anti-PKC-{alpha} (1:100) at room temperature for 2 h. Cells were washed three times for 5 min each with 0.1% gelatin in PBS and incubated for 60 min with Alexa-488 conjugated goat anti-mouse, followed by washing twice for 1 min in high-salt PBS and twice in regular PBS. Actin cytoskeleton was visualized by incubation with phalloidin-tetramethylrhodamine isothiocyanate (100 µg/ml, 45 min). The coverslips were then mounted in 50% glycerol in 0.2 M Tris·HCl, pH 8.0, containing 2.5% n-propyl gallate to retard quenching of the fluorescence. Moesin was detected with an epifluorescence microscope (TE 2000S, Nikon Instruments, Florence, Italy) equipped with a charge-coupled device (CCD) camera (MicroMax 512BFT, Princeton Instruments, Princeton, NJ) using a Delta RAM Highspeed Multiwavelength Illuminator for excitation (Photon Technology International, Lawrenceville, NJ). Each imagine was subjected to 40 cycles of three-dimensional deconvolution with Autodeblur software (Universal Imaging). Alternatively, cells were fixed with 4% paraformaldehyde in PBS and then double stained for colocalization studies with anti-phospho-moesin and F-actin. Fluorescent signal was detected with a confocal microscope (Leica TCS, SP2, Leica Microsystem).

Subcellular fractionation. Cellular fractions were obtained with the Qproteome Cell Compartment kit. Cytosolic, membrane, nuclear, and cytoskeletal proteins were obtained with specific extraction buffer according to the manufacturer's instructions (Qiagen, Milan, Italy).

Analysis of association with F-actin. The interaction of moesin with actin cytoskeleton was analyzed by its solubility in Triton X-100 as described previously (42). Briefly, CD8 cells were seeded 2 days before the experiments and grown to confluence. The Triton X-100-soluble fraction was extracted by a 1-min incubation with a buffer containing 80 mM PIPES, 5 mM EGTA, 1 mM MgCl2, 0.5% Triton X-100, and 50 mM NaF, pH 6.4, which preserves the cytoskeleton association of proteins. Proteins were then subjected to SDS-PAGE and transferred onto Immobilon-P (Millipore) by standard procedures.

Intracellular calcium measurements. Alterations of intracellular calcium concentration during hypotonic treatment were determined by a spectrofluorometric technique. CD8 cells were grown to confluence on glass coverslips and loaded for 30 min with fura-2 AM (10 µM). Coverslips were inserted into a specially designed cuvette for cell perfusion, and calcium was measured with an epifluorescence microscope (TE 2000S, Nikon Instruments) equipped with a CCD camera (MicroMax 512BFT, Princeton Instruments) using a DeltaRAM Highspeed Multiwavelength Illuminator for excitation (Photon Technology International) and a beam splitter (Optical Insight) on the emission side.

Chemical dithiobis(succinimidyl propionate) cross-linking. CD8 cells were seeded 2 days before the experiments and grown to confluence in 10-cm Petri dishes. Cells were left untreated or preincubated for 10 min with the hypotonic solution. Alternatively, cells were incubated for 5 min with 1 µM thapsigargin in the isotonic solution and then either left in the isotonic solution in presence of 1 mM EDTA or exposed for 10 min to the hypotonic shock in the presence of 1 mM EDTA. After treatments cells were then lysed for 90 min in ice in PBS containing 1% Triton X-100 and protease inhibitors (1 mM PMSF, 2 mg/ml leupeptin, 2 mg/ml pepstatin A). The lysates were then centrifuged at 12,000 g for 20 min and then incubated for 30 min at room temperature with gentle shaking in the presence or absence of 2 mM dithiobis(succinimidyl propionate) DSP. The cross-linking reaction was stopped with 10 mM Tris·HCl, pH 7.5. The total protein concentrations of the lysates were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Milan, Italy), following the manufacturer's instructions. The proteins were analyzed with the Nu-Page system (4–12% gel) under nonreducing conditions (Invitrogen, San Giuliano Milanese Italy).

Evaluation of phospho-moesin by immunoblotting. CD8 cells were left untreated or subjected to hypotonic shock for 10 min in the presence or the absence of the selective inhibitors of Rho kinase (Y-27632, 1 µM) or PKC-{alpha} (Gö-6976, 50 nM) (both from Calbiochem, Milan, Italy). Gö-6976 is known to selectively inhibit Ca2+-dependent PKC-{alpha} isozyme and PKC-betaI. However, since PKC-betaI is not expressed in CD8 cells (38), this inhibitor specifically affects PKC-{alpha}. Alternatively, cells were incubated for 5 min with 1 µM thapsigargin in the isotonic solution and then either left in the isotonic solution in the presence of 1 mM EDTA or exposed for 10 min to hypotonic shock in the presence of 1 mM EDTA. After treatments, cells were washed three times in PBS and lysed in a buffer containing 50 mM Tris, 110 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet P-40, and 2 mM phenylmethylsulfonyl fluoride, pH 8. Cell lysates were incubated on ice for 1 h and vortexed several times. Insoluble material was pelleted at 12,000 g, and protein content was determined with the Bio-Rad protein assay (Bio-Rad Laboratories), following the manufacturer's instructions. Equal amounts of proteins were separated with the Nu-Page system (12% gel; Invitrogen) and subjected to immunoblotting studies using specific antibodies against moesin and phospho-moesin.

Statistical analysis. All values are expressed as means ± SE. Student's t-test was used for statistical analysis. A statistically significant difference was assumed at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hypotonicity induces F-actin remodeling and redistribution of moesin in membrane protrusion. In response to hypotonic shock, cells swell and a profound alteration in cell architecture occurs. As a result, the actin cytoskeleton is remodeled to counteract cell shape modification (9, 12). When renal collecting duct CD8 cells were exposed to hypotonic medium for 10 min, compared with control, F-actin staining revealed a strong rearrangement of the actin network consisting of the reduction of the ventral stress fibers and the formation of F-actin protrusions at the cell cortex (Fig. 1A). These alterations were observed even at a shorter time (2 min; data not shown) as also described for other cell types (9).


Figure 1
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  		Fig. 1. A: effect of hypotonicity on actin reorganization. CD8 cells were left under basal conditions (control, Ctr) or exposed to a hypotonic solution for 10 min (Hypo). F-actin was stained with tetramethylrhodamine isothiocyanate-conjugated phalloidin and visualized by epifluorescence microscopy. Nuclei were stained with DAPI (blue color). Hypotonicity induces significant actin remodeling, consisting of the disappearance of ventral stress fibers and the formation of membrane protrusion at the cell border. B: cells were treated as described in A. After fixation, cells were incubated with anti-moesin antibody and fluorescence was visualized by epifluorescence microscopy. In control cells, most of the moesin staining was localized at the cell adhesion sites, while in hypotonicity-treated cells moesin was recruited in membrane protrusions at the cell cortex. Bar = 5 µm.

 
Membrane protrusions are thought to be sensory structures involved in the transduction of extracellular signals inside the cell. New intriguing evidence indicates that in the actin-containing membrane protrusions induced by hypotonicity, actin binding proteins and several regulatory proteins are recruited to restore normal cell volume. Proteins of the ERM family are key components of cortical actin cytoskeleton and contribute toward the structural organization of particular plasma membrane domains (19, 46). In CD8 cells, hypotonicity caused a dramatic modification in the localization of the ERM protein moesin (Fig. 1B). Compared with control cells, where moesin had a typical localization at cell-cell contacts, in cells exposed to hypotonicity most of the labeling was concentrated in F-actin-enriched membrane protrusions. This shift in moesin distribution on hypotonic shock was confirmed by cell fractionation followed by Western blotting analysis (Fig. 2). CD8 cells were subjected to cell fractionation under control conditions or after exposure to hypotonic solution for 10 min. While in control cells most of the moesin signal was detectable in the cytosol, in cells exposed to hypotonic shock moesin redistributed to the membrane fraction, with a concomitant decrease in the cytosol. Statistical analysis revealed that, after hypotonic shock, the membrane-to-cytosol ratio of the densitometric signal relative to the moesin band increased by 2.00 ± 0.18-fold (n = 4, compared with control 1.00 ± 0.088, P < 0.05; Fig. 2).


Figure 2
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  		Fig. 2. Moesin distribution in subcellular fractions. Cellular fractions were prepared as described in MATERIALS AND METHODS. Equal amounts of proteins (30 µg/lane) from a membrane-enriched fraction (M) and a cytosol fraction (C) from control (isotonic, Iso) and hypotonicity-treated (Hypo) cells were blotted and probed with anti-moesin antibody. Left: representative Western blotting experiment. Right: statistical analysis of signal intensity ratios relative to the immunodetected band in M and C fractions. Values are expressed as means ± SE (n = 4 experiments). In hypotonicity-treated cells, the M-to-C ratio was significantly increased compared with control (*P < 0.05).

 
Moesin interaction with F-actin increases during cell swelling. In their active state, ERM family proteins translocate from the cytosol to the plasma membrane, where they cross-link membrane with cytoskeletal elements (1, 6, 8, 19, 34). To evaluate whether hypotonicity alters the association of moesin with F-actin, we employed an extraction procedure using a buffer containing Triton X-100, which preserves (and does not extract) actin cytoskeleton and actin-associated proteins. Triton X-100-extracted material from both control and hypotonicity-exposed cells was blotted and revealed with moesin antibodies (Fig. 3A). Relative to the control, in hypotonicity-treated cells the amount of moesin dissociated from F-actin was decreased (0.48 ± 0.023-fold; n = 3, P < 0.05 compared with control), suggesting an increase in the F-actin-enriched fraction (Fig. 3A). Moesin abundance was therefore determined in a cytoskeleton-enriched fraction isolated with the Qproteome Cell Compartment kit. Equal amounts of proteins from control and hypotonicity-treated cells were blotted and probed with anti-moesin antibody. As expected, hypotonicity caused a significant increase in the amount of moesin in the cytoskeleton-enriched fraction (Fig. 3B) (densitometry of the signals: 3.05 ± 0.475-fold; n = 4, P < 0.05 compared with control).


Figure 3
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  		Fig. 3. A: evaluation of moesin dissociation from F-actin during cell swelling. Alteration of moesin association with F-actin was evaluated by its solubility in Triton X-100, which preserves the cytoskeleton and cytoskeleton-associated proteins. Cells were left under basal conditions or exposed to the hypotonic solution for 10 min. Triton X-100 extracts from control cells and from cells exposed to hypotonic solution were blotted and revealed with moesin antibodies. Relative to the controls, in hypotonicity-treated cells the amount of moesin dissociated from F-actin was significantly decreased. Right: statistical densitometric analysis of the immunodetected band. Values are means ± SE (n = 3 experiments; *P < 0.01). B: expression of moesin in cytoskeleton-enriched fraction. Moesin abundance was determined in a cytoskeleton-enriched fraction. Equal amounts of proteins (30 µg/lane) from control and hypotonicity-treated cells were blotted with anti-moesin antibody. Values are means ± SE (n = 4 experiments). Hypotonicity caused a significant increase in the abundance of moesin in the cytoskeletal enriched fraction. Right: statistical densitometric analysis, *P < 0.01.

 
The F-actin binding capability of moesin is influenced by several signals. This interaction can be stabilized by phosphorylation of a threonine residue in the COOH-terminal domain, within the actin binding site (24). PKC has been demonstrated to phosphorylate this conserved threonine residue, and consistently PKC is activated in response to hypotonicity in several cell types (2, 10, 39). In particular, it has been reported that dominant-negative PKC-{alpha} impairs RVD in HeLa cells (17). Interestingly, we found that in renal CD8 cells hypotonicity caused a significant increase of PKC-{alpha} detectable in the membrane fraction (Fig. 4A; densitometric analysis shown on right). Statistical analysis revealed that in hypotonicity-treated cells, the ratio of PKC-{alpha} between membrane and cytosol fractions increased significantly (1.65 ± 0.27 compared with control 100 ± 0.08; n = 5, P < 0.05). To further investigate the cellular localization of PKC-{alpha} under isotonic and hypotonic conditions, immunofluorescence experiments were performed. Figure 4B shows that hypotonic shock resulted in a relocalization of PKC-{alpha} at the plasma membrane, where it might exert its kinase activity. No membrane transposition, and thus activation, of PKC-{epsilon}, PKC-{iota}, PKC-{lambda}, or PKC-{delta}, all expressed in CD8 cells (38), occurred during hypotonicity (Fig. 5).


Figure 4
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  		Fig. 4. A: PKC-{alpha} distribution in subcellular fractions. Cellular fractions were prepared as described in MATERIALS AND METHODS. Equal amounts of proteins (60 µg/lane) from a membrane-enriched fraction (M) and a cytosol fraction (C) from control and hypotonic preincubated cells were probed with anti-PKC-{alpha} antibody. Right: statistical analysis of the immunodetected bands. Values are means ± SE (n = 5 experiments). In hypotonicity-treated cells, the ratio between PKC-{alpha} in the M and C fractions (M/C) was found to be significantly increased (1.64 ± 0.23-fold; n = 5) compared with control (*P < 0.05). B: cells were treated as described in A, fixed, and incubated with anti-PKC-{alpha} antibody (1:100 dilution). Fluorescent signal was detected with a confocal microscope (Leica TCS, SP2, Leica Microsystem). In control cells, most PKC-{alpha} was localized in the cytosol, while in hypotonicity-treated cells PKC-{alpha} was recruited in membrane protrusions at the cell cortex. Bar = 5 µm.

 

Figure 5
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  		Fig. 5. Representative Western blotting of PKC isoform distribution in subcellular fractions. Cellular fractions were prepared as described in MATERIALS AND METHODS. Equal amounts of proteins (60 µg/lane) from a membrane-enriched fraction and a cytosol fraction from control and hypotonic preincubated cells were blotted with anti-PKC-{epsilon}, anti-PKC-{iota}, anti-PKC-{lambda}, and anti-PKC-{delta} antibodies.

 
It is known that classic PKCs (PKC-{alpha}, PKC-betaI, PKC-betaII, PKC-{gamma}) are activated by diacylglycerol (DAG) in the presence of Ca2+ (24), and the role of Ca2+ during RVD in response to hypotonic shock is well established in many cell types (10, 45). We therefore verified in CD8 cells whether cell swelling was associated with an increase in intracellular calcium as described in other renal cells (45). Changes in intracellular calcium concentration were evaluated in cells loaded with 10 µM fura-2 AM. Figure 6A shows a representative response evoked by hypotonicity. Perfusion with hypotonic solution caused an increase in intracellular calcium (Fig. 6, A and C; fluorescence ratio: 1.49 ± 0.0081 compared with control 1 ± 0.003 at peak; P < 0.0001, n = 25) that was reversed within 20 min. To evaluate whether the increase in intracellular calcium was due to calcium entry from the extracellular fluid, cells were exposed to hypotonic solution containing no calcium. In this experimental condition the hypotonicity-induced calcium release was rapid and transient but lower than that observed in the presence of extracellular calcium (Fig. 6, B and C; fluorescence ratio 1.1 ± 0.003 compared with control 1 ± 0.003 at peak; P < 0.0001, n = 25). Cell preincubation with 1 µM thapsigargin did not prevent hypotonicity-induced elevation of intracellular calcium concentration, suggesting that hypotonic shock is accompanied by a calcium release from intracellular stores, as well as by calcium entry from the extracellular fluid (Fig. 6, B and C; fluorescence ratio 1.21 ± 0.002 compared with control 1 ± 0.003 at peak; P < 0.0001, n = 25). These data likely suggest that calcium-dependent PKC-{alpha} activation might be responsible for the modulation of the ERM protein moesin activity.


Figure 6
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  		Fig. 6. Hypotonicity effect on intracellular calcium concentration. A: representative trace of the time course of the emission ratio (340/380 excitation) in fura-2-loaded cells perfused with hypotonic buffer. B: evaluation of intracellular calcium level in fura-2-loaded cells in the presence or absence of external Ca2+ and/or thapsigargin (Thaps). C: values are means ± SE (n = 25, *P < 0.0001).

 
Cell swelling causes transition of moesin from oligomeric to active monomeric form. The NH2- and COOH-terminal domains of the ERM family proteins can associate through intra- and intermolecular interactions, and these interactions mask several binding sites on the protein (5, 28). To be active, ERM proteins must be in a monomeric form characterized by the COOH- and NH2-terminal domains both being exposed, and in this conformation they can interact with F-actin and membrane proteins (50).

We show here that hypotonicity promotes a transition of moesin from an oligomeric to a monomeric conformation (Fig. 7B). Cell lysates were prepared from isotonic and hypotonicity-treated cells, in the presence or absence of calcium, and incubated with 2 mM DSP for chemical cross-linking. Equal amount of proteins were blotted under nonreducing conditions and probed with moesin antibody. In the absence of DSP, all moesin was detected as monomers having the same abundance in control and in hypotonicity-treated cell extracts. Evaluation of the relative abundance of monomeric versus oligomeric forms in isotonic and hypotonicity-treated cells, using DSP, revealed that hypotonicity increased the monomer-to-oligomer ratio (0.78 ± 0.18 vs. 1.75 ± 0.28, n = 5; Fig. 7C). In contrast, the monomerization observed after hypotonic shock was reduced in cells where intracellular calcium stores were depleted in the absence of extracellular calcium (Fig. 7).


Figure 7
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  		Fig. 7. Identification of moesin oligomers/monomers by chemical cross-linking. Cell lysates were prepared as described in MATERIALS AND METHODS. Equal amounts of proteins (30 µg/lane) were resolved with the Nu-Page system (4–12% gel) under nonreducing condition and either stained with Coomassie blue (A) or immunoblotted with moesin antibody (B). C: statistical analysis of immunodetected bands. Values are means ± SE (n = 5 experiments; P < 0.05). Hypotonicity promotes the transition of moesin from an oligomeric to a monomeric conformation, while the monomerization was blunted in the absence of calcium. DSP, dithiobis(succinimidyl propionate).

 
It was reported previously that in LLC-PK1 cells moesin transition from oligomers to monomers at the plasma membrane is regulated by phosphorylation of a conserved threonine within the F-actin binding domain (14). Phosphorylation stabilizes ERM proteins in an open and active conformation (29). Interestingly, exposure of CD8 cells to hypotonic shock increased the amount of phospho-moesin (1.98 ± 0.21 vs. control 1.00 ± 0.05; Fig. 8). A similar increase was also observed in the presence of the Rho kinase inhibitor Y-27632 (1 µM), indicating that Rho kinase is not involved in the phosphorylation of moesin during hypotonicity (2.00 ± 0.27 vs. control 1.00 ± 0.05). In contrast, a selective PKC-{alpha} inhibitor, Gö-6976 (50 nM), prevents moesin phosphorylation, indicating that PKC-{alpha} might be the kinase committed to phosphorylate moesin during hypotonic shock (0.92 ± 0.16 vs. control 1.00 ± 0.05; Fig. 8). Interestingly, moesin phosphorylation did not increase on hypotonic treatment in the absence of calcium, likely indicating that calcium might play a role not only in promoting monomerization but also in regulating moesin phosphorylation. The total moesin content remained constant during 10 min of hypotonic shock, indicating that no degradation of the protein occurred (Fig. 8).


Figure 8
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  		Fig. 8. Evaluation of phospho-moesin abundance. Cells were treated as described in MATERIALS AND METHODS and then lysed. Top: representative Western blotting experiment. Equal amounts of proteins (30 µg/lane) were blotted and probed either with a specific antibody recognizing moesin or with an antibody selective for phospho-moesin. Bottom: statistical analysis of the immunodetected bands. Values are expressed as means ± SE (n = 4 experiments; *P < 0.001). Y-27632, Rho kinase inhibitor (1 µM); Gö-6976, PKC inhibitor (50 nM).

 
To clarify the dynamic interaction occurring between actin cytoskeleton and moesin during hypotonic shock, colocalization experiments were performed to simultaneously test the subcellular localization of phospho-moesin and F-actin under both basal and hypotonic conditions. Compared with the control condition, exposure of CD8 cells to the hypotonic solution caused a relocalization of phospho-moesin to the cell cortex in the membrane processes, where the actin cytoskeleton is tightly organized. A similar colocalization was observed when cells were preincubated with the selective inhibitor of Rho kinase Y-27632, suggesting that Rho kinase is not the kinase committed to control moesin phosphorylation during hypotonicity. In contrast, pretreatment with the PKC-{alpha} inhibitor Gö-6976 caused a significant decrease in phospho-moesin staining and prevented both actin patch formation at the cell border and moesin phosphorylation during hypotonic shock. In this experimental condition actin and phospho-moesin no longer colocalize (Fig. 9).


Figure 9
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  		Fig. 9. Colocalization of phospho-moesin and F-actin. Cells were processed as described in MATERIALS AND METHODS and costained under basal and hypotonic conditions in the absence or presence of Rho kinase inhibitor Y-27632 (1 µM) or PKC inhibitor Gö-6976 (50 nM), respectively. Fluorescent signal was detected with a confocal microscope (Leica TCS, SP2, Leica Microsystem). Bar = 5 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Regulation of cell volume is a fundamental feature, conserved through evolution, to maintain cellular volume constant in response to challenges in extracellular osmolarity. Animal cells respond to alteration of external osmolality first by changing their volume, which in turn activates several signals affecting cytoskeletal structure that might regulate the activity of volume-regulatory transporters (32).

In this study, we propose moesin, a member of the ERM family known to cross-link actin filaments with plasma membranes, as a new player functionally involved in regulating cytoskeleton organization to counteract cell swelling occurring in response to a hypotonic challenge.

This study was performed in a renal collecting duct cell line stably expressing the aquaporin (AQP)2 water channel (48). In the inner medulla, collecting duct cells are subjected to strong variations in external osmolarity generated by circulating vasopressin. In this cell line, we (42) recently provided the first evidence that moesin is functionally involved in actin remodeling facilitating AQP2 apical targeting. Moreover, we (43) have found that hypotonicity induces AQP2 internalization and cytosol-to-membrane translocation of the multifunctional protein ICln, known to be essential for the generation of ion currents activated during RVD. We show here that treatment with hypotonic solution resulted in a strong actin reorganization, consisting in the loss of ventral stress fibers and in the formation of F-actin patches at the cell cortex. Dynamic rearrangements of the actin cytoskeleton are central to cell morphological changes, and several pieces of evidence have highlighted the importance of the ERM protein signaling pathways in this process (25, 30). Interestingly, it has been reported that, in their active state, ERM proteins translocate to the plasma membrane, where they interact with both F-actin and membrane proteins (50). Consistent with these observations, we report here that during hypotonic shock moesin translocates at the cell border and interacts with actin, which is also recruited at the same sites. This interaction is paralleled with an increase in moesin phosphorylation. Phosphorylation of the conserved threonine residue within the actin binding domain of moesin is indeed critical for regulating the conformational change and functional activities of moesin (21). Among the kinases that have been shown to possibly phosphorylate moesin (Rho-kinase, phosphatidylinositol 4-phosphate 5-kinase, myotonic dystrophy kinase-related Cdc42-binding kinase, and PKC) (29, 31, 33, 36), we identify PKC-{alpha} as the kinase committed to stabilizing active moesin through phosphorylation. Preincubation of cells with a selective PKC-{alpha} inhibitor prevented hypotonicity-induced moesin phosphorylation at threonine 558. This result is in agreement with previous findings in HeLa cells, showing that PKC-{alpha} is the kinase activated during hypotonicity (17). PKC-{alpha} belongs to the conventional PKC isoenzyme, which requires calcium and DAG for activation, and it was previously shown to be expressed in CD8 cells (38). Cell response to hypotonic challenge has been reported to be associated with an increase in intracellular calcium in several cell types. In inner medullary collecting duct cells, hypotonicity caused a significant increase in intracellular calcium levels due to both entry from extracellular medium and release from intracellular stores (45). Similarly, in CD8 cells, hypotonicity shock also caused an increase in intracellular calcium levels due to release from intracellular stores and entry from the extracellular solution. Activation of PKC-{alpha} by calcium was found to be essential for moesin phosphorylation resulting in its rearrangement during hypotonicity. In fact, while in control cells the moesin monomer-to-polymer ratio increased in response to hypotonic shock, in cells with depleted intracellular calcium stores and in the absence of external calcium this effect was prevented, confirming the causal relationship among those phenomena. It must be emphasized that monomerization is essential for moesin activation since it exposes the phosphorylation site for PKC-{alpha}-dependent phosphorylation. Consistent with this finding, hypotonic treatment significantly increased the amount of phospho-moesin, which was abolished both in the absence of calcium and in the presence of the selective inhibitor of PKC-{alpha}. On the other hand, if cells are exposed to an hypotonic shock in the presence of the specific inhibitor of the calcium-dependent PKC-{alpha}, neither moesin phosphorylation nor recruitment to the cell border occurs and F-actin does not colocalize with phospho-moesin (Figs. 8 and 9). Together these results represent strong evidence that the physiological sequence during hypotonicity would be 1) rise in intracellular calcium, 2) relocation of the cytosolic PKC-{alpha} at the plasma membrane protrusions, and 3) phosphorylation of moesin and its relocation into F-actin patches.

To conclude, our data point to a novel role of moesin as a regulatory protein participating in the reorganization of F-actin in membrane protrusions induced by hypotonic shock. We provide here the first evidence that under hypotonicity moesin is recruited at the cell borders and interacts with actin in a phosphorylated state under control of the calcium-dependent kinase PKC-{alpha}. Together with previous findings demonstrating the functional involvement of moesin in actin remodeling, facilitating AQP2 trafficking, our data point to a complex role of this regulatory protein in signaling pathways and in cytoskeleton organization. Moesin is emerging as part of the volume-sensing system that helps to regulate the complex machinery that is activated by hypotonicity.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from Telethon (proposal no. GGP04202 to G. Valenti), from PRIN (Research Program of National Interest) to G. Valenti, from Centro di Eccellenza di Genomica in campo Biomedico ed Agrario (CEGBA), and from the Vigoni program (2005–2006).


   ACKNOWLEDGMENTS
 
We thank Anthony Green for proofreading and providing linguistic advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Valenti, Dipartimento di Fisiologia Generale e Ambientale, Via Amendola 165/A, 70126 Bari, Italy (e-mail: g.valenti{at}biologia.uniba.it)

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.


   REFERENCES
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 DISCUSSION
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