Am J Physiol Cell Physiol Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 294: C1046-C1055, 2008. First published February 13, 2008; doi:10.1152/ajpcell.00134.2007
0363-6143/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/C1046    most recent
00134.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nielsen, M.-B.
Right arrow Articles by Hoffmann, E. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nielsen, M.-B.
Right arrow Articles by Hoffmann, E. K.

RECEPTORS AND SIGNAL TRANSDUCTION

Effects of osmotic stress on the activity of MAPKs and PDGFR-β-mediated signal transduction in NIH-3T3 fibroblasts

M.-B. Nielsen, S. T. Christensen,* and E. K. Hoffmann*

Section of Cell and Developmental Biology, Department of Biology, University of Copenhagen, Copenhagen, Denmark

Submitted 3 April 2007 ; accepted in final form 5 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Signaling in cell proliferation, cell migration, and apoptosis is highly affected by osmotic stress and changes in cell volume, although the mechanisms underlying the significance of cell volume as a signal in cell growth and death are poorly understood. In this study, we used NIH-3T3 fibroblasts in a serum- and nutrient-free inorganic medium (300 mosM) to analyze the effects of osmotic stress on MAPK activity and PDGF receptor (PDGFR)-β-mediated signal transduction. We found that hypoosmolarity (cell swelling at 211 mosM) induced the phosphorylation and nuclear translocation of ERK1/2, most likely via a pathway independent of PDGFR-β and MEK1/2. Conversely, hyperosmolarity (cell shrinkage at 582 mosM) moved nuclear and phosphorylated ERK1/2 to the cytoplasm and induced the phosphorylation and nuclear translocation of p38 and phosphorylation of JNK1/2. In a series of parallel experiments, hypoosmolarity did not affect PDGF-BB-induced activation of PDGFR-β, whereas hyperosmolarity strongly inhibited ligand-dependent PDGFR-β activation as well as downstream mitogenic signal components of the receptor, including Akt and the MEK1/2-ERK1/2 pathway. Based on these results, we conclude that ligand-dependent activation of PDGFR-β and its downstream effectors Akt, MEK1/2, and ERK1/2 is strongly modulated (inhibited) by hyperosmotic cell shrinkage, whereas cell swelling does not seem to affect the activation of the receptor but rather to activate ERK1/2 via a different mechanism. It is thus likely that cell swelling via activation of ERK1/2 and cell shrinkage via activation of the p38 and JNK pathway and inhibition of the PDGFR signaling pathway may act as key players in the regulation of tissue homeostasis.

mitogen-activated protein kinases; platelet-derived growth factor receptor-β; volume sensor

THE MECHANISMS RESPONSIBLE for the maintenance of a constant cell volume under "resting" conditions and for the cell's capability of counteracting volume perturbation by volume recovery processes have been described in a wide variety of cells and have been studied in detail for many years (23, 26, 36). Increasing evidence now supports the conclusion that signaling in cell volume control cooperates with signaling in cell proliferation, cell migration, and apoptosis. However, we still know little as to the function of a change in cell volume as a direct signal in the regulation of these cellular parameters. As discussed by Hoffmann and Ussing (27), the term "volume regulation" may lead to the misconception that cells have one preferred volume, which is not the case since the "preferred" volume may highly depend on the differential and functional state of the cell, such as in secreting epithelia (21, 27) and in cells during growth and proliferation per se. Thus, cell proliferation is often stimulated by osmotic swelling and inhibited by osmotic shrinkage (2, 7, 14, 62), and cell shrinkage is an early event in and a hallmark of the apoptotic mode of programmed cell death (5, 18, 34, 43, 44, 50, 61), such that cell division depends on an increase in cell volume (see Refs. 36 and 62) and is delayed in shrunken cells (49). Indeed, it has been proposed that a change in cell volume acts as an immediate signal that impinges on the apoptotic death process (7, 13, 18, 39, 47, 48, 58, 73) as well as on progression through the cell cycle (36–38). Therefore, it is important to identify the level of cross-talk between signaling pathways in cell volume regulation, cell proliferation, and apoptosis to understand the potential role of changes in cell volume as a direct signal controlling cell growth and death in tissue homeostasis and developmental processes.

Growth control and apoptosis in mammalian cells are highly regulated by receptor tyrosine kinase receptors (RTKs), which, upon ligand-dependent activation, launch the activation of a series of downstream signal transduction pathways, including MAPKs and the cell survival-promoting protein kinase B (Akt) (33, 45). Generally, activation of p38 MAPK and JNK is associated with the inhibition of cell cycle progression and promotion of apoptosis, whereas activation of MEK1/2-ERK1/2 and phosphatidylinositol 3-kinase (PI3K)-Akt pathways inhibit apoptosis and stimulate proliferative responses (9, 12, 18, 20, 25, 67, 67, 70, 77). That osmotic stress activates MAPKs was first described in Saccharomyces cerevisiae (28) and later also in mammalian cells (67). With a few exceptions (see, e.g., Ref. 52), cell shrinkage by hyperosmotic stress is associated with the stimulation of p38 MAPK and JNK (3, 4, 15, 18–20, 31, 42, 46, 56, 61, 61, 69, 74, 75), whereas cell swelling by hypoosmotic stress stimulates ERK1/2 and Akt (31, 63, 73, 79) in concurrence with the conclusion that cell shrinkage is a hallmark of apoptosis and that cell swelling is associated with proliferative responses. Furthermore, changes in cell volume may impinge on the cross-talk between different MAPK cascades, such that p38 MAPK inhibits ERK1/2 following osmotic shrinkage in fibroblasts (18) and Ehrlich-Lettre ascites cells (55), and direct p38 MAPK-mediated inhibition of ERK1/2 has been demonstrated (78). MEK-independent ERK inhibition by p38 has also been demonstrated in heart cells (30).

A number of reports have also implicated a role of RTKs in osmosensing and volume control as upstream regulators of, e.g., MAPKs and the PI3K/Akt pathway. A prominent example is that of the EGF receptor (EGFR) family, including the EGFR in Swiss 3T3 fibroblasts and ErbB4 in cerebellar granule neurons, which are activated by cell swelling induced by hypoosmotic stress following the activation of PI3K/Akt and/or MEK1/2-ERK1/2 pathways (17, 41). Stimulation of the EGFR by cell swelling has also been reported in other cell types (41, 54). Conversely, cell shrinkage by hypertonic stress blocks EGFR-mediated activation of PI3K/Akt and MEK1/2-ERK1/2 pathways in kidney cells, although blockage of signaling is induced immediately downstream of Ras leaving ligand-dependent activation of the receptor unaffected by hypertonic stress (11). Probably these observations are also cell type specific, since hypertonic stress in hepatoma cells mediates the phosphorylation of EGFR, which is required for CD95-mediated initiation of apoptosis (59, 60). Indeed, it is important to realize that the activation or deactivation of MAPKs and other signal pathways during osmotic stress may also highly depend on culture conditions and the strength of the osmotic stress.

In the present study, we used NIH-3T3 fibroblasts to investigate the effect of osmotic stress on the activity of MAPKs and on signal transduction via the RTK PDGF receptor (PDGFR)-β, which plays an important role in cell proliferation, cell survival, cell migration, and angiogenesis in development and tissue homeostasis (46, 65). Significantly, experiments were performed in serum-depleted inorganic medium to eliminate the interference of signal molecules from serum, such as growth factors, that impinge on the activity of RTKs and downstream signal transduction pathways. Furthermore, the inorganic medium allowed us to directly evaluate the differences in the activation of signal pathways during cell swelling and cell shrinkage at which the differences are caused exclusively by differences in osmotic pressure. Thereby, the mechanisms of changes in cell volume can be studied simultaneously in one cell type to investigate the modulation of signal transduction pathways, including the possible causal relationship between MAPKs, PDGFR-β, and the Akt signaling pathway in an attempt to get closer to the understanding of cell volume as a signal in cell proliferation and apoptosis. A portion of these results has previously been presented in abstract form (24).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and antibodies. Unless otherwise stated, reagents were of analytical grade and obtained from Sigma (St. Louis, MO). Recombinant PDGF-BB was from Calbiochem and was used at 25 ng/ml. Primary antibodies [rabbit polyclonal anti-PDGFR-β and anti-phosphorylated (p-)PDGFR-β (p-Tyr857)] were obtained from Santa Cruz Biotechnology; rabbit polyclonal anti-p38 MAPK, anti-p-p38 MAPK (p-Thr180/p-Tyr182), anti-ERK1/2, anti-p-ERK1/2 (p-Thr202/p-Tyr204), anti-p-JNK (detecting both JNK1 and JNK2 phosphorylated at Thr183/Tyr185), and anti-p-MEK1/2 (Ser217/221) were from Cell Signaling Technology (Beverly, MA), as was the mouse monoclonal antibody against p-Akt (which detects Akt1, Akt2, and Akt3 phosphorylated at Ser473). Monoclonal mouse anti-β-actin was from Sigma. The secondary antibody for immunoflourescence microscopy analysis was Cy3-conjugated affinipure goat anti-rabbit IgG (Jackson ImmunoResearch). Secondary antibodies for Western blot analysis were alkaline phosphatase-conjugated goat anti-rabbit and goat anti-mouse IgG (Jackson ImmunoResearch).

Cell culture and saline solutions. NIH-3T3 cells were maintained in RPMI-1640 medium (Sigma) supplemented with 10% FCS and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) in a humidified incubator at 37°C and 5% CO2. Cells were passaged every 3–4 days and only passages 5–25 were used for experiments. Experiments were carried out in cultures with a confluency of 80–90% in the presence and absence of PDGF-BB. For experiments in Ringer solutions, the medium was removed, cells were washed fast twice with PBS, and isotonic, hypotonic, or hypertonic Ringer was then added (defined as time 0). The standard saline Ringer solution had an osmolarity of 300 mosM and contained (in mM) 143 NaCl, 5 KCl, 1 MgSO4, 1 Na2HPO4, 1 CaCl2, 3.3 MOPS, 3.3 TES, and 5 HEPES (pH 7.4). The standard hypertonic Ringer solution was prepared by either the addition of NaCl or sucrose to obtain a final osmolarity of 582 mosM. Sucrose is a mean to induce hypertonicity without changing the ionic strength of the medium. Hypotonic Ringer solution (211 mosM) was prepared by reducing the concentration of all components except MOPS, TES, and HEPES compared with the standard solution. Osmolarities of the saline solutions were regularly controlled using a freezing point osmometer. Unless otherwise indicated, experiments were carried out at 37°C.

Light microscopy. Cells incubated in isotonic, hypertonic, and hypotonic Ringer solutions were continuously examined with phase-contrast microscopy on a Nikon Diaphot 300 microscope to monitor changes in cell volume. Images were taken with a Nikon D1 digital camera and processed using Adobe Photoshop version 6.0.

SDS-PAGE and Western blot analysis. Cell lysates were prepared essentially as previously described (66). Briefly, cells grown to confluence in 78-cm2 culture dishes were washed with ice-cold PBS (136.89 mM NaCl, 2.68 mM KCl, 8.10 mM Na2HPO4, and 1.7 mM KH2HPO4), added to 200 µl of boiling lysis buffer (1% SDS and 10 mM Tris·HCl; pH 7.5), and transferred to Eppendorf tubes using a rubber policeman. Lysates were then homogenized through 27-gauge syringes and spinned down at 16,000 g to clear the homogenate. A 5-µl aliquot was removed for protein determination using the DC Protein Assay from Bio-Rad. Aliquots of ~20 µg protein (equal amounts in each well for a given experiment, calculated by protein content determination and verified by Ponceau S staining) were resolved under reducing conditions using 10% NuPAGE Bis-Tris gels and NuPAGE MOPS-SDS running buffer (NP0002) as previously described (8) using Fermentas protein standards and a NOVEX XCell system from Invitrogen. Separated proteins were electrotransferred to nitrocellulose membranes using the XCell II Blot Module (NOVEX), followed by staining in 1% Ponceau S red solution. Nitrocellulose membranes were blocked in 5% milk in Tris-buffered saline-Tween 20 [TBST; 10 mM Tris·HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20] plus 0.5% Na-azide for 2 h at room temperature and incubated overnight or for 2 h at room temperature with primary antibodies diluted in 5% nonfat dry milk and TBST. All antibodies were diluted to 1:200 except for anti-β-actin, which was used at 1:30,000. Membranes were washed four times for 5 min in TBST, incubated for 1 h with secondary antibodies diluted to 1:600 in 5% milk and TBST, and washed four times for 10 min in TBST prior to being developed with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium solution (KLP, Gaithersburg, MD). Developed blots were scanned, and the band intensity was estimated from arbitrary densitometric values obtained using UN-SCAN-IT software. Blots were further processed for publication in Adobe Photoshop version 6.0.

Immunofluorescence microscopy analysis. Immunofluorescence microscopy analysis was essentially performed as previously described (66). Primary antibodies were used at 1:50 dilution. Fluorescent secondary antibodies were diluted to 1:600, and 4',6-diamidino-2-phenylindole (Molecular Probes) was used at 1:1,000 dilution. Cells were visualized on an Eclipse E600 microscope (Nikon) with EPI-FL3 filters and a MagnaFire cooled charge-coupled device camera (Optronics, Goleta, CA), and digital images were processed using Adobe Photoshop version 6.0.

Statistical analysis. Significance was evaluated using a double-sided Student's t-test, with P < 0.05 taken to indicate a statistically significant difference. All experiments were conducted between three and six times.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of osmotic stress on activation of p38. The activation of p38 during hypertonic stress (582 mosM) induced by the addition of either NaCl or sucrose as well as hypotonic stress (211 mosM) stress in Ringer solution was initially investigated with antibodies directed against p38 and p-p38 using Western blot analysis. The phosphorylation level of p38 increased slightly after 0.5 min of hypertonic stress induced with NaCl, and this level increased to about fivefold after 10 min of osmotic stress (Fig. 1, A and D). A similar increase in p-p38 was observed in the presence of sucrose (Fig. 1, B and D). In contrast, no increase in p-p38 was observed upon hypotonic (211 mosM) stress during 30 min (Fig. 1, C and D). During a more dramatic osmotic stress (150 mosM), an increase in p-p38 could, however, be observed (data not shown), probably as a general stress response. We then analyzed the cellular localization of p38 and p-p38 by immunofluorescence microscopy after 5 and 30 min of hypertonic (NaCl, 582 mosM) and hypotonic (211 mosM) stress. As demonstrated, p38 primarily localized to the cytoplasm and outside the nucleus in isotonic cells, whereas cells treated with NaCl showed a more prominent nuclear localization of p38 both after 5 (Fig. 1E) and 30 min (Fig. 1G). In hypotonic cells, p38 localized in a pattern similar to that of isotonic cells. We next examined the localization and level of p-p38, which in isotonic cells localized to the cytoplasm and slightly to the nucleus (Fig. 1, F and H). Upon hypertonic stress induced by NaCl, the level of p-p38 dramatically increased and predominantly localized to the nucleus in accordance with the increased activation of p38, as demonstrated by Western blot analysis (Fig. 1A). There was a clear difference with respect to the nuclear localization of p-p38 between 5-min hypertonically treated cells (Fig. 1F) and 30-min hypertonically treated cells (Fig. 1H), showing that the effect cannot be just an effect of the hypertonicity on the antibody but is a real and gradual effect on the nuclear localization caused by hypertonic treatment. In contrast, in hypotonic cells, p38 localized in a pattern similar to that of isotonic cells and similar to that of nonphosphorylated p38 both after 5 and 30 min.


Figure 1
View larger version (72K):
[in this window]
[in a new window]

  		Fig. 1. Effect of osmotic stress on the phosphorylation and nuclear localization of p38 in NIH-3T3 cells. A–C: SDS-PAGE and Western blot analysis of the level of phosphorylated (p-)p38 in cells incubated in isotonic (iso; 300 mosM), hypertonic (NaCl; 582 mosM), hypertonic (sucrose; 582 mosM), or hypotonic (hypo; 211 mosM) Ringer solutions at various time intervals. D: intensity of p38 phosphorylation in hypertonic or hypotonic Ringer solutions relative to the isotonic control. *The level of p-p38 was significantly different from that of the isotonic control (P < 0.05). Equal amounts of protein were loaded in each well as calculated by protein content determination and verified by Ponceau S red staining. E: immunofluorescence microscopy analysis (IF) of the level and cellular distribution of p38 (red) in cells incubated in isotonic, hypertonic (NaCl; 582 mosM), and hypotonic (211 mosM) Ringer solutions for 5 min. F: IF of the level and cellular distribution p-p38 in cells incubated in isotonic (300 mosM), hypertonic (NaCl; 582 mosM), and hypotonic (211 mosM) Ringer solutions for 5 min. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; blue). G and H are similar to E and F but after incubation at the various osmolarities for 30 min.

 
Effects of osmotic stress on activation of ERK1/2. Activation of ERK1/2 during hypertonic stress (582 mosM) induced by the addition of either NaCl or sucrose as well as hypotonic stress (211 mosM) stress was initially investigated with antibodies directed against ERK1/2 and p-ERK1/2 using Western blot analysis. The phosphorylation level of ERK1/2 was unaffected or slightly reduced upon hypertonic stress (Fig. 2, A, B, and D), whereas hypotonic cells showed a small but significant increase in ERK1/2 phosphorylation after 0.5 min of incubation, and this increase in phosphorylation of ERK1/2 in hypotonic cells appeared throughout the experiment (Fig. 2, C and D). Concomitantly, immunofluorescence microscopy analysis showed that the level and nuclear localization of ERK1/2 characteristically decreased upon hypertonic stress and increased in hypotonic cells (Fig. 2E). In a series of parallel experiments, supplementation of serum in full medium generally induced a high level of ERK1/2 phosphorylation, which was largely unaffected by hypotonic stress (data not shown).


Figure 2
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 2. Effect of osmotic stress on phosphorylation and nuclear localization of ERK1/2 in NIH-3T3 cells. A–C: SDS-PAGE and Western blot analysis of the level of p-ERK1/2 in cells incubated in either isotonic (300 mosM), hypertonic (NaCl; 582 mosM), hypertonic (sucrose; 582 mosM), or hypotonic (211 mosM) Ringer solutions at various time intervals. D: intensity of ERK1/2 phosphorylation in hypertonic or hypotonic Ringer solutions relative to the isotonic control. *The level of p-Erk1/2 was significantly different from that of the isotonic control (P < 0.05). Equal amounts of protein were loaded in each well as calculated by protein content determination and verified by Ponceau S red staining. E: IF of the level and cellular distribution of p-Erk1/2 in cells incubated in isotonic (300 mosM), hypertonic (NaCl; 582 mosM), and hypotonic (211 mosM) Ringer solutions for 30 min. Nuclei were stained with DAPI (blue).

 
Effects of osmotic stress on activation of MEK1/2. To investigate the position of MEK1/2 as an upstream regulator of ERK1/2 during osmotic stress, we examined the level of p-MEK1/2 upon hypertonic (582 mosM) and hypotonic (211 mosM) stress. As shown in Fig. 3, there were no significant changes in the activation of MEK1/2 at either osmotic situation, indicating that ERK1/2 upon hypotonic treatment is activated independently of MEK1/2.


Figure 3
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 3. Effect of osmotic stress on the level of phosphorylation of MEK1/2 in NIH-3T3 cells. A–C: SDS-PAGE and Western blot analysis of the level of p-MEK1/2 in cells incubated in either isotonic (300 mosM), hypertonic (NaCl; 582 mosM), hypertonic (sucrose; 582 mosM), or hypotonic (211 mosM) Ringer solutions at various time intervals. D: intensity of MEK1/2 phosphorylation in hypertonic or hypotonic Ringer solutions relative to the isotonic control (n = 5). Equal amounts of protein were loaded in each well as calculated by protein content determination and verified by Ponceau S red staining.

 
Effects of osmotic stress on activation of JNK1/2. Previously, JNK1/2 has been suggested as a MAPK participating in osmotic stress responses (see Introduction). To investigate this in further detail, cells were subjected to hypertonic stress (582 mosM) induced by NaCl and hypotonic stress (211 mosM) with antibodies directed against p-JNK1/2 using Western blot analysis. Characteristically, hypotonic cells showed no increase in the phosphorylation of JNK1/2, whereas hypertonic stress significantly increased the phosphorylation level of JNK1/2 at 46 kDa after 30 min of incubation (Fig. 4).


Figure 4
View larger version (36K):
[in this window]
[in a new window]

  		Fig. 4. Effect of osmotic stress on the level of phosphorylation of JNK1/2 in NIH-3T3 cells. A and B: SDS-PAGE and Western blot analysis of the level of p-JNK1/2 in cells incubated in either isotonic (300 mosM), hypertonic (NaCl; 582 mosM), or hypotonic (211 mosM) Ringer solutions at various time intervals. D: intensity of JNK1/2 phosphorylation in hypertonic or hypotonic Ringer solutions relative to the isotonic control. Equal amounts of protein were loaded in each well as calculated by protein content determination and verified by Ponceau S red staining. *The level of p-JNK1/2 was significantly different from that of the isotonic control (P < 0.05) (n = 3).

 
Activation of PDGFR-β and its mitogenic downstream components is strongly inhibited in hypertonic cells. To investigate the effect of hypertonicity on PDGFR-mediated signal transduction, cells were subjected to either isotonic or hypertonic sucrose-Ringer solution (582 mosM) in the presence and absence of PDGF-BB followed by Western blot analysis with antibodies directed against PDGFR-β and p-PDGFR-β as well as the phosphorylated forms of ERK1/2, MEK1/2, Akt, and p38. After 30 s of incubation, PDGF-BB clearly increased the level of p-PDGFR-β about fivefold in isotonic cultures, whereas hypertonic cells showed no increase in p-PDGFR-β (Fig. 5, A and D). The level of p-PDGFR-β in isotonic cells further increased ~20-fold after 2 and 5 min of incubation, followed by a decrease in receptor phosphorylation after 10 and 30 min of incubation (Fig. 5, A–D), signifying the conventional step in RTK downregulation. In contrast, hypertonic cells showed a very moderate increase in receptor phosphorylation only after 10 and 30 min of incubation. Concomitantly, activation of the mitogenic ERK1/2-MEK1/2 pathway by PDGF-BB after 2, 5, and 10 min of incubation was dramatically reduced in hypertonic cells but increased after 30 min of PDGF-BB stimulation (Fig. 5, E and F) parallel to a moderate increase in phosphorylation of p38 after 10 min in hypertonic cells in the presence of PDGF-BB (Fig. 5, B, C, and G). In addition, PDGF-BB-mediated activation of mitogenic Akt was strongly reduced in hypertonic cells up to 30 min after the addition of the ligand, as shown in the Western blot of p-Akt in Fig. 6A and from the mean intensity of p-Akt after the addition of PDGF-BB in isotonic and hypertonic medium, respectively, in Fig. 6B. Thus, hypertonicity strongly inhibits and delays the early and major ligand-dependent activation of PDGFR-β and the mitogenic downstream components of the receptor in signal transduction.


Figure 5
View larger version (45K):
[in this window]
[in a new window]

  		Fig. 5. Effect of hypertonic stress on ligand-dependent activation of PDGF receptor (PDGFR)-β and PDGFR-β-mediated signal transduction in NIH-3T3 cells. A–C: SDS-PAGE and Western blot analysis of the level of PDGFR-β, p-PDGFR-β, p-MEK1/2, p-ERK1/2, and p-p38 in isotonic (300 mosM) and hypertonic (hyper; sucrose, 582 mosM) Ringer solutions after 0.5, 5, and 30 min of incubation with and without PDGF-BB. Equal amounts of protein were loaded in each well as calculated by protein content determination and verified by Ponceau S red staining. D–G: intensity of protein phosphorylation after the addition of PDGF-BB (25 ng/ml) in isotonic or hypertonic Ringer solutions. Values are given relative to the values in the isotonic control without PDGF-BB.

 

Figure 6
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 6. Effect of hypertonic stress on PDGF-BB-mediated activation of Akt in NIH-3T3 cells. A: SDS-PAGE and Western blot analysis of the level of p-Akt in isotonic (300 mosM) and hypertonic (high sucrose, 582 mosM) Ringer solutions at various time intervals. B: intensity of Akt phosphorylation in hypertonic Ringer solution relative to the isotonic control as a function of time after the addition of PDGF-BB. *The effect of PDGF-BB on the level of p-Akt was significantly lower in the hypertonic medium than in the isotonic control (P < 0.05).

 
PDGFR-β is not activated by cell swelling induced by hypotonic stress. With the aim to examine the effect of hypotonic stress on the activation of PDGFR-β, we performed Western blot analysis on the level of receptor phosphorylation after incubation of cells in hypotonic (211 mosM) Ringer solution, where cells are clearly swollen compared with isotonic cells (Fig. 7A). As evaluated by Western blot analysis, the level of p-PDGFR-β was kept at a low level in both isotonic and hypotonic cells, in clear contrast to the level of p-ERK1/2, which increased about threefold after cells were incubated in hypotonic Ringer solution (Fig. 7, B and C). These results support the conclusion that PDGFR-β is not activated by hypotonic stress and that ERK1/2 in hypotonic cells is activated independently of both the receptor and MEK1/2. To investigate whether hypotonicity affects PDGFR-β activation with PDGF-BB, the intensity of PDGFR-β, ERK1/2, and p38 phosphorylaton was measured in hypotonic Ringer solution relative to the isotonic control as a function of time after the addition of PDGF-BB (25 ng/ml). There were no significant differences in any of the groups. Thus, hypotonicity (cell swelling) does not affect the affinity of PDGFR-β for the agonist.


Figure 7
View larger version (43K):
[in this window]
[in a new window]

  		Fig. 7. Effect of hypotonic stress and cell swelling on the activation of PDGFR-β in NIH-3T3 cells. A: phase-contrast microscopy of cells incubated for 10 min in isotonic (300 mosM) and hypotonic (211 mosM) Ringer solutions. B: SDS-PAGE and Western blot analysis of the level of p-PDGFR-β and p-ERK1/2 after 10 min of incubation in isotonic (300 mosM) and hypotonic (211 mosM) Ringer solutions. C: intensity of PDGFR-β and ERK1/2 phosphorylation in hypotonic Ringer solution relative to the isotonic control. Equal amounts of protein were loaded in each well as calculated by protein content determination and verified by Ponceau S red staining. *The level of p-ERK1/2 was significantly different from that of the isotonic control (P < 0.05). D: intensity of PDGFR-β, ERK1/2, and p38 phosphorylaton in hypotonic Ringer solution relative to the isotonic control as a function of time after the addition of PDGF-BB (25 ng/ml). There were no significant differences in any of the groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It is well known that MAPKs and other members of protein kinases in signal transduction, such as the PI3K/Akt pathway, are major targets of osmotic stress in eukaryotic cells (25). However, we still know little as to the precise mechanisms that control either the activation or deactivation of signal transduction components largely because different cell types in cultures produce different results and because the culture conditions chosen to perform the experimentation may vary significantly from one research group to another. In this report, we used NIH-3T3 fibroblasts in inorganic Ringer solution to study changes in protein kinase activity in signal transduction exclusively by differences in osmotic pressure, i.e., independently of variations in nutrient composition and in background signaling originating from growth factors and other ligands present in serum.

Differential changes in activation of MAPKs upon osmotic stress. In our study, we initially found that ERK1/2 and p38 act oppositely in terms of their activation upon osmotic stress in fibroblasts. Prominently, ERK1/2 is phosphorylated and targeted to the nucleus upon hypotonic Ringer solution (Fig. 2), indicating that cell swelling facilitates progression through the cell cycle, since nuclear translocation of ERK1/2 is a critical step to transduce cell growth (6). In hypertonic Ringer solution, phosphorylated ERK1/2 remained at a level comparable with that of isotonic controls, as evaluated by Western blot analysis. However, immunofluorescence microscopy revealed that hypertonic stress moves nuclear p-Erk1/2 to cytoplasmic puncta, supporting the conclusion that a change in cell volume is a critical step in growth signaling through ERK1/2. Importantly, these observations may be compromised in fibroblasts cultured in the presence of serum, which greatly stimulates the activation of ERK1/2; we have previously shown under these circumstances that cell shrinkage significantly decreases the phosphorylation of ERK1/2. Conversely, hypertonic stress strongly induced an early activation of p38 by phosphorylation and nuclear translocation of p-p38 (Fig. 1), whereas hypotonic stress had no effect on either the level of protein phosphorylation and cellular distribution of p-p38. Similar observations have been previously demonstrated for p38 and ERK1/2 in hypertonic growth medium in the presence of serum in fibroblasts (18), suggesting that cell shrinkage blocks the early activation of receptor-mediated signal transduction in cell survival and growth. Concomitantly, hypertonic stress induced the late activation of JNK, as evaluated by the level of phosphorylation of this MAPK using Western blot analysis (Fig. 4). With respect to the mechanism of p38 activation, studies in HEK-293 cells (76) and fibroblasts (18) have indicated that shrinkage activation of p38 involves Cdc42 and/or Rac, small GTP-binding proteins (G proteins of the Rho family), which have been directly shown to be activated by osmotic shrinkage (40). The physiological consequences of p38 and JNK activation by hyperosmolarity have mainly been studied in long-time experiments where p38 and JNK normally counteract cell proliferation and activate the apoptotic process (9, 18, 57, 64, 77). p38 is also involved in adaptive transcriptional regulation in response to osmotic stress (67), which correlates well with the present finding of a translocation to the nucleus of p-p38. Finally, p38 is so rapidly modulated after cell shrinkage that it can also play a role in the regulation of osmolyte transport during regulatory volume increase, which has also been shown to be the case in some cell types (56, 61, 69).

The mechanisms by which ERK1/2 in fibroblasts is activated upon hypotonic stress are presently unknown. One of the most common pathways leading to ERK1/2 activation is the signaling cascade initiated by growth factor receptors, such as PDGFR, via the small G protein Ras and MEK1/2. However, the hypososmotic activation of ERK1/2 seems not to be caused by activation of this pathway, since neither MEK1/2 (Fig. 3) nor PDGFR-β (Fig. 7) were activated by phosphorylation upon hypotonic stress in Ringer solution. In human embryonic intestinal epithelial cells, ERK1/2 is phosphorylated upon cell swelling via Ras-independent activation of leukotriene D4 (LTD4) (53), and we have previously demonstrated that LTD4 is released after cell swelling in Ehrlich ascites tumor cells and is responsible for the activation of the swelling-activated K+ channel (22, 29, 35, 71). Indeed, ERK1/2 is involved in acute volume regulation by activation of volume regulatory channels (16, 68), adding further complexity to the role of ERK1/2 during changes in cell volume upon osmotic stress.

In conclusion, we prepared a serum- and nutrient-free medium to demonstrate that cell swelling in hypotonic medium induces the phosphorylation and nuclear translocation of ERK1/2 via a pathway independent of signaling via MEK1/2. Conversely, cell shrinkage in hypertonic medium inhibited the nuclear localization of p-ERK1/2 and stimulated the phosphorylation of p38 and JNK1/2 as well as translocation of p38 to the nucleus.

Activation of PDGFR-β and its mitogenic downstream components is inhibited after shrinkage in hypertonic medium but unchanged in hypotonic medium. To demonstrate the effect of hypertonic stress on ligand-dependent activation of RTK in cultures with a high signal-to-noise ratio, we investigated the activity of PDGFR-β and its signal transduction in fibroblasts in Ringer solution.

PDGF signaling controls a series of cellular responses in cell survival, apoptosis, proliferation, and migration during embryonic and postnatal development as well as in tissue homeostasis (1, 51, 72). Activation of its receptors by dimerization of PDGFR-{alpha} and/or PDGFR-β is followed by the phosphorylation of tyrosines on the cytosolic side of the receptors at specific docking sites for other proteins that induce distinct cascades of signal transduction such as PI3K in the Akt pathway and Grb2 in the Ras-Raf-MEK1/2-ERK1/2 pathway (72). In our analysis, we found that hypertonic stress effectively blocks the early and major PDGF-BB-mediated activation of PDGFR-β in fibroblasts and consequently ligand-dependent activation of MEK1/2-ERK1/2 and PI3K/Akt pathways (Fig. 5), supporting the conclusion that shrinkage blocks the activation of receptor-mediated signal transduction in cell survival and growth. It is presently unknown by which mechanisms hypertonic stress affects the activity of the receptor, but our data add further evidence to the role of RTKs in sensing osmotic stress and/or in the registration of changes in cell volume that impinge upon signaling in development and tissue homeostasis. Previously, members of the EGFR family have been shown to become activated upon cell swelling by hypotonic stress in fibroblasts (17, 54), keratinocytes (32), kidney cells, and cerebellar granule neurons (41, 54), in which an increase in cell volume activates EGFRs at a level comparable with that of its natural ligands. Since we were unable to detect any changes in PDGFR-β activity in fibroblasts upon cell swelling induced by hypotonic stress in either the absence (Fig. 7, B and C) or presence of PDGF-BB (Fig. 7D), we suggest that diverse types of RTKs may respond differently to changes in cell volume and/or osmotic stress, although it is currently unknown whether ligand-dependent activation of the EGFR is affected by hypertonic stress in these cells.

Hypertonic stress has a major impact on ligand-dependent activation of PDGFR-β, and it is likely that inhibition of its downstream components in signal transduction is brought about in combination with a direct inhibition of both PDGFR-β and mechanisms downstream of the receptor. Hypertonic stress has previously been shown to reduce Akt activity by inhibiting the specific phosphorylation of Akt and/or by promoting PP2A-mediated dephosphorylation of Akt (46), but certainly inhibition of ligand-dependent receptor autophosphorylation prevents the receptor docking of PI3K and activation of the PI3K/Akt pathway as well. The docking and activation of other receptor adaptor proteins in PDGFR-β-mediated signal transduction in fibroblasts, such as Grb2, PLC-{gamma}, Src, and Shp2, may be affected in a similar manner in hypertonic cells, preventing receptor transduction. Another important observation is that hypertonic stress-induced phosphorylation of p38 is increased in PDGF-BB-stimulated cells and concomitantly with a decrease in the activation of Akt, MEK1/2, and ERK1/2, whereas p38 phosphorylation in isotonic cells is maintained at a low level in both the absence and presence of the ligand (Fig. 5). These observations indicate the presence of functional cross-talk between different MAPK pathways during osmotic stress in fibroblasts, in which inhibition of PDGFR-mediated signal transduction components, such as ERK1/2, is likely to be associated with an increase in p38 activity in fibroblasts.

The findings that signaling through RTKs are strongly affected by an increase in osmotic pressure open up a new realm of opportunities in investigating cellular signaling in development and tissue homeostasis. In this scenario, PDGFR-β may potentially function as part of a volume sensory unit involved after cell shrinkage. PDGFR-β-mediated signaling through the PI3K/Akt pathway is an important regulator of the balance between cell survival and cell death, by which Akt promotes cell survival by phosphorylating and inactivating a number of proteins involved in apoptosis (10). We have previously demonstrated that inhibition of Akt and MEK1/2-ERK1/2 by hypertonic stress in serum-stimulated fibroblasts is associated with cell death by apoptosis (18). It is therefore important to realize that an osmotic change in the extracellular milieu may act as a key player in the regulation of tissue homeostasis and in developmental processes, since deficiencies in PDGFR signaling lead to a plethora of human diseases and disorders such as neurodegenerative diseases and cancer (1, 51).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Danish Natural Sciences Research Foundation Grant 21-04-0535 (to E. K. Hoffmann and S. T. Christensen), Carlsberg Foundation Grant 0894-10 (to E. K. Hoffmann), Danish Cancer Society Grant DP05072 (to E. K. Hoffmann and S. T. Christensen), and Novo Nordic (to E. K. Hoffmann).


   ACKNOWLEDGMENTS
 
We are indebted to Birthe J. Hansen and Ecaterina Magnussen for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. K. Hoffmann, Section of Cell and Developmental Biology, Dept. of Biology, The August Krogh Bldg., Univ. of Copenhagen, 13 Universitetsparken, Copenhagen DK-2100, Denmark (e-mail: ekhoffmann{at}bio.ku.dk)

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.

* S. T. Christensen and E. K. Hoffmann contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Alvarez RH, Kantarjian HM, Cortes JE. Biology of platelet-derived growth factor and its involvement in disease. Mayo Clin Proc 81: 1241–1257, 2006.[Abstract/Free Full Text]

2. Anbari K, Schultz RM. Effect of sodium and betaine in culture media on development and relative rates of protein synthesis in preimplantation mouse embryos in vitro. Mol Reprod Dev 35: 24–28, 1993.[CrossRef][Web of Science][Medline]

3. Bildin VN, Wang Z, Iserovich P, Reinach PS. Hypertonicity-induced p38MAPK activation elicits recovery of corneal epithelial cell volume and layer integrity. J Membr Biol 193: 1–13, 2003.[CrossRef][Web of Science][Medline]

4. Bode JG, Gatsios P, Ludwig S, Rapp UR, Haussinger D, Heinrich PC, Graeve L. The mitogen-activated protein (MAP) kinase p38 and its upstream activator MAP kinase kinase 6 are involved in the activation of signal transducer and activator of transcription by hyperosmolarity. J Biol Chem 274: 30222–30227, 1999.[Abstract/Free Full Text]

5. Bortner CD, Cidlowski JA. A necessary role for cell shrinkage in apoptosis. Biochem Pharmacol 56: 1549–1559, 1998.[CrossRef][Web of Science][Medline]

6. Brunet A, Roux D, Lenormand P, Dowd S, Keyse S, Pouyssegur J. Nuclear translocation of p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J 18: 664–674, 1999.[CrossRef][Web of Science][Medline]

7. Burg MB. Response of renal inner medullary epithelial cells to osmotic stress. Comp Biochem Physiol A 133: 661–666, 2002.[CrossRef][Medline]

8. Christensen ST, Guerra C, Wada Y, Valentin T, Angeletti RH, Satir P, Hamasaki T. A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila. J Biol Chem 276: 20048–20054, 2001.[Abstract/Free Full Text]

9. Chuang SM, Wang IC, Yang JL. Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis 21: 1423–1432, 2000.[Abstract/Free Full Text]

10. Coffer PJ, Jin J, Woodgett JR. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 335: 1–13, 1998.[Web of Science][Medline]

11. Copp J, Wiley S, Ward MW, van der GP. Hypertonic shock inhibits growth factor receptor signaling, induces caspase-3 activation, and causes reversible fragmentation of the mitochondrial network. Am J Physiol Cell Physiol 288: C403–C415, 2005.[Abstract/Free Full Text]

12. De Nadal E, Alepuz PM, Posas F. Dealing with osmostress through MAP kinase activation. EMBO Rep 3: 735–740, 2002.[CrossRef][Web of Science][Medline]

13. Dmitrieva NI, Michea LF, Rocha GM, Burg MB. Cell cycle delay and apoptosis in response to osmotic stress. Comp Biochem Physiol A 130: 411–420, 2001.[CrossRef][Medline]

14. Dubois JM, Rouzaire-Dubois B. The influence of cell volume changes on tumour cell proliferation. Eur Biophys J 33: 227–232, 2004.[Web of Science][Medline]

15. Duzgun SA, Rasque H, Kito H, Azuma N, Li W, Basson MD, Gahtan V, Dudrick SJ, Sumpio BE. Mitogen-activated protein phosphorylation in endothelial cells exposed to hyperosmolar conditions. J Cell Biochem 76: 567–571, 2000.[CrossRef][Web of Science][Medline]

16. Ebner HL, Fiechtner B, Pelster B, Krumschnabel G. Extracellular signal regulated MAP-kinase signalling in osmotically stressed trout hepatocytes. Biochim Biophys Acta 1760: 941–950, 2006.[Medline]

17. Franco R, Lezama R, Ordaz B, Pasantes-Morales H. Epidermal growth factor receptor is activated by hyposmolarity and is an early signal modulating osmolyte efflux pathways in Swiss 3T3 fibroblasts. Pflügers Arch 447: 830–839, 2004.[CrossRef][Web of Science][Medline]

18. Friis MB, Friborg CR, Schneider L, Nielsen MB, Lambert IH, Christensen ST, Hoffmann EK. Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts. J Physiol 567: 427–443, 2005.[Abstract/Free Full Text]

19. Galvez AS, Ulloa JA, Chiong M, Criollo A, Eisner V, Barros LF, Lavandero S. Aldose reductase induced by hyperosmotic stress mediates cardiomyocyte apoptosis: differential effects of sorbitol and mannitol. J Biol Chem 278: 38484–38494, 2003.[Abstract/Free Full Text]

20. Gillis D, Shrode LD, Krump E, Howard CM, Rubie EA, Tibbles LA, Woodgett J, Grinstein S. Osmotic stimulation of the Na+/H+ exchanger NHE1: relationship to the activation of three MAPK pathways. J Membr Biol 181: 205–214, 2001.[Web of Science][Medline]

21. Greger R, Heitzmann D, Hug MJ, Hoffmann EK, Bleich M. The Na+2Cl–K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage. Pflügers Arch 438: 165–176, 1999.[CrossRef][Web of Science][Medline]

22. Hoffmann EK. LTD4 activates charybdotoxin-sensitive and -insensitive K+ channels channels in Ehrlich ascites tumor cells. Pflügers Arch 438: 263–268, 1999.[CrossRef][Web of Science][Medline]

23. Hoffmann EK, Dunham PB. Membrane mechanisms and intracellular signalling in cell volume regulation. Int Rev Cytol 161: 173–262, 1995.[Web of Science][Medline]

24. Hoffmann EK, Nielsen M, Christensen ST. Effects of osmotic stress on the activity of receptor tyrosine kinases and MAP kinases in HIH3T3-fibroblasts (Abstract). Mol Biol Cell 15: 14A, 2004.

25. Hoffmann EK, Pedersen SF. Sensors and signal transduction pathways in vertebrate cell volume regulation. Contrib Nephrol 152: 54–104, 2006.[Web of Science][Medline]

26. Hoffmann EK, Simonsen LO. Membrane mechanisms in volume and pH regulation in vertebrate cells. Physiol Rev 69: 315–382, 1989.[Free Full Text]

27. Hoffmann EK, Ussing HH. Membrane mechanisms in volume regulation in vertebrate cells and epithelia. In: Membrane Transport in Biology, edited by Giebisch GH, Schafer JA, Ussing HH, Kristensen P. Berlin, Germany: Springer-Verlag, 1992, p. 317–399.

28. Hohmann S. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66: 300–372, 2002.[Abstract/Free Full Text]

29. Hougaard C, Niemeyer MI, Hoffmann EK, Sepúlveda FV. K+ currents activated by leukotriene D4 or osmotic swelling in Ehrlich ascites tumor cells. Pflügers Arch 440(2): 283–294, 2000.[Web of Science][Medline]

30. House SL, Branch K, Newman G, Doetschman T, Schultz JEJ. Cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2 is mediated by the MAPK cascade. Am J Physiol Heart Circ Physiol 289: H2167–H2175, 2005.[Abstract/Free Full Text]

31. Kim RD, Darling CE, Cerwenka H, Chari RS. Hypoosmotic stress activates p38, ERK 1 and 2, and SAPK/JNK in rat hepatocytes. J Surg Res 90: 58–66, 2000.[CrossRef][Web of Science][Medline]

32. Kippenberger S, Loitsch S, Guschel M, Muller J, Kaufmann R, Bernd A. Hypotonic stress induces E-cadherin expression in cultured human keratinocytes. FEBS Lett 579: 207–214, 2005.[CrossRef][Web of Science][Medline]

33. Kodaki T, Woscholski R, Parker PJ. V-Ras activates phosphatidylinositol 3-kinase. Biochem Soc Trans 23: 195S, 1995.[Medline]

34. Krick S, Platoshyn O, Sweeney M, Kim H, Yuan JX. Activation of K+ channels induces apoptosis in vascular smooth muscle cells. Am J Physiol Cell Physiol 280: C970–C979, 2001.[Abstract/Free Full Text]

35. Lambert IH, Hoffmann EK, Christensen P. Role of prostaglandins and leukotrienes in volume regulation by Ehrlich ascites tumor cells. J Membr Biol 98: 247–256, 1987.[CrossRef][Web of Science][Medline]

36. Lang F, Busch GL, Ritter M, Völkl H, Waldegger S, Gulbins E, Häussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247–306, 1998.[Abstract/Free Full Text]

37. Lang F, Lepple-Wienhues A, Szabó I, Siemen D, Gulbins E. Cell volume in cell proliferation and apoptotic cell death. In: Cell Volume Regulation, edited by Lang F. Basel, Germany: Karger, 1998, p. 158–168.

38. Lang F, Ritter M, Gamper N, Huber S, Fillon S, Tanneur V, Lepple-Wienhues A, Szabo I, Gulbins E. Cell volume in the regulation of cell proliferation and apoptotic cell death. Cell Physiol Biochem 10: 417–428, 2000.[Web of Science][Medline]

39. Lang KS, Fillon S, Schneider D, Rammensee HG, Lang F. Stimulation of TNF alpha expression by hyperosmotic stress. Pflügers Arch 443: 798–803, 2002.[CrossRef][Web of Science][Medline]

40. Lewis A, Di Ciano C, Rotstein OD, Kapus A. Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization. Am J Physiol Cell Physiol 282: C271–C279, 2002.[Abstract/Free Full Text]

41. Lezama R, Diaz-Tellez A, Ramos-Mandujano G, Oropeza L, PasantesMorales H. Epidermal growth factor receptor is a common element in the signaling pathways activated by cell volume changes in isosmotic, hyposmotic or hyperosmotic conditions. Neurochem Res 30: 1589–1597, 2005.[CrossRef][Web of Science][Medline]

42. Liedtke CM, Cole TS. Activation of NKCC1 by hyperosmotic stress in human tracheal epithelial cells involves PKC-delta and ERK. Biochim Biophys Acta 1589: 77–88, 2002.[Medline]

43. Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97: 9487–9492, 2000.[Abstract/Free Full Text]

44. Maeno E, Shimizu T, Okada Y. Normotonic cell shrinkage induces apoptosis under extracellular low Cl conditions in human lymphoid and epithelial cells. Acta Physiol (Oxf) 187: 217–222, 2006.[CrossRef][Medline]

45. Marshall CJ. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev 4: 82–89, 1994.[CrossRef][Medline]

46. Meier R, Thelen M, Hemmings BA. Inactivation and dephosphorylation of protein kinase Balpha (PKBalpha) promoted by hyperosmotic stress. EMBO J 17: 7294–7303, 1998.[CrossRef][Web of Science][Medline]

47. Moran J, Hernandez-Pech X, Merchant-Larios H, Pasantes-Morales H. Release of taurine in apoptotic cerebellar granule neurons in culture. Pflügers Arch 439: 271–277, 2000.[CrossRef][Web of Science][Medline]

48. Nylandsted J, Gyrd-Hansen M, Danielewicz A, Fehrenbacher N, Lademann U, Hoyer-Hansen M, Weber E, Multhoff G, Rohde M, Jaattela M. Heat shock protein 70 promotes cell survival by inhibiting lysosomal membrane permeabilization. J Exp Med 200: 425–435, 2004.[Abstract/Free Full Text]

49. O'Neill WC. Physiological significance of volume-regulatory transporters. Am J Physiol Cell Physiol 276: C995–C1011, 1999.[Abstract/Free Full Text]

50. Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, Morishima S. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 532: 3–16, 2001.[Abstract/Free Full Text]

51. Ostman A. PDGF receptors-mediators of autocrine tumor growth and regulators of tumor vasculature and stroma. Cytokine Growth Factor Rev 15: 275–286, 2004.[CrossRef][Web of Science][Medline]

52. Pan Z, Capo-Aponte JE, Zhang F, Wang Z, Pokorny KS, Reinach PS. Differential dependence of regulatory volume decrease behavior in rabbit corneal epithelial cells on MAPK superfamily activation. Exp Eye Res 84: 978–990, 2007.[CrossRef][Web of Science][Medline]

53. Paruchuri S, Hallberg B, Juhas M, Larsson C, Sjölander A. Leukotriene D4 activates MAPK through a Ras-independent but PKC{varepsilon}-dependent pathway in intestinal epithelial cells. J Cell Sci 115: 1883–1893, 2002.[Abstract/Free Full Text]

54. Pasantes-Morales H, Lezama RA, Ramos-Mandujano G. Tyrosine kinases and osmolyte fluxes during hyposmotic swelling. Acta Physiol (Oxf) 187: 93–102, 2006.[CrossRef][Medline]

55. Pedersen SF, Darborg BV, Rasmussen M, Nylandsted J, Hoffmann EK. The Na+/H+ exchanger, NHE1, differentially regulates mitogen-activated protein kinase subfamilies after osmotic shrinkage in Ehrlich Lettre Ascites cells. Cell Physiol Biochem 20: 735–750, 2007.[CrossRef][Web of Science][Medline]

56. Pederson SF, Varming C, Christensen ST, Hoffmann EK. Mechanisms of activation of NHE by cell shrinkage and by calyculin A in Ehrlich ascites tumor cells. J Membr Biol 189: 67–81, 2002.[CrossRef][Web of Science][Medline]

57. Porras A, Zuluaga S, Black E, Valladares A, Alvarez AM, Ambrosino C, Benito M, Nebreda AR. P38 alpha mitogen-activated protein kinase sensitizes cells to apoptosis induced by different stimuli. Mol Biol Cell 15: 922–933, 2004.[Abstract/Free Full Text]

58. Qin S, Ding J, Takano T, Yamamura H. Involvement of receptor aggregation and reactive oxygen species in osmotic stress-induced Syk activation in B cells. Biochem Biophys Res Commun 262: 231–236, 1999.[CrossRef][Web of Science][Medline]

59. Reinehr R, Becker S, Hongen A, Haussinger D. The Src family kinase Yes triggers hyperosmotic activation of the epidermal growth factor receptor and CD95. J Biol Chem 279: 23977–23987, 2004.[Abstract/Free Full Text]

60. Reinehr R, Schliess F, Haussinger D. Hyperosmolarity and CD95L trigger CD95/EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation. FASEB J 17: 731–733, 2003.[Abstract/Free Full Text]

61. Roger F, Martin PY, Rousselot M, Favre H, Feraille E. Cell shrinkage triggers the activation of mitogen-activated protein kinases by hypertonicity in the rat kidney medullary thick ascending limb of the Henle's loop. Requirement of p38 kinase for the regulatory volume increase response. J Biol Chem 274: 34103–34110, 1999.[Abstract/Free Full Text]

62. Rouzaire-Dubois B, Malo M, Milandri JB, Dubois JM. Cell size-proliferation relationship in rat glioma cells. Glia 45: 249–257, 2004.[CrossRef][Web of Science][Medline]

63. Sadoshima J, Qiu Z, Morgan JP, Izumo S. Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling-induced ERK activation and c-fos gene expression in cardiac myocytes. EMBO J 15: 5535–5546, 1996.[Web of Science][Medline]

64. Sarkar D, Su ZZ, Lebedeva IV, Sauane M, Gopalkrishnan RV, Valerie K, Dent P, Fisher PB. mda-7 (IL-24) Mediates selective apoptosis in human melanoma cells by inducing the coordinated overexpression of the GADD family of genes by means of p38 MAPK. Proc Natl Acad Sci USA 99: 10054–10059, 2002.[Abstract/Free Full Text]

65. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 103: 211–225, 2000.[CrossRef][Web of Science][Medline]

66. Schneider L, Clement CA, Teilmann SC, Pazour GJ, Hoffmann EK, Satir P, Christensen ST. PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts. Curr Biol 15: 1861–1866, 2005.[CrossRef][Web of Science][Medline]

67. Sheikh-Hamad D, Gustin MC. MAP kinases and the adaptive response to hypertonicity: functional preservation from yeast to mammals. Am J Physiol Renal Physiol 287: F1102–F1110, 2004.[Abstract/Free Full Text]

68. Shen MR, Chou CY, Browning JA, Wilkins RJ, Ellory JC. Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease. J Physiol 537: 347–362, 2001.[Abstract/Free Full Text]

69. Shen MR, Chou CY, Hsu KF, Ellory JC. Osmotic shrinkage of human cervical cancer cells induces an extracellular Cl–-dependent nonselective cation channel, which requires p38 MAPK. J Biol Chem 277: 45776–45784, 2002.[Abstract/Free Full Text]

70. Shrode LD, Krump E, Grinstein S. Activation of protein kinases upon volume changes: role in cellular homeostasis. Contrib Nephrol 123: 79–93, 1998.[Web of Science][Medline]

71. Stutzin A, Hoffmann EK. Swelling-activated ion channels: functional regulation in cell-swelling, proliferation and apoptosis. Acta Physiol (Oxf) 187: 27–42, 2006.[CrossRef][Medline]

72. Tallquist M, Kazlauskas A. PDGF signaling in cells and mice. Cytokine Growth Factor Rev 15: 205–213, 2004.[CrossRef][Web of Science][Medline]

73. Terada Y, Inoshita S, Hanada S, Shimamura H, Kuwahara M, Ogawa W, Kasuga M, Sasaki S, Marumo F. Hyperosmolality activates Akt and regulates apoptosis in renal tubular cells. Kidney Int 60: 553–567, 2001.[CrossRef][Web of Science][Medline]

74. Uhlik MT, Abell AN, Johnson NL, Sun WY, Cuevas BD, Lobel-Rice KE, Horne EA, Dell'Acqua ML, Johnson GL. Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock. Nat Cell Biol 5: 1104–1110, 2003.[CrossRef][Web of Science][Medline]

75. Umenishi F, Schrier RW. Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene. J Biol Chem 278: 15765–15770, 2003.[Abstract/Free Full Text]

76. Wesselborg S, Bauer MKA, Vogt M, Schmitz ML, Schulze-Osthoff K. Activation of transcription factor NF-kappaB and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways. J Biol Chem 272: 12422–12429, 1997.[Abstract/Free Full Text]

77. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326–1331, 1995.[Abstract/Free Full Text]

78. Zhang H, Shi XQ, Hampong M, Blanis L, Pelech S. Stress-induced inhibition of ERK1 and ERK2 by direct interaction with p38 MAP kinase. J Biol Chem 276: 6905–6908, 2001.[Abstract/Free Full Text]

79. Zhang Z, Yang XY, Cohen DM. Hypotonicity activates transcription through ERK-dependent and -independent pathways in renal cells. Am J Physiol Cell Physiol 275: C1104–C1112, 1998.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
A. C. P. Thirone, P. Speight, M. Zulys, O. D. Rotstein, K. Szaszi, S. F. Pedersen, and A. Kapus
Hyperosmotic stress induces Rho/Rho kinase/LIM kinase-mediated cofilin phosphorylation in tubular cells: key role in the osmotically triggered F-actin response
Am J Physiol Cell Physiol, March 1, 2009; 296(3): C463 - C475.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
E. K. Hoffmann, I. H. Lambert, and S. F. Pedersen
Physiology of Cell Volume Regulation in Vertebrates
Physiol Rev, January 1, 2009; 89(1): 193 - 277.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/4/C1046    most recent
00134.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nielsen, M.-B.
Right arrow Articles by Hoffmann, E. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nielsen, M.-B.
Right arrow Articles by Hoffmann, E. K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.