Hypertonic shock inhibits growth factor receptor signaling, induces caspase-3 activation, and causes reversible fragmentation of the mitochondrial network

Jeremy Copp,1 Sandra Wiley,2 Manus W. Ward,2 and Peter van der Geer1

1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla; and 2MitoKor, San Diego, California

Submitted 17 February 2004 ; accepted in final form 23 September 2004


    ABSTRACT
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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Hyperosmotic stress can be encountered by the kidney and the skin, as well as during treatment of acute brain damage. It can lead to cell cycle arrest or apoptosis. Exactly how mammalian cells detect hyperosmolarity and how the cell chooses between cell cycle arrest or death remains to be established. It has been proposed that hyperosmolarity is detected directly by growth factor receptor protein tyrosine kinases. To investigate this, we tested whether growth factors and osmotic stress cooperate in the activation of signaling pathways. Receptors responded normally to the presence of growth factors, and we observed normal levels of GTP-bound Ras under hyperosmotic conditions. In contrast, activation of Raf, Akt, ERK1, ERK2, and c-Jun NH2-terminal kinase was strongly reduced. These observations suggest that hyperosmotic conditions block signaling directly downstream of active Ras. It is thought that apoptotic cell death due to environmental stress is initiated by cytochrome c release from the mitochondria. Visualization of cytochrome c using immunofluorescence showed that hypertonic conditions result in a breakup of the mitochondrial network, which is reestablished within 1 h after hypertonic medium is replaced with isotonic medium. When we carried out live imaging, we observed that the mitochondrial membrane potential disappeared immediately after the onset of hyperosmotic shock. Our observations provide new insights into the hypertonic stress response pathway. In addition, they show that signaling downstream of Ras and mitochondrial dynamics can easily be manipulated by the exposure of cells to hyperosmotic conditions.

protein tyrosine kinases; Ras; mitogen-activated protein kinase; hyperosmotic shock


SIGNALING DOWNSTREAM of growth factor receptor protein tyrosine kinases controls a number of important processes, including proliferation, cell survival, differentiation, and migration (33, 56). Activated growth factor receptors autophosphorylate on one or more tyrosine residues. These phosphorylation sites act to either regulate the kinase activity of the receptor or function as binding sites for cellular signaling proteins (56). It is through the interactions with these proteins that receptors turn on downstream signaling pathways.

One well-characterized signaling pathway is the Ras-MAP kinase cascade (42). Activated receptors bind directly or indirectly to the adaptor protein Grb2. Grb2 is associated with the guanine-nucleotide exchange factor Sos, which is an activator of Ras (44, 45). Activated, GTP-bound Ras recruits Raf to the inner leaflet of the plasma membrane, where Raf is activated. After its activation, Raf activates MEK, and MEK turns on ERK1 and ERK2 (42).

Raf activation is a highly complex process that is not yet completely understood. It involves several protein-protein interactions and protein phosphorylation events (12, 58). Several protein kinases have been implicated in the phosphorylation of Raf, including the p21 (Rac/Cdc42)-activated kinase (PAK), protein kinase C (PKC), and Akt. The phosphorylation of Ser338 by both PAK and PKC correlates with Raf activation.

A second important signaling pathway activated downstream of receptor protein tyrosine kinases is the phosphatidylinositol (PI) 3-kinase-Akt pathway. There is evidence that activation of PI 3-kinase is at least partly dependent on the presence of active Ras (37, 54). PI 3-kinase activation results in the production of PI 3,4,5-trisphosphate, which leads to the recruitment of Akt to the inner leaflet of the plasma membrane, where it is activated by PDK-1 and PDK-2 (1, 2). Akt promotes cell survival by phosphorylating and inactivating a number of proteins that are involved in apoptosis (17).

The cells of the mammalian kidney are frequently presented with hyperosmotic conditions due to the accumulation of both NaCl and urea (5, 6). It is known that several signaling proteins that are commonly activated downstream of growth factor receptors are also turned on by hyperosmotic stress, including phospholipase C{gamma} (PLC{gamma}), PI 3-kinase, and Shc (19, 50, 73). In addition, it was found that hyperosmotic stress turns on the Ras-MAP kinase pathway, resulting in the activation of ERK1 and ERK2 (18, 34, 63, 66). In addition to ERK1 and ERK2, stress-activated MAP kinases, including p38MAPK and c-Jun NH2-terminal kinase (JNK), are known to be activated in response to hyperosmotic stress (26, 31). It remains unclear which molecules in mammalian cells respond directly to hyperosmotic stress.

In cultured cells, hyperosmotic stress can block progression through the cell cycle (40, 47). Blocking the cell cycle is thought to provide the cells with time to adjust to the increasing osmolarity of their environment. Alternatively, hyperosmotic stress can stimulate the cells to initiate apoptosis (47, 72). Apoptosis is a cellular program that results in cell death (28). This program can be initiated by activation of death receptors such as members of the TNF receptor family (28). Alternatively, cellular stress can cause the release of cytochrome c from the mitochondria (29). Cytochrome c, together with the apoptotic protease-activating factor (Apaf) activates caspase-9, which can activate effector caspases (29). Changes in mitochondrial membrane potential are often found to be associated with apoptosis (53). Intermediate levels of osmotic stress appear to induce cell cycle arrest, whereas higher levels induce apoptosis. Exactly how the cell chooses between these two alternatives remains unresolved.

How mammalian cells sense changes in osmolarity in their environment remains poorly understood. Some studies suggest that growth factor receptors themselves are directly involved in sensing osmotic stress (15, 50, 55). Because the signaling in response to hyperosmotic shock resembles that downstream of activated growth factor receptors, we wanted to investigate whether growth factors and hyperosmotic shock can cooperate in the activation of cellular signaling pathways. Surprisingly, we found that activation of MAP kinase downstream of growth factor receptors is inhibited by hyperosmotic conditions and that this inhibition occurs in a transient manner. In addition, we found that hyperosmotic stress inhibits survival signaling downstream of activated growth factor receptors in a sustained manner and that it can lead to the activation of the cellular apoptotic machinery and mitochondrial fragmentation.


    EXPERIMENTAL PROCEDURES
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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 REFERENCES
 
Cells lines, antibodies, and other reagents. Vero cells were grown in minimal essential medium with Earle’s salts containing 10% fetal bovine serum. Monoclonal anti-phosphotyrosine antibody 4G10, anti-Met DO-24, anti-Met DL-21, anti-phospho-Raf-1 (Ser338), and a polyclonal serum against p85 were purchased from Upstate Biotechnology (Lake Placid, NY). A polyclonal serum against Raf-1 (E-10) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against phospho-p38MAPK, phospho-JNK, phospho-MEK1/2, and phospho-mTor and a monoclonal antibody against phospho-ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA). A monoclonal antibody against Ras was obtained from Transduction Laboratories (Lexington, KY). A polyclonal antibody against the carboxy-terminal six amino acids of the epidermal growth factor (EGF) receptor was a gift from Dr. Jack Kyte. The MEK1 inhibitor PD-98059 was purchased from New England Biolabs (Beverly, MA). EGF and nerve growth factor (NGF) were obtained from Amgen (Thousand Oaks, CA). Hepatocyte growth factor (HGF) was obtained from Sigma Aldrich (St. Louis, MO).

Immunoprecipitation. Cells were rinsed twice with cold PBS and lysed in 1 ml of 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 500 µM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (PLC-lysis buffer) per 10-cm tissue culture dish. Lysates were cleared by centrifugation at 10,000 rpm in a microcentrifuge at 4°C, incubated with 5 µl of polyclonal antiserum or 1 µl of monoclonal antibody for 1 h on ice, and subsequently incubated for 1 h with 100 µl of 10% protein A-Sepharose or anti-mouse IgG-Sepharose for 1 h at 4°C on an agitator. Sepharose beads were collected by centrifugation and washed four times with PLC-lysis buffer. Immunoprecipitates were boiled for 3 min in 62.5 mM Tris-Cl (pH 6.8), 10% glycerol, 5% {beta}-mercaptoethanol, 5 mM DTT, 2.3% SDS, and 0.025% bromphenol blue (SDS-sample buffer) and resolved by SDS-PAGE.

Immunoblotting. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes using a Bio-Rad semidry blotting apparatus at 50 mA per gel for 60 min at room temperature. Immunoblotting was performed per the manufacturer’s protocol. Reactive proteins were visualized using ECL (Amersham Biosciences, Piscataway, NJ).

Ras activation assay. GST-Ras-binding domain (RBD) was expressed in Escherichia coli and purified on glutathione-Sepharose beads. The beads were washed several times in Tris-buffered saline containing 1% Triton X-100 (TBST) and stored at 4°C in TBST containing 0.02% azide. For affinity precipitation, lysates were incubated with GST-RBD prebound to glutathione-agarose (~10 µl beads containing ~10–20 µg of protein) for 1 h at 4°C on an agitator. Bound proteins were resolved by SDS-PAGE on a 12% acrylamide gel and subjected to Western blot analysis with an anti-Ras monoclonal antibody.

Caspase-3 assay. Vero cells were plated in 96-well plates 24 h before the experiment. Cells were treated with EGF (1.5 nM) in the presence or absence of 150 mM NaCl added to the medium. After 6 h of incubation, the media were aspirated and the cells were incubated with PBS containing 0.03% digitonin and 23.5 µM DEVD-amc (Calbiochem, La Jolla, CA). The appearance of fluorescent cleaved substrate was followed kinetically in an FMAX 96-well fluorescent plate reader (355-nm excitation/460-nm emission). Caspase-3 activity was normalized for cell numbers after staining with 0.01 mg/ml propidium iodide and detection with a 544-nm excitation/612-nm emission filter pair.

Fluorescence microscopy. Vero cells were plated onto polylysine-coated glass coverslips 24 h before treatment. NaCl (150 mM) was added to the growth media for the times indicated. To monitor recovery, the cells were incubated with NaCl for 1 h, washed twice with normal growth medium to remove salt, and incubated with normal growth medium for the times indicated. All incubations were performed in a tissue culture incubator. The cells were then fixed with 3% paraformaldehyde at room temperature for 15 min, washed with PBS, and permeabilized and blocked for 15 min in PBS containing 0.1% Tween 20 (PBST) with 0.3% Triton X-100 and 3% BSA. Coverslips were incubated for 1 h at room temperature with the anti-cytochrome c antibody (clone 6H2.B4; BD Biosciences Pharmingen, San Diego, CA) diluted 1:400 in PBST containing 3% BSA and 2% normal goat serum in a humidified chamber. Coverslips were washed three times for 5 min with PBST at room temperature and incubated for 30 min at room temperature with goat anti-mouse-Alexa 488 (Molecular Probes, Eugene, OR) diluted 1:500 in PBST containing 3% BSA and 2% normal goat serum. Coverslips were washed as before, and nuclei were visualized by staining with Hoechst 33258 during the last wash (Molecular Probes). Coverslips were mounted onto glass slides with Airvol 205 (Air Products and Chemical, Allentown, PA) containing Dabco (Sigma Aldrich) and stored in the dark at 4°C. Immunostained cells were visualized with a Nikon Eclipse E800 microscope and a Hamamatsu camera using Simple PCI imaging software (Compix, Cranberry Township, PA).

To visualize mitochondria using fluorescently labeled adenine nucleotide translocator 3 (ANT3) or cyclophilin D (CypD), Vero cells were transiently transfected with pcDNA3-ANT3-yellow fluorescent protein (YFP) or enhanced cyan fluorescent protein (pECFP)-N1-CypD using FuGENE (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s protocol. Forty-eight hours after transfection, the cells were treated, fixed, and immunostained for cytochrome c. The samples were further processed and analyzed as described above.

Visualization of mitochondrial membrane potential in intact cells. Vero cells were incubated in 120 mM NaCl, 3.1 mM KCl, 0.4 mM KH2PO4, 5 mM NaHCO3, 1.2 mM Na2SO4, 1.3 mM CaCl2, and 20 mM HEPES (medium) containing 100 nM tetramethylrhodamine methylester (TMRM) for 30 min before being mounted in a perfusion chamber and placed on a 37°C temperature-controlled stage. TMRM (100 nM) was maintained in the medium throughout the experiment. Imaging was performed using a 75-W xenon lamp-based monochromator (Photonics, Martinsried, Germany). The emission light was detected using a charge-coupled device camera (Hamamatsu, Hamamatsu City, Japan), and data acquisition was controlled using Simple PCI imaging software. Cells were excited at 540 nm, and the emission was collected through a 610-nm/75-nm filter. Images were collected every 30 s for a period of 15 min. NaCl (30 mM) was added to the cells after 3 min, and the cells were returned to normal (120 mM) NaCl after 13 min.


    RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tyrosine phosphorylation of a 40-kDa protein in response to EGF is inhibited in the presence of hyperosmotic levels of NaCl. Polypeptide growth factors and hyperosmotic stress activate several common signaling pathways. To find out whether these stimuli act cooperatively, we examined protein tyrosine phosphorylation after stimulation of Vero cells with EGF under normal and hyperosmotic conditions. Vero cells were used in these experiments because they are an established cell line that is derived from the kidney of an African green monkey and because they naturally express receptors for EGF and HGF. Vero cells were grown to confluence, starved overnight in serum-free medium, and stimulated for 1 min with EGF immediately after different amounts of NaCl were added to the medium. Whole cell lysates were analyzed by immunoblotting with anti-phosphotyrosine (Fig. 1A). The data indicate that tyrosine phosphorylation of most proteins was unaffected by the addition of NaCl. Interestingly, the tyrosine phosphorylation of one protein with an apparent molecular mass of ~40 kDa (p40) appeared to be sensitive to the addition of NaCl. The phosphorylation of p40 decreased significantly when 100 mM NaCl was added and was abolished completely when 200 mM NaCl was added to the medium. To find out whether activation of the EGF receptor itself was affected by the addition of NaCl to the medium, we analyzed receptor immunoprecipitates by immunoblotting with anti-phosphotyrosine and anti-EGF receptor (Fig. 1, B and C). The data show that EGF receptor tyrosine phosphorylation was unaffected by the addition of NaCl. These results suggest that the presence of hyperosmotic concentrations of NaCl specifically inhibits the ability of activated EGF receptors to cause tyrosine phosphorylation of p40.



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Fig. 1. Epidermal growth factor (EGF)-induced tyrosine phosphorylation of a 40-kDa protein is inhibited by hyperosmotic concentrations of NaCl. Vero cells were left unstimulated (lane 1) or were stimulated for 1 min with EGF (lanes 2–7) in the presence or absence of increasing concentrations of NaCl added to the growth medium immediately before stimulation. Whole cell lysates were analyzed by immunoblotting with anti-phosphotyrosine (phospho-Tyr) (A). Anti-EGF receptor immunoprecipitates from the same cells were analyzed by immunoblotting with anti-phosphotyrosine (B) and anti-EGF receptor (EGFR; C). Data are representative of several independent experiments. WCL, whole cell lysates; IP, immunoprecipitation.

 
NaCl inhibits activation of ERK1 and ERK2 in response to EGF. To investigate the possibility that p40 represents either ERK1 or ERK2, we stimulated Vero cells with EGF for 1 min directly after addition of increasing amounts of NaCl to the medium. Whole cell lysates were analyzed by immunoblotting with an antibody that recognizes the activated forms of ERK1 and ERK2 (Fig. 2A). An anti-ERK2 blot was included as a control (Fig. 2B). Our results show robust activation of ERK1 and ERK2 in response to EGF. ERK activation was reduced when cells were stimulated after the addition of 100 mM NaCl to the medium and was completely inhibited after the addition of 200 mM NaCl to the medium. To prove that p40 represents either ERK1 or ERK2, we analyzed EGF-induced tyrosine phosphorylation of p40 in the presence of either NaCl or the MEK inhibitor PD-98059. Whole cell lysates were analyzed by immunoblotting with an antibody against phosphotyrosine (Fig. 2C) and with an antibody against active ERK1 and ERK2 (Fig. 2D). Stimulation in the presence of either 50 µM PD-98059 or 200 mM NaCl resulted in the complete loss of both p40 and ERK phosphorylation (Fig. 2, C and D). These results indicate that p40 represents either ERK1 or ERK2.



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Fig. 2. EGF-induced activation of ERK1 and ERK2 is inhibited in the presence of hyperosmotic concentrations of NaCl. A and B: Vero cells were left unstimulated (lane 1) or were stimulated for 1 min with EGF (lanes 2–7) in the presence or absence of increasing concentrations of NaCl added to the medium immediately before stimulation. Whole cell lysates were analyzed by immunoblotting with an antibody that recognizes active ERK1 and ERK2 (A) and an antibody that recognizes ERK2 (B). C and D: untreated Vero cells (lanes 1 and 2) or Vero cells treated with either NaCl (lanes 3 and 5) or the MEK inhibitor PD-98059 (lanes 4 and 6) were left unstimulated (lanes 1, 5, and 6) or were stimulated with EGF (lanes 2–4). Whole cell lysates were analyzed by immunoblotting with an antibody against phosphotyrosine (C) or an antibody that recognizes active ERK1 and ERK2 (D). Data are representative of several independent experiments.

 
It is well established that osmotic shock leads to the activation of stress-activated MAP kinases, including p38MAPK and JNK (26, 31). To confirm that these cells were responding normally to a hyperosmotic environment, we examined both p38MAPK and JNK activation. The results show that p38MAPK was turned on by hyperosmotic stress and that activation was maximal upon addition of 300 mM NaCl (Fig. 3A). EGF alone did not stimulate significant p38MAPK activity. Interestingly, EGF-induced activation of JNK was inhibited in the presence of hyperosmotic concentrations of NaCl (Fig. 3B). Thus the addition of NaCl to the growth medium inhibits the EGF-dependent activation of JNK as well as ERK1 and ERK2.



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Fig. 3. EGF-induced activation of JNK is inhibited in hypertonic medium. Vero cells were left unstimulated (lane 1) or were stimulated for 1 min with EGF (lanes 2–7) in the presence or absence of increasing concentrations of NaCl added to the medium immediately before stimulation. Whole cell lysates were analyzed by immunoblotting with an antibody that recognizes the active form of p38MAPK (A) and an antibody that recognizes the active form of JNK (B). Data are representative of several independent experiments.

 
Hyperosmotic concentrations of NaCl inhibit activation of ERK1 and ERK2 in response to various polypeptide growth factors. To find out whether the effects of NaCl are specific for signaling downstream of the EGF receptor, we examined ERK activation in response to either HGF or NGF. To study HGF-induced ERK activation, Vero cells were grown to confluence, starved overnight, and stimulated for 1 min with HGF directly after the addition of NaCl to the medium. Whole cell lysates were analyzed by immunoblotting with an antibody against activated ERK. Vero cells stimulated with EGF were analyzed in parallel as controls. The results show that activation of ERK1 and ERK2 in response to HGF was completely inhibited by the addition of 200 mM NaCl (Fig. 4A). Similarly, NGF-induced activation of ERK1 and ERK2 in PC-12 cells was strongly inhibited after the addition of 200 mM NaCl (Fig. 4B). Our results indicate that the presence of hyperosmotic levels of NaCl inhibits the activation of ERK1 and ERK2 in response to a variety of growth factors in both Vero and PC-12 cells.



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Fig. 4. ERK1 and ERK2 activation by various growth factors is blocked by the addition of 200 mM NaCl to the growth medium. A: Vero cells were left unstimulated in the absence (lanes 1 and 4) or presence (lane 7) of 200 mM NaCl added to the medium or were stimulated for 1 min with EGF (lanes 2 and 3) or hepatocyte growth factor (HGF; lanes 5 and 6) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 200 mM NaCl added to the medium. Whole cell lysates were analyzed by immunoblotting with an antibody that recognizes active ERK1 and ERK2. B: PC-12 cells were left unstimulated in the absence (lanes 1 and 4) or presence (lane 7) of 200 mM NaCl added to the medium or were stimulated with EGF (lanes 2 and 3) or nerve growth factor (NGF; lanes 5 and 6) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 200 mM NaCl added to the medium. Whole cell lysates were analyzed by immunoblotting with an antibody that recognizes active ERK1 and ERK2. Data shown are representative of several independent experiments.

 
Inhibition of growth factor-stimulated MAP kinase activation by sorbitol. To examine whether the reduction in growth factor-induced ERK1 and ERK2 activation is due to hyperosmolarity in general or a specific action of NaCl, we examined growth factor-stimulated MAP kinase activation in the presence of sorbitol. Vero cells were stimulated for 1 min with EGF after the addition of various amounts of sorbitol to the medium, and whole cell lysates were probed for the presence of activated ERK1 and ERK2. The results show that activation of ERK1 and ERK2 was strongly reduced after the addition of 200 mM sorbitol and almost completely absent after the addition of 300 mM sorbitol to the medium (Fig. 5). Sorbitol did not inhibit receptor activation (data not shown). Thus growth factor-induced MAP kinase activation is inhibited by hyperosmotic shock.



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Fig. 5. Growth factor-stimulated MAP kinase activation is inhibited by sorbitol. Vero cells were left unstimulated (lane 1) or were stimulated for 1 min with EGF (lanes 2–7) in the presence or absence of increasing concentrations of sorbitol added to the medium immediately before stimulation. Whole cell lysates were analyzed by immunoblotting with an antibody that recognizes active ERK1 and ERK2 (A) and an antibody that recognizes ERK2 (B). Data are representative of several independent experiments.

 
Growth factor-induced activation of Raf and MEK, but not Ras, is blocked by hyperosmotic shock. To determine which step in the activation of MAP kinase is blocked, we examined the activation of MEK, Raf, and Ras in response to EGF in the absence or presence of 200 mM NaCl added to the medium. MEK activation was evaluated by blotting whole cell lysates with an antibody that recognizes the active form of MEK. The results show that MEK remained inactive when cells were stimulated after the addition of 200 mM NaCl to the medium (Fig. 6A). Raf activation was measured by probing Raf immunoprecipitates with antibodies against active (Fig. 6B) and total Raf (Fig. 6C). The data show a lack of Raf activation when cells were stimulated after the addition of 200 mM NaCl. Active Ras was measured using a GST-fusion protein containing the RBD of Raf that binds specifically to GTP-bound Ras (20, 61) (Fig. 6D). Our results show that Ras activation was unaffected by the addition of NaCl. These data suggest that hyperosmotic shock inhibits Raf activation in the presence of active, GTP-bound Ras.



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Fig. 6. Hyperosmotic conditions inhibit growth factor-induced Raf activation. Vero cells were left untreated (lanes 1 and 4) or were stimulated for 1 min with EGF (lanes 2 and 3) or HGF (lanes 5 and 6) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 200 mM NaCl added to the medium. Whole cell lysates were analyzed by immunoblotting with anti-phospho-MEK antibody (A). Anti-Raf immunoprecipitates were analyzed by immunoblotting with anti-phospho-Raf (B) and anti-Raf antibodies (C). Raf-Ras-binding domain (RBD)-bound proteins were analyzed by immunoblotting with anti-Ras (D). Data are representative of several independent experiments.

 
Inhibition of growth factor receptor signaling to the Ras-MAP kinase pathway is transient. Our data conflict with those described in several previously published reports showing the activation of signaling pathways downstream of growth factor receptors in response to hyperosmotic stress. In many of these reports, the osmotic agent was left on the cells for at least 5 min, if not longer (15, 55, 63). To reconcile our results with those previously published, we wanted to test whether the effects we observed on signaling were transient. Vero cells were placed in media containing an additional 200 mM NaCl for the indicated times and then either left unstimulated or stimulated with EGF for 1 min. Whole cell lysates were analyzed for the presence of activated ERK1 and ERK2 (Fig. 7). The results show that during extended preincubation under hypertonic conditions, the cells recovered their ability to turn on ERK1 and ERK2. Furthermore, hypertonic stimulation for 10 or 30 min resulted in growth factor-independent ERK activation. However, the levels of MAP kinase activation seen in response to hypertonic conditions alone were significantly lower than those seen in response to growth factors. We observed similar results when these same whole cell lysates were probed with antibodies that recognize the active forms of MEK and JNK (data not shown). These results indicate that the hyperosmotic stress-induced block in growth factor-dependent MAP kinase activation is transient. Apparently, cells have the ability to adapt to the hypertonicity of their environment.



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Fig. 7. Inhibition of growth factor-dependent ERK1 and ERK2 activation is transient. Vero cells were treated with an additional 200 mM NaCl added to the medium for increasing amounts of time and were left unstimulated (lanes 1 and 6–9) or were stimulated for 1 min with EGF (lanes 2–5). Whole cell lysates were probed by immunoblotting with an antibody that recognizes active ERK1 and ERK2 (A) and an antibody that recognizes ERK2 (B). Data are representative of several independent experiments.

 
Hyperosmolarity inhibits the activation of Akt in response to EGF. To determine whether the PI 3-kinase-Akt pathway is inhibited by hyperosmolarity, we measured growth factor-induced Akt activation under normal and hyperosmotic conditions. Vero cells were stimulated for 1 min with EGF in the absence or presence of 200 mM NaCl, added immediately before stimulation, and cell lysates were analyzed by immunoblotting with an antibody that recognizes the active form of Akt (Fig. 8A). As a control, these lysates were also probed for total levels of Akt (Fig. 8B). The data show that Akt activation downstream of activated growth factor receptors was blocked after the addition of 200 mM NaCl to the medium. To determine what concentration of NaCl is required to inhibit Akt, we tested the effect of several different concentrations of NaCl on Akt activation (Fig. 8C). As with ERK1 and ERK2, we found that Akt activity was significantly inhibited in the presence of 100 mM NaCl and completely abolished with concentrations of 200 mM or higher (Fig. 8C).



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Fig. 8. Hypertonic conditions inhibit activation of Akt downstream of growth factor receptors. Vero cells were left unstimulated (lanes 1 and 4) or were stimulated for 1 min with EGF (lanes 2 and 3) in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 200 mM NaCl added to the medium. Whole cell lysates were analyzed by immunoblotting with anti-phospho-Akt (A), anti-Akt (B), and anti-phospho-mTor antibodies (D). C: Vero cells were left unstimulated (lane 1) or were stimulated for 1 min with EGF (lanes 2–7) in the presence or absence of increasing concentrations of NaCl added to the growth medium immediately before stimulation. Whole cell lysates were analyzed by immunoblotting with anti-phospho-Akt antibodies. Data are representative of several independent experiments.

 
To confirm that both Akt activation and its subsequent downstream signaling are inhibited under hypertonic conditions, we analyzed the phosphorylation of mTor, a known substrate of Akt (49). Signaling downstream of mTor also has been implicated in sensing osmotic stress (21, 51). Our results show that mTor was phosphorylated in response to EGF and that this phosphorylation was inhibited in hypertonic medium (Fig. 8D). These data show that activation of the PI 3-kinase-Akt-mTor signaling pathway is inhibited by hyperosmotic conditions.

Inhibition of survival signaling by hyperosmotic shock is sustained and leads to apoptosis. Because the inhibition of signaling to ERK1, ERK2, and JNK by hyperosmotic stress is transient, we wanted to test whether the same held true for Akt activation. Vero cells were stimulated for 1 min with EGF after preincubation under hyperosmotic conditions for various amounts of time, and whole cell lysates were analyzed by immunoblotting with an antibody that recognizes phosphorylated Akt (Fig. 9A). ERK activation was analyzed in parallel (Fig. 9B). In contrast to what we observed with ERK1 and ERK2, Akt activation was inhibited regardless of the amount of time the cells were present in hyperosmotic medium. NaCl alone did not lead to any detectable Akt phosphorylation (results not shown). Our data show that EGF cannot induce Akt activation, no matter how prolonged the exposure to hypertonic medium. ERK1 and ERK2 were activated in response to EGF after pretreatment under hypertonic conditions for 1–8 h, although not to the same degree as when the cells were stimulated in isotonic medium. These results indicate that the ability of Vero cells to adapt to the hypertonicity of their environment does not pertain to the activation of survival signaling.



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Fig. 9. Inhibition of cell survival signaling by osmotic stress occurs in a prolonged manner. Vero cells were treated with an additional 200 mM NaCl added to the growth medium for increasing amounts of time and were left unstimulated (lane 1) or were stimulated for 1 min with EGF (lanes 2–7). Whole cell lysates were normalized for total protein concentration and probed by immunoblotting with an antibody that recognizes active Akt (A) and an antibody that recognizes active ERK1 and ERK2 (B). Data are representative of several independent experiments. C: Vero cells were stimulated with EGF for 6 h in the absence or presence of 200 mM NaCl. After 6 h, caspase-3 activity was determined by measuring the cleavage of a specific substrate (DEVD-amc). Results are means of 4 independent determinations, with SD shown as error bars. The results show a significant increase in caspase-3 activity (P < 0.01).

 
Survival signaling downstream of Ras appears to be inhibited by hyperosmotic shock. Because cell viability is dependent on proper signaling through this pathway, we next investigated whether hyperosmotic shock could induce apoptotic cell death. Caspase-3 is a known mediator of apoptosis that is activated by various apoptotic signaling cascades (65). Its activation is a common marker for detecting cells that are undergoing apoptosis. Vero cells were stimulated with EGF for 6 h in normal control medium or in medium to which 200 mM NaCl had been added. The cells were then permeabilized and incubated with a fluorescence-labeled caspase-3 substrate, and the appearance of the cleaved fluorescent product was measured. Our results show that caspase-3 activation occurred when cells were stimulated under hyperosmotic conditions (Fig. 9C). These results indicate that viability is compromised when cells are maintained under hyperosmotic conditions.

Hyperosmotic conditions lead to mitochondrial fragmentation in Vero cells. Cellular stress is believed to result in the release of caspase activators, such as cytochrome c, from the mitochondria (29). Because Akt is thought to counteract this process, we tested whether hyperosmotic stress results in the release of cytochrome c from the mitochondria. Vero cells were seeded on coverslips, incubated in growth medium supplemented with 150 mM NaCl for various amounts of time, fixed, and stained with an antibody against cytochrome c. Control Vero cells showed a typical reticular cytochrome c immunostaining pattern (Fig. 10A). Surprisingly, during the incubation in medium supplemented with 150 mM NaCl, the mitochondrial network started to disintegrate. The effects were clearly visible at 5 and 10 min after the onset of hypertonic conditions, and mitochondrial fragmentation was complete within 1 h. To find out whether this response was reversible, we incubated Vero cells in medium supplemented with 150 mM NaCl, and the cells were then washed and allowed to recover for increasing amounts of time in isotonic medium. The results show that mitochondrial fragmentation in response to hyperosmotic stress was completely reversible (Fig. 10B). The network started to recover within 5 min after hypertonic medium was replaced with isotonic medium, and most cells had recovered within 1 h. These results indicate that hyperosmotic stress induces a reversible fragmentation of the mitochondrial network.



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Fig. 10. Hyperosmotic stress induces reversible mitochondrial fragmentation. A: Vero cells seeded on coverslips were incubated in medium supplemented with 150 mM NaCl for the times indicated, fixed, and stained with an antibody against cytochrome c. B: to monitor recovery, Vero cells were incubated in medium supplemented with 150 mM NaCl for 1 h and allowed to recover for the times indicated before the cells were fixed and stained with an antibody against cytochrome c. Data are representative of several independent experiments.

 
To confirm the results described above, we visualized mitochondria using fluorescently labeled versions of ANT3 and CypD, ANT-YFP, and CFP-CypD. ANT is located in the inner mitochondrial membrane, whereas CypD can be found in the mitochondrial matrix (8, 35). Vero cells transiently expressing ANT3-YFP or CFP-CypD were left untreated, were incubated for 1 h under hyperosmotic conditions, or were incubated for 1 h under hyperosmotic conditions followed by a 1-h recovery in isotonic medium. The cells were subsequently immunostained for cytochrome c and visualized using fluorescence microscopy (Fig. 11, A and B). The results show a disintegration of the mitochondrial network with all markers used. The data were quantified using cytochrome c immunofluorescence (Fig. 11C). These observations show that the mitochondrial network of nearly all Vero cells disintegrates after incubation under hyperosmotic conditions for 1 h.



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Fig. 11. Visualization of mitochondrial fragmentation using fluorescently labeled adenine nucleotide translocator (ANT) and cyclophilin D (CypD). Vero cells transiently expressing ANT3-yellow fluorescent protein (YFP) (A) or cyan fluorescent protein (CFP)-CypD (B) grown on coverslips were left untreated (control), incubated for 1 h in medium supplemented with 150 mM NaCl (salt), or incubated for 1 h in medium supplemented with 150 mM NaCl and then allowed to recover for 1 h in isotonic medium (salt/wash). The cells were subsequently fixed, stained with an antibody against cytochrome c, and visualized using fluorescence microscopy. Data are representative of several independent experiments. CytC, cytochrome c. C: mitochondrial fragmentation was quantified by counting cells with fragmented or reticular mitochondria. Three samples of each condition were analyzed, and at least 100 cells were counted in each sample. Data are means, with SD shown as error bars. Changes in the mitochondrial morphology after incubation under hypertonic conditions and after recovery in isotonic medium were significant (P < 0.01).

 
To learn more about this process, we visualized the mitochondria while NaCl was added to the cells. This was done by incubating the cells with TMRM, a fluorescent dye that is driven to accumulate inside the mitochondria by the mitochondrial membrane potential. Vero cells were pretreated with TMRM for 30 min and observed by fluorescence microscopy before, during, and after addition of NaCl to the medium. Control cells showed bright mitochondrial staining (Fig. 12A). Addition of 100 mM NaCl to the medium resulted in the immediate disappearance of mitochondrial staining, suggesting that the mitochondrial membrane potential dissipates instantaneously in response to hyperosmotic stress (results not shown). Adding as little as 30 mM NaCl to the medium caused disappearance of the membrane potential within minutes (Fig. 12, B and C). Replacing the hypertonic medium with isotonic control medium resulted in complete recovery (Fig. 12D). These data show that the mitochondrial membrane potential is exquisitely sensitive to osmotic conditions of the extracellular medium.



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Fig. 12. Reversible disappearance of the mitochondrial membrane potential in response to mild hyperosmotic stress. Vero cells were seeded on coverslips, incubated with tetramethylrhodamine methylester, and visualized using fluorescence microscopy during the experiment. Cells are shown before the addition of NaCl (A), 30 s after the 30 mM NaCl was added to the medium (B), 6 min after of NaCl was added (C), and 10 min after the hypertonic medium was replaced with isotonic control medium (D). Data are representative of several independent experiments.

 

    DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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Hyperosmotic conditions can be encountered in the kidney and the skin, as well as during certain types of medical treatment (5, 6, 24, 57, 67). It is known that these conditions can lead to cell cycle delay or apoptosis (23). Exactly how cells detect these conditions and how they decide between cell cycle delay or apoptosis remains unresolved.

A variety of signaling proteins have been implicated in the detection of hyperosmotic conditions. These include growth factor receptor protein tyrosine kinases, cytokine receptors, Shc, PI 3-kinase, PLC{gamma}, PKC, Akt, Ras, Raf, MEK, and several MAP kinase family members (15, 50, 55, 62). On the basis of these observations, it has been speculated that hyperosmotic conditions lead directly to the activation of growth factor receptors and the activation of the pathways normally turned on by these receptors. However, this appears to conflict with the fact that growth factor receptors usually stimulate cell cycle progression and promote cell survival (36). In addition, growth factor receptor activation in response to hyperosmotic shock appears to peak between 15 and 30 min after the onset of hyperosmotic conditions (55). This suggests that growth factor receptor activation may be a secondary response and that these receptors do not directly detect hyperosmolarity in the environment.

Because many of the pathways activated by growth factor receptors are also turned on by hyperosmotic stress, we wanted to know whether growth factors and hyperosmotic shock cooperate in the activation of downstream signaling pathways. In an attempt to learn something about the early events induced by hyperosmotic shock, we stimulated cells with EGF for 1 min immediately after the onset of hyperosmotic conditions. In contrast to what we expected, we observed that hyperosmotic conditions blocked growth factor-induced activation of MAP kinase. This resulted from an apparent block in the ability to turn on Raf. This conclusion is supported by experiments showing normal activation of the EGF receptor and normal levels of GTP-bound Ras but strongly reduced levels of active Raf, active MEK, and active ERK1 and ERK2 in the presence of hyperosmotic concentrations of NaCl. The block in Raf activation occurred within 1 min after the onset of hyperosmotic conditions, suggesting that the cells respond very fast to these conditions. We also observed a block in EGF-induced JNK activation, whereas NaCl-induced p38MAPK activation was normal, peaking when 300 mM NaCl was added to the medium. However, within 10 min, cells recovered their ability to activate these signaling pathways. Our observations suggest strongly that hyperosmotic conditions rapidly and transiently turn on a signaling cascade that interferes with signaling downstream of growth factor receptors.

The activation of Raf is a complex process with several key steps that include the translocation of Raf to the plasma membrane, the interaction of Raf with accessory proteins such as Sur-8, KSR (kinase suppressor of Ras), and CNK (connector enhancer of KSR), and changes in Raf phosphorylation (48, 58). Although it was initially thought that that the interaction with active Ras was both necessary and sufficient for recruitment of Raf to the membrane, recent evidence suggests that CNK may play a role in this process (64). KSR is thought to act as a scaffold for Raf, MEK, and MAP kinase and may also link Raf to upstream activators (3, 38). It is possible that one or more of these proteins is targeted by the osmotic stress-activated pathway.

Several phosphorylation sites have been reported to contribute to Raf activation, including Ser338, Tyr341, and Thr491 and Ser494 in the activation loop (16, 22, 41, 43, 71). Using an anti-serum that is specific for Raf phosphorylated on Ser338, we have shown that the phosphorylation of this site was inhibited in hyperosmotic media. We also observed that Raf normally shifts in mobility during SDS-PAGE after stimulation with EGF. This shift in mobility was blocked when cells were stimulated under hyperosmotic conditions (results not shown). Both PKC and PAK have been implicated as Ser338 kinases, and both are turned on downstream of activated growth factor receptor protein tyrosine kinases (9, 11, 32). We are currently investigating whether activation of either PKC or PAK downstream of the EGF receptor is affected by hyperosmotic shock.

We observed that, in addition to the Ras-MAP kinase pathway, EGF-dependent activation of Akt was also blocked by hyperosmotic shock. Akt is activated downstream of PI 3-kinase, and full PI 3-kinase activation results in part from an interaction with active Ras (37, 54, 68). This observation is consistent with the idea that it is indeed the activation of signaling proteins immediately downstream of Ras that is affected by hyperosmotic shock. However, the inhibition of Akt is sustained, indicating that there are key differences in how hyperosmotic shock affects these signaling pathways. Interestingly, PI 3-kinase signaling pathways have been linked to both upregulating Raf activation through Cdc42, Rac, and Pak signaling (13, 60) and downregulating Raf via Akt (75). Activation of PI 3-kinase signaling itself is regulated by binding both Cdc42 and Rac (7, 10, 74). It will be of interest to analyze growth factor-induced signaling downstream of these other small G proteins for their sensitivity to hyperosmotic shock in an effort to better understand how Raf is affected by PI 3-kinase-regulated signaling pathways.

Activation of both the Ras-MAP kinase pathway and the PI 3-kinase-Akt pathway are thought to be important for cell survival (39, 52). Consistent with sustained inhibition of Akt, we observed that increasing the osmolarity of the medium by adding 200 mM NaCl resulted in cell death by apoptosis. This finding is consistent with previous observations (27, 59).

Our observations have implications for the use of osmotic agents to control cerebral edema and to alleviate intracranial pressure caused by the swelling that often accompanies traumatic brain injury, stroke, and brain tumors (24, 57, 67). Our studies show that, along with causing apoptotic cell death, several signaling pathways are affected under hyperosmotic conditions. If growth factor receptor signaling is important for proper healing, then the use of osmotic agents like mannitol and hypertonic saline may not be the best way to promote full recovery. Our observations support earlier work suggesting that hyperosmotic treatment after brain injury leads to further loss of neurons in the damaged area (25).

In general, there are two different pathways that lead to cell death by apoptosis. Activation of death receptors causes activation of caspase-8, and activated caspase-8 relays the signal to downstream effector caspases (4). Alternatively, cellular stress results in the release of apoptotic activators from the mitochondria. These include cytochrome c, which is involved in the activation of caspase-9, an activator of caspase-3 (29). Surprisingly, when cells that were incubated under hypertonic conditions were stained for cytochrome c, we did not observe cytochrome c release. This suggests that cytochrome c release may not act as the intracellular trigger for hypertonic stress-induced apoptosis.

Instead of cytochrome c release, we observed that the hyperosmotic stress induced the disintegration of the mitochondrial reticulum and mitochondrial dysfunction. This is consistent with previous reports describing disruption of the mitochondrial membrane potential in response to osmotic stress (21, 46). We have demonstrated that this process happens almost immediately and that it is exquisitely sensitive to changes in osmolarity, occurring in as little as 30 mM NaCl.

It is known that mitochondria form a highly dynamic network that is constantly subject to fusion and fission events (69, 70). This dynamic behavior is thought to be important for distribution of mitochondria to daughter cells during cell division, provision of ATP to all parts of the cell, and maintenance of the mitochondrial genome (69, 70). In addition, mitochondrial fusion is essential for spermatogenesis in Drosophila (30). Several dynamin-like and non-dynamin-like GTPases that are involved in mitochondrial fission and fusion, respectively, have been identified (69, 70). However, exactly how this process is regulated remains unresolved. We found that incubation of cells for 1 h in medium supplemented with 150 mM NaCl resulted in mitochondrial fragmentation. The mitochondrial morphology under hypertonic conditions was very similar to that observed in cells lacking mitofusin 1 or mitofusin 2 (14). This suggests that the disintegration of the mitochondrial network in response to hyperosmotic shock may be caused by inhibition of mitofusins. We observed that the cells were able to reestablish a normal-looking mitochondrial network within 1 h after being returned to isotonic medium. This shows that mitochondrial fusion is a relatively fast process. Finally, we observed that hypertonic conditions caused an immediate dissipation of the mitochondrial membrane potential. This suggests that the disintegration of the mitochondrial network could be secondary to the disruption of chemiosmotic energy production and that the mitochondrial membrane potential is exquisitely sensitive to the osmolarity of the extracellular milieu.

In conclusion, we have shown that hyperosmotic conditions, which can be encountered in a variety of situations, block signaling by growth factor receptor protein tyrosine kinases immediately downstream of Ras. This can now be used as a tool for further analysis of both Raf and Akt activation that occurs downstream of Ras. We also have shown that the mitochondrial membrane potential can be turned off quickly and reversibly by hyperosmotic conditions. Finally, our results show that mitochondrial fusion is a rapid process that can be manipulated by the addition of hyperosmotic concentrations of NaCl to the medium.


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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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This research was supported in part by National Cancer Institute Grant 2R01 CA-78629. J. Copp was supported by National Institutes of Health Training Grants T32 CA-09523 and 5T 329M07240.


    ACKNOWLEDGMENTS
 
Present addresses: J. Copp, Molecular and Cell Biology Laboratory, Salk Institute, La Jolla, CA 92037; S. Wiley, Dept. of Pharmacology, Univ. of California, San Diego, La Jolla, CA 92037.


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. van der Geer, Dept. of Chemistry and Biochemistry, Univ. of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0359 (E-mail: geer{at}ucsd.edu)

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.


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