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Cell Swelling Activates Stress-Activated Protein Kinases, p38 MAP Kinase and JNK, in Renal Epithelial A6 Cells
Biochemical and Biophysical Research Communications 266, 547–550 (1999) Article ID bbrc.1999.1843, available online at http://www.idealibrary.com on
Cell Swelling Activates Stress-Activated Protein Kinases, p38 MAP Kinase and JNK, in Renal Epithelial A6 Cells Naomi Niisato,* Martin Post,* Willy Van Driessche,† and Yoshinori Marunaka* ,1 *Lung and Cell Biology Programmes, Hospital for Sick Children, University of Toronto, Toronto, Ontario M5G 1X8, Canada; and †Laboratorium voor Fysiologie, K. U. Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium
Received November 9, 1999
Osmotic shock is well recognized as one of the factors activating stress-activated protein kinases (SAPKs), p38 MAP kinase and c-Jun N-terminal kinases (JNKs). In renal epithelial A6 cells, hypo-osmotic shock transiently activated SAPKs with maximal activation at 5 min. A6 cells showed a regulatory volume decrease (RVD) after swelling when the cells were exposed to a hypo-osmotic solution. In contrast, activation of SAPKs was maintained over 90 min after hypo-osmotic shock in the presence of 5-nitro-2-(3phenylpropylamino)benzoic acid (NPPB, a Cl 2 channel blocker), which completely blocked the RVD and kept the cells continuously swelling. Exposure of the cells to a high K 1 iso-osmotic solution containing nystatin, which induces continuous cell swelling, also continuously activated SAPKs. Furthermore, membrane deformation induced by chlorpromazine activated SAPKs. These results suggest that changes in membrane tension by cell swelling or chlorpromazine, but not osmolality, are important steps for activation of SAPKs in A6 cells. © 1999 Academic Press
Two mitogen-activated protein (MAP) kinase family members have been identified to be activated by cellular stress (chemical, heat and osmotic shock, UV radiation) and cytokines, and have therefore been termed stress-activated protein kinases or SAPKs (1, 2). SAPK1 (also termed c-Jun N-terminal kinases (JNKs)) isoforms phosphorylate and activate transcription factors such as c-Jun, ATF2 and Elk1. SAPK2 (also termed p38 MAP kinase) activates two protein kinases, termed MAP kinase-activated protein kinase 2 (MAPKAP2) and MAPKAP3. Although many reports have shown that hyperosmotic shock activates JNK and p38 MAP kinase (3– 1 To whom correspondence should be addressed at Lung Biology, Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Fax: 1 1 (Country code) 416-813-5771. E-mail: [email protected].
5), little is known about the effects of hypo-osmotic shock on activation of JNKs and p38 kinase. In intestine 407 cell (6) and cardiac myocytes (7), hypo-osmotic shock activates p38 kinase and JNKs, respectively. However, the mechanism by which hypo-osmotic shock activates these kinases is not fully understood. The present study focused on the role of cell volume in activation of SAPKs, p38 kinase and JNKs, in renal epithelial A6 cells by hypo-osmotic shock. We report here that cell swelling-induced changes in membrane tension are important for activation of p38 kinase and JNKs in renal epithelial cells under iso-osmotic and hypo-osmotic conditions. MATERIAL AND METHODS Solutions. The iso-osmotic solution (255 mOsm/kg H 2O) contained the following ion concentrations (in mM); 120 NaCl, 3.5 KCl, 1 CaCl 2, 1 MgCl 2, 5 glucose, 10 N-2-hydroxyethyl-piperazine-N-2ethanesulfonic acid (HEPES). The hypo-osmotic solution (135 mOsm/kg H 2O) contained the following ion concentrations (in mM); 55 NaCl, 3.5 KCl, 1 CaCl 2, 1 MgCl 2, 5 glucose, 10 HEPES. The iso-osmotic high K solution contained the following ion concentrations (in mM); 123.5 KCl, 1 CaCl 2, 1 MgCl 2, 5 glucose, 10 HEPES. The pH of solutions used in the present study was adjusted to 7.4 by NaOH. Bathing solutions were stirred with air. Cell culture. A6 cells were derived from Xenopus laevis distal nephron and purchased from American Type Culture Collection (ATCC). Briefly, A6 cells (passage 72– 84) were grown on plastic culture flasks in NCTC-109 medium modified for amphibian cells supplemented with 10% fetal bovine serum (osmolality 5 255 mOsm/kg H 2O) (8 –10). The flasks were kept in a humidified incubator at 27°C with 2.0% CO 2 in air. Cells were seeded onto Nunc filters at density of 5 3 10 4 cells/well and were cultured for 9 –13 days. Western blotting. A6 cells were lysed by lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl 2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM NaF, 10 mM pyrophosphate, 200 mM Na-orthovanadate, 250 mg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 100 kallikrein inactivator units/ml aprotinin, pH 7.4) after various treatments. Cells were homogenized by sonication and centrifuged at 12,000 3 g for 10 min at 4°C to remove insoluble debris. The cell lysates containing 20 mg of protein were boiled in SDS sample buffer (60 mM Tris-HCl, 2% (w/v) SDS, 5% (v/v) glycerol, pH 6.8) and then subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to nitrocellulose membranes. Nonspecific binding was blocked
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FIG. 1. Hypo-osmotic shock-induced activation of p38 MAPK. After exposure of A6 cells to hypo-osmotic solution (55 mM NaCl solution) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with antiphospho-p38 MAPK (A) and anti-p38 MAPK (B) antibodies. Molecular weights in kDa are shown at the right.
by incubation in 5% (w/v) non-fat milk in Tris-buffered saline (TBS) containing 0.1% (v/v) Tween 20 (TBST) for 60 min. Membranes were immunoblotted with anti-phospho-p38, anti-p38, anti-phospho-JNK or anti-JNK antibodies (New England BioLabs Inc, Mississauga, Ontario, CA) in 5% (w/v) bovine serum albumin in TBST at 4°C. After overnight incubation, the membrane was washed with TBST and incubated for 60 min at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (New England BioLabs Inc, Mississauga, Ontario, CA) in 5% (w/v) non-fat milk in TBST. After washing with TBST, blots were detected with LumiGLO chemiluminescent reagent (New England BioLabs Inc, Mississauga, Ontario, CA). Measurement of cell volume. We applied the same method to confluent monolayer A6 cells as previously reported (11). Cell height was used as an index for cell volume. The cell height was measured using fluorescent biotin-coated microbeads (model L-5251, Molecular Probes, Eugene, OR) which were seeded on the apical and basolateral membranes of the monolayer. Materials. Medium NCTC-109 and fetal bovine serum were purchased from GIBCO (Grand Island, NY). Nunc filters (Nunc Tissue Culture Inserts) was obtained from Nunc (Roskilde, Denmark). Chlorpromazine and nystatin was purchased from SIGMA (St. Louis, MO). NPPB was obtained from Research Biochemicals International (Natick, MA). Data presentation. All data shown in the present study are represented as means 6 SE. Where SE bars are not visible, they are smaller than the symbol.
sure of A6 cells to a hypo-osmotic solution. The phosphorylation of p38 MAP kinase returned to basal levels within 30 min. Figure 1B shows the total p38 MAP kinase, indicating that hypo-osmotic shock induces no significant changes in the total p38 MAP kinase. As reported by other investigators (5, 13), we also observed that hyposmolality transiently activated p42/ p44 MAP kinase, which is not a member of SAPKs. We focused the present study on the regulation of SAPKs, therefore we just studied the regulatory mechanism of SAPKs, p38 MAP kinase and JNKs, by changes in cell volume. Next, we examined the effect of hypo-osmotic shock on activation of p46 and p54 JNKs using an antibody specific for the Thr183/Tyr185-phosphorylated form of JNKs (14). Hypo-osmotic shock caused a transient and marked activation of JNKs, especially p46 JNK (Fig. 2A). p54 JNK was only slightly activated (Fig. 2A). Figure 2B shows the total JNKs, indicating that hypoosmotic shock induces no significant changes in the total JNKs. These results indicate that hypo-osmotic shock transiently activates both p38 MAP kinase and JNKs in renal epithelial A6 cells. NPPB Caused Sustained Activation of SAPKs To study the regulation of cell volume in monolayered A6 cells by hypo-osmotic shock, we measured time-dependent changes in cell volume when A6 cells were exposed to a hypo-osmotic solution. As De Smet et al. (15) have reported in A6 cells, changing extracellular solution from an iso-osmotic to a hypo-osmotic solution causes rapid cell swelling, followed by RVD to recover to an original cell volume (Fig. 3). This RVD is generally caused by K 1/Cl 2 release, and a blockade of either K 1 or Cl 2 channel attenuates RVD (15, 16). In renal A6 cells, NPPB (a Cl 2 channel blocker) inhibited RVD response, resulting in continued cell swelling (Fig. 3). Hypo-osmotic shock-induced activation of SAPKs was transient and the time-course of activation of
RESULTS AND DISCUSSION Hypo-osmotic Shock Activates Stress-Activated Protein Kinases (p38 Kinase and JNKs) Since hyper-osmotic shock, chemical stress, UV irradiation and cytokines can activate stress activated protein kinases (SAPKs), we asked whether hypo-osmotic shock might activate SAPKs in renal epithelial A6 cells. First, we assessed phosphorylation of p38 MAP kinase at Thr180/Tyr182 sites using an antibody specific for the Thr180/Tyr182-phosphorylated form of p38 MAP kinase (12), which is activated form of p38 MAP kinase. As shown in Fig. 1A, hypo-osmotic shock caused a rapid and transient activation in p38 MAP kinase which reached a peak around 5 min after expo-
FIG. 2. Hypo-osmotic shock-induced activation of JNK. After exposure of A6 cells to hypo-osmotic solution (55 mM NaCl solution) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phosphoJNK (A) and anti-JNK (B) antibodies. Molecular weights in kDa are shown at the right.
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FIG. 3. Effects of NPPB on hypo-osmotic shock-induced change in cell volume. The change in cell volume was measured in the presence (NPPB, a thin solid line) and absence (control, a thick solid line) of 100 mM NPPB (bilateral application). Time 0 was indicated when the extracellular solution was changed from an iso-osmotic to a hypo-osmotic solution. NPPB was applied at 215 min. In the presence of NPPB, the regulatory volume decrease was completely blocked. The dash-line shows the mean 6 SE.
SAPKs by hypo-osmotic shock was almost identical to that of cell volume change. Therefore, we assessed the relationship between activation of SAPKs and changes in cell volume in A6 cells. When we blocked RVD by addition of NPPB, activation of SAPKs was maintained over 90 min (Fig. 4). These results suggest that cell swelling induces activation of SAPKs. To confirm the hypothesis that cell swelling but not hypo-osmotic shock is essential for activation of SAPKs, we examined whether SAPKs are activated by cell swelling under iso-osmotic condition. A high K 1 iso-osmotic solution containing nystatin, which causes cell swelling (17) mimicked the action of NPPB on SAPKs (Fig. 5). These results strongly suggest that cell swelling but not hypo-osmotic shock is important for activation of SAPKs.
FIG. 4. Effects of NPPB on hypo-osmotic shock-induced p38 MAPK and JNK activation. After exposure of A6 cells to a hypoosmotic solution (55 mM NaCl solution) in the presence or absence of 100 mM NPPB (bilateral application) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phospho-p38 MAPK (A) and anti-phosphoJNK (B) antibodies. Numbers to the right indicate molecular weights in kDa.
FIG. 5. Activation of p38 MAPK and JNK by high K 1 solution containing nystatin. After exposure of A6 cells to an isotonic high K 1 solution (123.5 mM KCl solution) containing 50 mM nystatin (bilateral application) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phospho-p38 MAPK (A) and anti-phospho-JNK (B) antibodies. Numbers to the right indicate molecular weights in kDa.
Chlorpromazine-Induced Activation of SAPKs Finally, we examined whether cell swelling induces activation of SAPKs via membrane deformation. In S. cerevisiae, PKC1 activation by low osmolality is mimicked by chlorpromazine (18), a cationic amphipath which is incorporated into the inner leaflet of the plasma membrane (19). It has also been reported that chlorpromazine causes stretch of the membrane and stimulation of the stretch-activated ion channel (20). Bilateral treatment with 100 mM chlorpromazine caused a gradual activation of SAPKs even in the isoosmotic solution (Fig. 6). Although maximal phosphorylation of SAPKs was approximately 5 min by hypoosmotic shock or high K 1 solution, chlorpromazineinduced phosphorylation of SAPKs reached maximal values within 30 min. The latter may be due to a longer time for chlorpromazine to enter the cells. Based on these results, we conclude that cell swelling-induced change in membrane tension plays a crucial role in activation of SAPKs in renal epithelial A6 cells.
FIG. 6. Activation of p38 MAPK and JNK by chlorpromazine. After exposure of A6 cells to an iso-osmotic solution (120 mM NaCl solution) in the presence or absence of 100 mM chlorpromazine (CP, bilateral application) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phospho-p38 MAPK (A) and anti-phospho-JNK (B) antibodies. Numbers to the right indicate molecular weights in kDa.
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ACKNOWLEDGMENTS The authors acknowledge the assistance of Ms. E. Waelkens for executing the cell volume measurement. This work was supported by grants-in-aid from the Kidney Foundation of Canada, the Medical Research Council of Canada (25045) to Y. Marunaka, and Grant G.0235.95 of the FWO-V (Belgium) to W. Van Driessche. N. Niisato is a recipient of Research Fellowship of The Hospital for Sick Children Research Institute. Y. Marunaka is a recipient of a Scholar of the Medical Research Council of Canada and the Premier’s Research Excellence Award.
REFERENCES 1. Cuenda, A., Cohen, P., Buee-Scherrer, V., and Goedert, M. (1997) EMBO J. 16, 295–305. 2. Tan, Y., Rouse, J., Zhang, A., Cariati, S., Cohen, P., and Comb, M. J. (1996) EMBO J. 15, 4629 – 4642. 3. Zhang, Z., and Cohen, D. M. (1996) Am. J. Physiol. 271, F1234 – F1238. 4. Berl, T., Siriwardana, G., Ao, L., Butterfield, L. M., and Heasly, L. E. (1997) Am. J. Physiol. 272, F305–F311. 5. Sinning, R., Schliess, F., Kubitz, R., and Haussinger, D. (1997) FEBS Lett. 400, 163–167. 6. Tilly, B. C., Gaestel, M., Engel, K., Edixhoven, M. J., and De Jonge, H. R. (1996) FEBS Lett. 395, 133–136. 7. Sadoshima, J., Qui, Z., Morgan, J. P., and Izumo, S. (1996) EMBO J. 15, 5535–5546.
8. Niisato, N., and Marunaka, Y. (1999) J. Physiol. (Lond.) 518, 417– 432. 9. Marunaka, Y., Shintani, Y., Downey, G. P., and Niisato, N. (1997) J. Gen. Physiol. 110, 327–336. 10. Marunaka, Y., Niisato, N., and Shintani, Y. (1998) J. Membr. Biol. 161, 235–245. 11. De Smet, P., Simaels, J., Declercq, P. E., and Van Driessche, W. (1995) J. Gen. Physiol. 106, 525–542. 12. Zervos, A. S., Faccio, L., Gatto, J. P., Kyriskis, J. M., and Brent, R. (1995) Proc. Natl. Acad. Sci. USA 92, 10531–10534. 13. Tilly, B. C., Van den Berghe, N., Tertoolen, L. G. J., Edixhoven, M. J., and De Jonge, H. R. (1993) J. Biol. Chem. 268, 19919 –19922. 14. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156 –160. 15. De Smet, P., Simaels, J., and Van Driessche, W. (1995) Pflu¨gers Arch. 430, 936 –944. 16. Zhang, J. J., and Jacob, T. J. (1997) J. Physiol. (Lond.) 499, 379 –389. 17. Henson, J. H., Rosener, C. D., Gaetano, C. J., Mendola, R. J., Forrest, J. N., Jr., Holy, J., and Kleinzeller, A. (1997) J. Exp. Zool. 279, 415– 424. 18. Kamada, Y., Jung, U. S., Piotrowski, J., and Levin, D. E. (1995) Genes Dev. 9, 1559 –1571. 19. Sheetz, M. P., and Singer, S. J. (1974) Proc. Natl. Acad. Sci. USA 71, 4457– 4461. 20. Martinac, B., Adler, J., and Kung, C. (1990) Nature 348, 261–263.
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Cell Swelling Activates Stress-Activated Protein Kinases, p38 MAP Kinase and JNK, in Renal Epithelial A6 Cells Naomi Niisato,* Martin Post,* Willy Van Driessche,† and Yoshinori Marunaka* ,1 *Lung and Cell Biology Programmes, Hospital for Sick Children, University of Toronto, Toronto, Ontario M5G 1X8, Canada; and †Laboratorium voor Fysiologie, K. U. Leuven, Campus Gasthuisberg, B-3000 Leuven, Belgium
Received November 9, 1999
Osmotic shock is well recognized as one of the factors activating stress-activated protein kinases (SAPKs), p38 MAP kinase and c-Jun N-terminal kinases (JNKs). In renal epithelial A6 cells, hypo-osmotic shock transiently activated SAPKs with maximal activation at 5 min. A6 cells showed a regulatory volume decrease (RVD) after swelling when the cells were exposed to a hypo-osmotic solution. In contrast, activation of SAPKs was maintained over 90 min after hypo-osmotic shock in the presence of 5-nitro-2-(3phenylpropylamino)benzoic acid (NPPB, a Cl 2 channel blocker), which completely blocked the RVD and kept the cells continuously swelling. Exposure of the cells to a high K 1 iso-osmotic solution containing nystatin, which induces continuous cell swelling, also continuously activated SAPKs. Furthermore, membrane deformation induced by chlorpromazine activated SAPKs. These results suggest that changes in membrane tension by cell swelling or chlorpromazine, but not osmolality, are important steps for activation of SAPKs in A6 cells. © 1999 Academic Press
Two mitogen-activated protein (MAP) kinase family members have been identified to be activated by cellular stress (chemical, heat and osmotic shock, UV radiation) and cytokines, and have therefore been termed stress-activated protein kinases or SAPKs (1, 2). SAPK1 (also termed c-Jun N-terminal kinases (JNKs)) isoforms phosphorylate and activate transcription factors such as c-Jun, ATF2 and Elk1. SAPK2 (also termed p38 MAP kinase) activates two protein kinases, termed MAP kinase-activated protein kinase 2 (MAPKAP2) and MAPKAP3. Although many reports have shown that hyperosmotic shock activates JNK and p38 MAP kinase (3– 1 To whom correspondence should be addressed at Lung Biology, Hospital for Sick Children, University of Toronto, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Fax: 1 1 (Country code) 416-813-5771. E-mail: [email protected].
5), little is known about the effects of hypo-osmotic shock on activation of JNKs and p38 kinase. In intestine 407 cell (6) and cardiac myocytes (7), hypo-osmotic shock activates p38 kinase and JNKs, respectively. However, the mechanism by which hypo-osmotic shock activates these kinases is not fully understood. The present study focused on the role of cell volume in activation of SAPKs, p38 kinase and JNKs, in renal epithelial A6 cells by hypo-osmotic shock. We report here that cell swelling-induced changes in membrane tension are important for activation of p38 kinase and JNKs in renal epithelial cells under iso-osmotic and hypo-osmotic conditions. MATERIAL AND METHODS Solutions. The iso-osmotic solution (255 mOsm/kg H 2O) contained the following ion concentrations (in mM); 120 NaCl, 3.5 KCl, 1 CaCl 2, 1 MgCl 2, 5 glucose, 10 N-2-hydroxyethyl-piperazine-N-2ethanesulfonic acid (HEPES). The hypo-osmotic solution (135 mOsm/kg H 2O) contained the following ion concentrations (in mM); 55 NaCl, 3.5 KCl, 1 CaCl 2, 1 MgCl 2, 5 glucose, 10 HEPES. The iso-osmotic high K solution contained the following ion concentrations (in mM); 123.5 KCl, 1 CaCl 2, 1 MgCl 2, 5 glucose, 10 HEPES. The pH of solutions used in the present study was adjusted to 7.4 by NaOH. Bathing solutions were stirred with air. Cell culture. A6 cells were derived from Xenopus laevis distal nephron and purchased from American Type Culture Collection (ATCC). Briefly, A6 cells (passage 72– 84) were grown on plastic culture flasks in NCTC-109 medium modified for amphibian cells supplemented with 10% fetal bovine serum (osmolality 5 255 mOsm/kg H 2O) (8 –10). The flasks were kept in a humidified incubator at 27°C with 2.0% CO 2 in air. Cells were seeded onto Nunc filters at density of 5 3 10 4 cells/well and were cultured for 9 –13 days. Western blotting. A6 cells were lysed by lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM MgCl 2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM NaF, 10 mM pyrophosphate, 200 mM Na-orthovanadate, 250 mg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 100 kallikrein inactivator units/ml aprotinin, pH 7.4) after various treatments. Cells were homogenized by sonication and centrifuged at 12,000 3 g for 10 min at 4°C to remove insoluble debris. The cell lysates containing 20 mg of protein were boiled in SDS sample buffer (60 mM Tris-HCl, 2% (w/v) SDS, 5% (v/v) glycerol, pH 6.8) and then subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to nitrocellulose membranes. Nonspecific binding was blocked
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0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 266, No. 2, 1999
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FIG. 1. Hypo-osmotic shock-induced activation of p38 MAPK. After exposure of A6 cells to hypo-osmotic solution (55 mM NaCl solution) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with antiphospho-p38 MAPK (A) and anti-p38 MAPK (B) antibodies. Molecular weights in kDa are shown at the right.
by incubation in 5% (w/v) non-fat milk in Tris-buffered saline (TBS) containing 0.1% (v/v) Tween 20 (TBST) for 60 min. Membranes were immunoblotted with anti-phospho-p38, anti-p38, anti-phospho-JNK or anti-JNK antibodies (New England BioLabs Inc, Mississauga, Ontario, CA) in 5% (w/v) bovine serum albumin in TBST at 4°C. After overnight incubation, the membrane was washed with TBST and incubated for 60 min at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (New England BioLabs Inc, Mississauga, Ontario, CA) in 5% (w/v) non-fat milk in TBST. After washing with TBST, blots were detected with LumiGLO chemiluminescent reagent (New England BioLabs Inc, Mississauga, Ontario, CA). Measurement of cell volume. We applied the same method to confluent monolayer A6 cells as previously reported (11). Cell height was used as an index for cell volume. The cell height was measured using fluorescent biotin-coated microbeads (model L-5251, Molecular Probes, Eugene, OR) which were seeded on the apical and basolateral membranes of the monolayer. Materials. Medium NCTC-109 and fetal bovine serum were purchased from GIBCO (Grand Island, NY). Nunc filters (Nunc Tissue Culture Inserts) was obtained from Nunc (Roskilde, Denmark). Chlorpromazine and nystatin was purchased from SIGMA (St. Louis, MO). NPPB was obtained from Research Biochemicals International (Natick, MA). Data presentation. All data shown in the present study are represented as means 6 SE. Where SE bars are not visible, they are smaller than the symbol.
sure of A6 cells to a hypo-osmotic solution. The phosphorylation of p38 MAP kinase returned to basal levels within 30 min. Figure 1B shows the total p38 MAP kinase, indicating that hypo-osmotic shock induces no significant changes in the total p38 MAP kinase. As reported by other investigators (5, 13), we also observed that hyposmolality transiently activated p42/ p44 MAP kinase, which is not a member of SAPKs. We focused the present study on the regulation of SAPKs, therefore we just studied the regulatory mechanism of SAPKs, p38 MAP kinase and JNKs, by changes in cell volume. Next, we examined the effect of hypo-osmotic shock on activation of p46 and p54 JNKs using an antibody specific for the Thr183/Tyr185-phosphorylated form of JNKs (14). Hypo-osmotic shock caused a transient and marked activation of JNKs, especially p46 JNK (Fig. 2A). p54 JNK was only slightly activated (Fig. 2A). Figure 2B shows the total JNKs, indicating that hypoosmotic shock induces no significant changes in the total JNKs. These results indicate that hypo-osmotic shock transiently activates both p38 MAP kinase and JNKs in renal epithelial A6 cells. NPPB Caused Sustained Activation of SAPKs To study the regulation of cell volume in monolayered A6 cells by hypo-osmotic shock, we measured time-dependent changes in cell volume when A6 cells were exposed to a hypo-osmotic solution. As De Smet et al. (15) have reported in A6 cells, changing extracellular solution from an iso-osmotic to a hypo-osmotic solution causes rapid cell swelling, followed by RVD to recover to an original cell volume (Fig. 3). This RVD is generally caused by K 1/Cl 2 release, and a blockade of either K 1 or Cl 2 channel attenuates RVD (15, 16). In renal A6 cells, NPPB (a Cl 2 channel blocker) inhibited RVD response, resulting in continued cell swelling (Fig. 3). Hypo-osmotic shock-induced activation of SAPKs was transient and the time-course of activation of
RESULTS AND DISCUSSION Hypo-osmotic Shock Activates Stress-Activated Protein Kinases (p38 Kinase and JNKs) Since hyper-osmotic shock, chemical stress, UV irradiation and cytokines can activate stress activated protein kinases (SAPKs), we asked whether hypo-osmotic shock might activate SAPKs in renal epithelial A6 cells. First, we assessed phosphorylation of p38 MAP kinase at Thr180/Tyr182 sites using an antibody specific for the Thr180/Tyr182-phosphorylated form of p38 MAP kinase (12), which is activated form of p38 MAP kinase. As shown in Fig. 1A, hypo-osmotic shock caused a rapid and transient activation in p38 MAP kinase which reached a peak around 5 min after expo-
FIG. 2. Hypo-osmotic shock-induced activation of JNK. After exposure of A6 cells to hypo-osmotic solution (55 mM NaCl solution) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phosphoJNK (A) and anti-JNK (B) antibodies. Molecular weights in kDa are shown at the right.
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FIG. 3. Effects of NPPB on hypo-osmotic shock-induced change in cell volume. The change in cell volume was measured in the presence (NPPB, a thin solid line) and absence (control, a thick solid line) of 100 mM NPPB (bilateral application). Time 0 was indicated when the extracellular solution was changed from an iso-osmotic to a hypo-osmotic solution. NPPB was applied at 215 min. In the presence of NPPB, the regulatory volume decrease was completely blocked. The dash-line shows the mean 6 SE.
SAPKs by hypo-osmotic shock was almost identical to that of cell volume change. Therefore, we assessed the relationship between activation of SAPKs and changes in cell volume in A6 cells. When we blocked RVD by addition of NPPB, activation of SAPKs was maintained over 90 min (Fig. 4). These results suggest that cell swelling induces activation of SAPKs. To confirm the hypothesis that cell swelling but not hypo-osmotic shock is essential for activation of SAPKs, we examined whether SAPKs are activated by cell swelling under iso-osmotic condition. A high K 1 iso-osmotic solution containing nystatin, which causes cell swelling (17) mimicked the action of NPPB on SAPKs (Fig. 5). These results strongly suggest that cell swelling but not hypo-osmotic shock is important for activation of SAPKs.
FIG. 4. Effects of NPPB on hypo-osmotic shock-induced p38 MAPK and JNK activation. After exposure of A6 cells to a hypoosmotic solution (55 mM NaCl solution) in the presence or absence of 100 mM NPPB (bilateral application) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phospho-p38 MAPK (A) and anti-phosphoJNK (B) antibodies. Numbers to the right indicate molecular weights in kDa.
FIG. 5. Activation of p38 MAPK and JNK by high K 1 solution containing nystatin. After exposure of A6 cells to an isotonic high K 1 solution (123.5 mM KCl solution) containing 50 mM nystatin (bilateral application) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phospho-p38 MAPK (A) and anti-phospho-JNK (B) antibodies. Numbers to the right indicate molecular weights in kDa.
Chlorpromazine-Induced Activation of SAPKs Finally, we examined whether cell swelling induces activation of SAPKs via membrane deformation. In S. cerevisiae, PKC1 activation by low osmolality is mimicked by chlorpromazine (18), a cationic amphipath which is incorporated into the inner leaflet of the plasma membrane (19). It has also been reported that chlorpromazine causes stretch of the membrane and stimulation of the stretch-activated ion channel (20). Bilateral treatment with 100 mM chlorpromazine caused a gradual activation of SAPKs even in the isoosmotic solution (Fig. 6). Although maximal phosphorylation of SAPKs was approximately 5 min by hypoosmotic shock or high K 1 solution, chlorpromazineinduced phosphorylation of SAPKs reached maximal values within 30 min. The latter may be due to a longer time for chlorpromazine to enter the cells. Based on these results, we conclude that cell swelling-induced change in membrane tension plays a crucial role in activation of SAPKs in renal epithelial A6 cells.
FIG. 6. Activation of p38 MAPK and JNK by chlorpromazine. After exposure of A6 cells to an iso-osmotic solution (120 mM NaCl solution) in the presence or absence of 100 mM chlorpromazine (CP, bilateral application) for the indicated time, cell lysates (20 mg) were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-phospho-p38 MAPK (A) and anti-phospho-JNK (B) antibodies. Numbers to the right indicate molecular weights in kDa.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS The authors acknowledge the assistance of Ms. E. Waelkens for executing the cell volume measurement. This work was supported by grants-in-aid from the Kidney Foundation of Canada, the Medical Research Council of Canada (25045) to Y. Marunaka, and Grant G.0235.95 of the FWO-V (Belgium) to W. Van Driessche. N. Niisato is a recipient of Research Fellowship of The Hospital for Sick Children Research Institute. Y. Marunaka is a recipient of a Scholar of the Medical Research Council of Canada and the Premier’s Research Excellence Award.
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