From the Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80262
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ABSTRACT |
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The c-Jun NH2-terminal protein kinases (JNKs), as well as the extracellular signal-regulated protein kinases (ERKs) and p38 mitogen-activated protein kinase, are activated in renal cells in response to extracellular hypertonicity. To determine whether activation of JNKs by hypertonicity is isoform-specific, renal inner medullary collecting duct cells were stably transfected with cDNA's encoding hemagglutinin (HA)-tagged JNK1 and JNK2 isoforms, and the expressed kinases were immunoprecipitated with an anti-HA antibody. Whereas both recombinant kinases were equivalently expressed, only immunoprecipitates from the HA-JNK2 cells displayed hypertonicity-inducible JNK activity. Furthermore, expression of dominant-negative JNK2 (HA-JNK2-APF) in stable clones inhibited hypertonicity-induced JNK activation by 40-70%, whereas expression of dominant-negative JNK1 (HA-JNK1-APF) had no significant inhibitory effect. Independent HA-JNK2-APF (but not HA-JNK1-APF) clones displayed greatly reduced viability relative to neomycin controls after 16 h of exposure to 600 mosM/kg hypertonic medium with percent survival of 20.5 ± 2.7 and 31.5 ± 7.3 for two independent HA-JNK2-APF clones compared with 80.1 ± 1.0 for neomycin controls (p < 0.001, n = 5, mean ± S.E.). However, neither JNK mutant blocked either regulatory volume increase or hypertonicity-induced enhancement of uptake of inositol, an organic osmolyte putatively involved in long term adaptation to hypertonicity. These results define JNK2 as the primary hypertonicity-activated JNK isoform in IMCD-3 cells and demonstrate its central importance in cellular survival in a hypertonic environment by a mechanism independent of acute regulatory volume increase as well as regulation of organic osmolyte uptake.
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INTRODUCTION |
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The cells of the inner medulla of the mammalian nephron are uniquely exposed to large fluctuations in extracellular tonicity due to the changes that occur during diuretic and antidiuretic states. One means of adaptation to a hypertonic environment that has been well described is the intracellular accumulation of "non-perturbing" osmolytes that occurs either by uptake via sodium-coupled transporters (1) or by the generation of sorbitol through the action of aldose reductase on glucose (2). This process involves increased transcription of transporter genes (3, 4) mediated by the osmotic response element that resides in the promoter region of these genes (5). The signaling pathways that impinge upon the osmotic response element remain undefined.
Transcriptional regulation is often mediated by mitogen-activated protein (MAP)1 kinase pathways (6), which are stimulated by diverse extracellular signals including hypertonicity (7, 8). In this regard, cells of renal origin display osmotic activation of multiple members of the MAP kinase family, including the extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal kinases (JNKs), and p38 MAP kinases (9-11). However, we have recently shown that induction of osmolyte transport by hypertonicity is not significantly impacted by pharmacologic inhibition of the ERK pathways (11). The marked activation of the JNKs and more modest activation of p38 MAP kinase (11), which is the mammalian counterpart of the osmoregulated HOG-1 in yeast (12), highlights the JNK pathway as being of significance with regard to osmoregulation in the kidney. However, the existence of three distinct JNK genes with several splice variants produced from each gene hampers the clear dissection of the role of JNK in osmoregulation in eukaryotic cells, since different isoforms may play different physiological roles. For example, in small cell lung cancer cells both JNK1 and JNK2 are activated by exposure to UV light but only the JNK1 isoform appears to regulate UV-induced apoptosis (13). Additionally, whereas JNK activation has been linked to induction of apoptosis in some cell types (14), its physiological role in the cellular response to osmotic challenge has not yet been elucidated. The present study was therefore undertaken to define the JNK isoforms regulated by hypertonicity in renal inner medullary collecting duct cells and to determine whether such activation plays a role in cellular adaptation to a hypertonic environment.
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EXPERIMENTAL PROCEDURES |
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Materials-- Cell culture media and serum were from Life Technologies, Inc. Recombinant GST-c-Jun(1-79) and ATF-2NT(1-254) were expressed in Escherichia coli and purified using glutathione-agarose (Sigma) and Ni+-nitrilotriacetic acid-agarose (Qiagen, Studio City, CA), respectively, as described previously (15). Anti-JNK antisera were from Santa Cruz Biotechnology. Radioisotopes were from NEN Life Science Products. The osmolarity of all solutions used was checked with an Advanced Instruments Model 3MO Micro-Osmometer.
Cell Culture-- The established inner medullary collecting duct cell line IMCD-3 is an immortalized line generously provided by Dr. Steve Gullans (Boston, MA) (16). The cells were routinely propagated in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 nutrient mixture, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Generation of JNK1 and JNK2 Stable Transfectants--
The
HA-JNK1, HA-JNK1-APF, HA-JNK2, and HA-JNK2-APF cDNAs (17-19) were
ligated between the HindIII and HpaI sites of the
retroviral vector pLNCX (20) and packaged into replication-defective
retrovirus using 293T cells and the retrovirus component expression
plasmids SV--A-MLV and
SV-
-env
-MLV as described previously (21,
22). IMCD-3 cells were cultured for 24 h in virus-containing
conditioned medium that had been filtered through a 0.45 µ filter and
supplemented with 8 µg/ml polybrene. Positive infectants were
selected in 500 µg/ml G418, cloned, and further characterized by
Western blots using anti-HA antisera, as well as by kinase activity
assay (see below). Experiments were performed on cells that had been
passaged less than 6 times.
Assay of JNK Kinase Activity by Immunoprecipitation--
To
determine activity of transfected HA-JNK's, IMCD-3 cells in 100-mm
tissue culture dishes were incubated at 37 °C for 10 min in
DMEM:F-12 medium alone or medium supplemented with 150 mM
NaCl (600 mosM final). They were then washed three times in isosmotic phosphate-buffered saline, and lysed in 0.2-0.5 ml of lysis
buffer (50 mM -glycerophosphate (pH 7.2), 0.5% Triton
X-100, 0.1 mM sodium vanadate, 2 mM
MgCl2, 1 mM EGTA, 1 mM
dithiothreitol, 2 µg/ml leupeptin, 4 µg/ml aprotinin). The lysate
was centrifuged at 4 °C for 10 min (10,000 × g) to
remove nuclei and cell debris, and the supernatants were adjusted to
100-200 µg of protein in 0.5 ml, to which was added 5 µl of mouse
monoclonal antiserum directed against the influenza HA epitope
(Boehringer Mannheim), and 100 µl of 10% protein G-Sepharose
(Pharmacia). After 2 h of rocking incubation at 4 °C, the
immunoprecipitates were washed three times in lysis buffer and
resuspended in 40 µl of 50 mM
-glycerophosphate (pH
7.2), 0.1 mM sodium vanadate, 20 mM
MgCl2, 200 µM [
-32P]ATP
(5000 cpm/pmol), and 100 µg/ml recombinant NH2-terminal domain of ATF-2 (ATF-2NT) (6) for 20 min at 30 °C. The reaction was
stopped by the addition of SDS sample buffer, and the lysates were
heated in a boiling water bath for 5 min and subsequently subjected to
SDS-polyacrylamide gel electrophoresis on a 10% polyacrylamide gel and
followed by autoradiography. The bands corresponding to phosphorylated
ATF-2NT were excised and counted in a liquid scintillation counter.
Assay by Mono Q Fast Performance Liquid Chromatography of
Dominant-Negative JNK Inhibition of Endogenous JNK Activity--
JNK
activity was also assessed following fractionation on Mono Q fast
performance liquid chromatography as described previously (11).
Portions of cell lysates prepared as described above (0.5 ml, 1.0-1.5
mg of protein) were applied to a Pharmacia HR5/5 Mono Q anion exchange
column equilibrated in 50 mM -glycerophosphate (pH 7.2),
0.1 mM sodium vanadate, 1 mM EGTA, and 1 mM dithiothreitol, and eluted with a 30-ml gradient of
0-600 mM NaCl in the same buffer. Fractions (1 ml) were
collected, and 20-µl aliquots were mixed with 20 ml of 50 mM
-glycerophosphate (pH 7.2), 0.1 mM sodium
vanadate, 20 mM MgCl2, 200 mM
[
-32P]ATP (5,000 counts/min
1
pmol
1), 50 mg/ml IP-20 (TTYADFIASGRTGRRNAIHD), and 100 µg/ml recombinant ATF-2NT. The IP-20, a protein kinase A inhibitor,
is routinely included in all protein kinase assays. The kinase
reactions were incubated for 30 min at 30 °C and terminated with 10 µl of SDS sample buffer, and ATF-2NT phosphorylation was assessed
following SDS-polyacrylamide gel electrophoresis by liquid
scintillation counting of the excised ATF-2NT bands.
Determination of Hypertonicity-induced Cell Lethality-- Subconfluent IMCD-3 cells on 12-well tissue culture plates were treated for 16 h with 1 ml of DMEM:F-12 medium or medium to which sufficient NaCl had been added to bring final osmolality to 400, 500, or 600 mosM/kg. Supernatants were collected, and remaining adherent cells were lifted in Hank's balanced salt solution containing 5 mg/ml porcine trypsin and an appropriate amount of NaCl so that the trypsin solution was isotonic with the treatment medium. Cell suspensions were washed once with the isotonic Hank's balanced salt solution and mixed 1:1 (v/v) with 0.4% trypan blue immediately before counting on a hemocytometer.
Assay of Inositol Uptake-- Inositol uptake was measured as in Veis et al. (23). Briefly, IMCD-3 cells were treated for 16 h with inositol-free DMEM (Life Technologies, Inc.) supplemented with 1% bovine serum albumin, fraction V (U. S. Biochemical Corp.), after which 5 µl of [3H]inositol (specific activity 20 Ci/mmol) was added into 0.5 ml of medium for 2 h. The cells were then vigorously washed three times in isotonic phosphate-buffered saline and lysed in 100 µl lysis buffer. The lysate was centrifuged at 10,000 × g, and separate aliquots of the supernatant were either counted in a liquid scintillation counter or analyzed for protein concentration.
Measurement of Mean Cell Volume-- Cell volume regulation was studied by observing the mean cell volume at various times after hypertonic treatment. Approximately 10 million cells were trypsinized, resuspended in DMEM to inactivate trypsin, centrifuged for 1 min at 2,000 × g, and resuspended in 4 ml of buffer E, consisting of 10 mM HEPES (pH 7.3), 140 mM NaCl, 4 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, 1 mM KH2PO4, and 300 mM glucose and having an osmolarity of approximately 300 mosM. After 30 min of equilibration, sufficient NaCl was added to bring the osmolarity to 600 mosM/kg, and the mean cell volume of 10,000 cells per time point was measured using a Coulter Multisizer, Coulter Sample Stand II, and Multisizer AccuComp version 1.19 software, utilizing an aperture tube diameter of 100 µm. Changes in cell volume over time are expressed as relative volume by normalizing to mean cell volume measurements taken on cells before addition of hypertonic NaCl.
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RESULTS AND DISCUSSION |
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Selective Osmotic Regulation of JNK2-- To ascertain whether the activation of JNK by hypertonicity is isoform specific, we prepared IMCD-3 cell clones stably expressing HA-tagged JNK1 and JNK2. The expression of both HA-tagged JNK isoforms was confirmed by Western blot (Fig. 1A). However, whereas neither the neomycin control (devoid of HA-tagged JNK) nor HA-JNK1 was stimulated by hypertonicity, the HA-JNK2 showed a consistent activation in each of three experiments; one is shown in Fig. 1B. A similar specificity was noted for activation of HA-JNK2 and not HA-JNK1 by UV light.2
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Inhibition of JNK2 Signaling Sensitizes IMCD-3 Cells to Hypertonicity-induced Lethality-- To characterize the role of JNK2 in response to osmotic stress, we developed stably transfected IMCD-3 cell clones expressing nonphosphorylatable mutants of JNK1 (JNK1-APF) and JNK2 (JNK2-APF). In these mutants, the phosphorylation site, Thr-Pro-Tyr (TPY), is altered to Ala-Pro-Phe (APF), rendering the expressed kinase incapable of being phosphorylated, and hence incapable of being activated (18, 24). These constructs are predicted to behave as competitive inhibitors of activation of cellular JNK1 and JNK2, respectively, by competing for binding of upstream MAP kinase kinases which normally phosphorylate and thereby activate the JNKs.
Fig. 2A shows the expression of these constructs in two clones each of HA-JNK1-APF and HA-JNK2-APF.To demonstrate whether these constructs inhibit hypertonicity-stimulated JNK activity, GST-c-Jun was employed in JNK activity assays of lysates of cells exposed to hypertonicity. Utilization of this substrate measures activity of all JNK isoforms.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Rick Roman and Greg Fitz, Department of Gastroenterology, University of Colorado Health Sciences Center, for assistance with cell volume measurements.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK-19928 and GM-48826.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.
To whom correspondence should be addressed: Box C-281, University
of Colorado School of Medicine, 4200 E. 9th Ave., Denver, CO 80262. Tel.: 303-315-7204; Fax: 303-315-4852; E-mail:
Tomas.Berl{at}UCHSC.edu.
1 The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; IMCD, inner medullary collecting duct cell; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; ATF-2NT, NH2-terminal domain of ATF-2.
2 P. Wojtaszek and T. Berl, unpublished observations.
3 P. Wojtaszek, P. Squier, J. J. Cohen, and T. Berl, unpublished observations.
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REFERENCES |
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