Divisions of Nephrology and Molecular Medicine, Oregon Health Sciences University and Portland Veterans Affairs Medical Center, Portland, Oregon 97201
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ABSTRACT |
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Acute hypotonic shock (50% dilution of medium with sterile water, but not with isotonic NaCl) activated the extracellular signal response kinase (ERK) mitogen-activated protein (MAP) kinases in renal medullary cells, as measured by Western analysis with a phospho-ERK-specific antibody and by in vitro kinase assay of epitope-tagged ERKs immunoprecipitated from stable HA-ERK transfectants. Hypotonicity also activated the transcription factor and ERK substrate Elk-1 in a partially PD-98059-sensitive fashion, as assessed by chimeric reporter gene assay. Consistent with these data, hypotonic stress activated transcription of the immediate-early gene transcription factor Egr-1 in a partially PD-98059-sensitive fashion. Hypotonicity-inducible Egr-1 transcription was mediated in part through 5'-flanking regions containing serum response elements and in part through the minimal Egr-1 promoter. Elimination of the Ets motifs adjacent to key regulatory serum response elements in the Egr-1 promoter diminished the effect of hypotonicity but failed to abolish it. Interestingly, hypotonicity also transiently activated p38 and c-Jun NH2-terminal kinase 1, as determined by immunoblotting with anti-phospho-MAP kinase antibodies. Taken together, these data strongly suggest that hypotonicity activates immediate-early gene transcription in renal medullary cells via MAP kinase kinase-dependent and -independent mechanisms.
urea; kidney; signal transduction; p38; stress-activated protein kinase; c-Jun amino-terminal kinase
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INTRODUCTION |
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ALTHOUGH UBIQUITOUS among prokaryotes and simple
eukaryotes, exposure to hypotonicity in higher eukaryotes is generally
limited to relatively few epithelia in the absence of systemic
disturbances in water balance. In response to water loading or
diuresis, cells of the renal medulla encounter a markedly hypotonic
milieu (20). In addition, the total solute concentration of a markedly
hypertonic renal medullary interstitium, achievable in the face of avid
water conservation and comprising a total solute burden >2,000
mosM, may fall by >50% in <1 h after diuresis (5). In
response to such physiological anisotonicity, cell volume is acutely
regulated through rapid influx or efflux of inorganic ions (e.g.,
K+ and
Cl). Thereafter,
accumulation or dumping of osmotically active organic solutes called
osmolytes ensues (20). In aggregate, these sequential mechanisms
achieve the regulatory volume decrease (RVD) essential for maintenance
of cell integrity in response to hypotonicity.
The molecular mechanisms underlying RVD have received increasing
attention; inorganic ion and organic solute efflux pathways have been
characterized. Swelling-responsive inorganic ion currents have been
observed in diverse systems from prokaryotes to mammalian cells
(reviewed in Ref. 8). Among renal epithelia,
Cl and cation currents have
been detected in cells of the proximal tubule (62), cortical collecting
duct (48, 61), and inner medullary collecting duct (IMCD) (31, 60) in
response to hypotonicity. Stretch-activated cation and anion channels,
implicated in RVD, have also been described in renal epithelia
(reviewed in Ref. 44). With respect to organic solutes,
hypotonicity-inducible efflux of sorbitol (18, 21, 39) and inositol
(26, 27, 33, 50, 51) has been extensively characterized in cultured cells of the renal medulla and brain, respectively. The signaling events leading to activation of inorganic ion and organic solute efflux
pathways remain obscure. Prior investigations have broadly implicated
phosphorylation events (17, 37, 41, 55, 57) and a mitogen-activated
protein kinase (MAPK) (57).
MAPKs transduce cellular signals (such as those perceived at the cell membrane in response to stressors and peptide mitogens) into nuclear transactivation events. Members of the extracellular signal response kinase (ERK) family of MAPKs are phosphorylated and activated by MAPK or ERK kinases (MEKs); on activation, ERKs phosphorylate and activate the transcription factor Elk-1, among other substrates (24). Activated Elk-1 interacts with the serum response factor (SRF) to mediate transcription through serum response elements (SREs) and adjacent Ets motif sequences contained within the 5'-flanking region of several genes (reviewed in Ref. 24), including the immediate-early gene (IEG) Egr-1 (59). Hypertonic stress inducible by NaCl activates ERKs (25, 54) but does not appear to substantially affect Egr-1 transcription or translation (13). Elevated concentrations of the cell-permeant solute urea, in contrast, activate ERKs and Elk-1 (10) and markedly enhance IEG transcription (11, 13). The effect of hypotonic stress and concomitant cell swelling on this key osmoregulatory signal transduction pathway remains unexplored.
It is shown here that, in cultured cells of the murine renal IMCD, mIMCD3, hypotonic stress induced phosphorylation and activation of ERK MAPKs. ERK activation conferred activation of the transcription factor and ERK substrate Elk-1. Hypotonicity also induced transcription of the ERK-responsive IEG Egr-1 in a partially MEK-dependent fashion. A MEK-independent and less specific pathway of transcriptional activation was also upregulated by hypotonic stress and affected multiple minimal promoters, including those of Egr-1 and thymidine kinase (TK).
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METHODS |
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Cell culture and hypotonic or urea treatment. mIMCD3 cells were maintained in DMEM-F-12 medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS (JRH, Lenexa, KS), as described previously (42). Cells were growth suppressed in DMEM-F-12 without serum for 24 h before treatment with medium supplemented with sterile water or urea. Unless indicated, hypotonic treatment represented a 50% final dilution achieved by the removal of 5 ml of medium from a 10-ml dish and replacement with 5 ml of sterile tissue-culture water. For all experiments, controls were treated with comparable medium dilution with sterile 150 mM NaCl; in no case was such sham treatment different from control. When used, urea was added to a final concentration of 200 mosM. When used, inhibitor pretreatment was performed for 30 min; inhibitors then remained in the culture medium for the duration of the experiment. PD-98059 (Calbiochem) stock solution was prepared in DMSO; DMSO served as vehicle control.
Epitope-tagged ERKs, stable transfection, and in vitro kinase assay. HA-ERK1 was prepared as previously described (10) from cDNA encoding human ERK1 (kindly provided by D. Charest and S. L. Pelech); HA-ERK2 was provided by M. Weber. Stable transfectants were prepared as described previously (10), and activation of each ERK isoform was quantitated via in vitro kinase assay, using myelin basic protein as a substrate, through a modification of published protocols (Ref. 10 and references therein).
Western analysis. Detergent lysates were prepared, subjected to electrophoresis, and transferred to polyvinylidinedifluoride, as previously described (12). Equal amounts of protein [in µg; quantitated via the DC assay (Bio-Rad) according to the manufacturer's directions] were loaded per gel lane. All incubations were performed at 25°C with gentle rocking. Membranes were blocked with 5% nonfat dry milk (NFDM) in PBS-0.1% Tween for 1.5 h and washed three times with PBS-0.1% Tween-1% NFDM; incubated with a 1:500 dilution of anti-phospho-ERK (New England BioLabs), anti-ERK (Zymed Laboratories, S. San Francisco, CA), anti-phospho-p38 (New England BioLabs), anti-phospho-stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK; New England BioLabs), anti-p38 (Santa Cruz), or anti-JNK (Santa Cruz) in PBS-1% NFDM for 1 h and washed three times with PBS-0.1% Tween-1% NFDM; and incubated with a 1:4,000 dilution of the horseradish peroxidase-coupled appropriate secondary antibody (Pierce, Rockford, IL) in PBS-0.1% Tween-1% NFDM and washed three times with PBS-0.3% Tween-1% NFDM. Detection was via enhanced chemiluminescence according to the manufacturer's directions (Amersham, Arlington Heights, IL).
Transient transfection and reporter gene analysis.
mIMCD3 cells were transiently transfected via electroporation (13);
luciferase and -galactosidase activity were monitored as previously
described (13). Values are means ± SE. The construction of
Egr-1-Luc (632-Luc), plasmids TK13, TK14, TK23, and TK24 (see Fig. 5),
and plasmids B, C, D, E, and G (see Fig. 4) has been previously
described (13). Elk-1/GAL4 chimera and 5XGAL4-Luc were kindly provided
by R. Maurer (Oregon Health Sciences University, Portland, OR).
Cell volume assay. Cell volume was determined by a modification of published methods (29). Monolayers in 12-well dishes were washed with equilibration buffer (in mM: 122 NaCl, 3.3 KCl, 0.4 MgSO4, 1.3 CaCl2, 1.2 potassium phosphate, 10 glucose, and 10 HEPES, pH 7.4) and equilibrated with this medium (0.5 ml/well) for 30 min. The indicated osmotic stressor was then added, and cells were pulsed with 14C-labeled 3-oxomethyl-D-glucose (0.3 µCi/ml; DuPont-NEN) for 10 min commencing 10 min before the completion of the timed interval of osmotic stress. At the conclusion of the interval of interest (i.e., 30 min), dishes were placed on ice and monolayers were washed twice with ice-cold stop buffer [in mM: 290 sucrose, 10 Tris · NO3, 0.5 Ca(NO3)2, and 0.2 phloretin, pH 7.4]. After the final wash, cells were solubilized in 1 ml of 1 M NaOH and incubated for 15 min at 25°C before they were counted in liquid scintillant. In this and other assays, statistical significance was assigned to P < 0.05 with respect to the indicated control (e.g., vehicle treatment) of data from three or more independent experiments compared via t-test (Excel, Microsoft).
Cell viability assay. mIMCD3 monolayers (in 24-well dishes and maintained in serum-free medium for 24 h) were treated for the indicated times with hypotonic stress or sham treatment (medium dilution with 150 mM NaCl). Cells were washed with ice-cold PBS and lysed in situ with 500 µl of 0.5% Triton X-100. Plates were spun at 1,000 g for 10 min at 4°C. ATP content in 5 µl of lysate supernatant was quantitated using the ATP determination kit (Molecular Probes, Eugene, OR) by addition to 200 µl of complete assay buffer containing 1× reaction buffer [in mM: 25 tricine (pH 7.8), 5 MgSO4, 0.1 EDTA, 0.1 sodium azide], 1.5 mM dithiothreitol, and 1.875 µg/ml luciferase. Reaction was initiated by injection of 100 µl of luciferin stock (0.5 mM luciferin in 1× reaction buffer) in a Berthold Lumat LB-9501 luminometer. Data were normalized to protein content per well, as determined by the DC protein assay (Bio-Rad) according to the manufacturer's directions.
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RESULTS |
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Hypotonic stress activates ERK without influencing ERK abundance. The ability of hypotonicity to induce phosphorylation of the ERK family of MAPKs was evaluated. Detergent lysates prepared from control and hypotonically stressed mIMCD3 cells were subjected to Western analysis with a commercially available antibody (New England BioLabs) recognizing only the phosphorylated (active) form of ERK1 and ERK2. In control experiments, phosphorylation of ERK1 and ERK2 was promptly upregulated by the known ERK activator urea (10) in this cell line (Fig. 1). Hypotonic stress (50% medium dilution) also resulted in a pronounced upregulation of ERK1 and ERK2 phosphorylation at 5 min of treatment, then phosphorylation promptly returned to near-basal levels (Fig. 1).
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Hypotonicity-inducible ERK activation is abrogated by the MEK inhibitor PD-98059. To elucidate the role of ERK signaling in hypotonic stress-inducible gene transcription (see below), the ability of the known MEK inhibitor PD-98059 (1) to block hypotonicity-associated ERK activation was investigated. Cells were pretreated with PD-98059 (50 µM) before hypotonic stress or urea stress. The compound blocked >95% of hypotonic stress-associated activation of ERK1 and ERK2 (Fig. 3). Therefore, at 50 µM, PD-98059 is an effective inhibitor of hypotonicity-inducible ERK activation.
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Hypotonic stress activates the Elk-1 transcription factor, a substrate of ERK, in an ERK-dependent fashion. Because activated ERK can activate the transcription factor Elk-1, the ability of hypotonic stress to activate Elk-1 was examined in a chimeric reporter gene assay (43). Cells were cotransfected with a luciferase reporter gene driven by tandem repeats of the yeast GAL4 consensus sequence as well as with an expression vector encoding the activation domain of Elk-1 linked to the DNA-binding domain of GAL4. When transfected cells were treated with water (Fig. 4A), fivefold upregulation of reporter gene activity, and hence Elk-1 activity, was observed. There was no effect of hypotonic stress on luciferase expression from pXP2 vector alone (data not shown). To ascertain the dependence of this phenomenon on ERK activation, cells were pretreated with PD-98059. PD-98059 (50 µM) had no effect on basal Elk-1 activity and inhibited the hypotonicity-inducible increment in Elk-1-dependent transcription by 62% (Fig. 4A).
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Hypotonicity-inducible transcription of the IEG Egr-1 is partially sensitive to PD-98059. Because the IEG transcription factor Egr-1 is transcriptionally activated by ERKs in other contexts, the ability of hypotonic stress to activate transcription of this gene was evaluated in transient transfection with a luciferase reporter gene linked to 1.2 kb of the murine Egr-1 5'-flanking sequence, as previously described (13). Hypotonic stress (Fig. 4B) activated Egr-1 transcription approximately fourfold. Pretreatment with PD-98059 (50 µM for 30 min) had no effect on basal Egr-1 transcription but inhibited hypotonicity-inducible Egr-1 transcription by 72% (Fig. 4B).
Promoter elements conferring hypotonicity responsiveness to the Egr-1 gene. To determine which, if any, of the previously described promoter elements in the murine Egr-1 5'-flanking sequence present in construct Egr-1-Luc (59) was responsible for conferring hypotonicity responsiveness, a series of deletion mutants was examined in the presence and absence of hypotonic treatment (Fig. 5). These mutants (Fig. 5, left) were designed to include or exclude two putative activator protein-1 (AP-1) sites and five SREs. With the entire 1.2 kb of the murine 5'-flanking sequence intact (Egr-1-Luc, construct A), hypotonicity-inducible reporter gene activity was increased sevenfold relative to control (Fig. 5, middle). In the absence of SRE/Ets motifs (but in the presence of the AP-1 sites, construct B), induction by water was significantly decreased (4.5-fold; Fig. 5, right), and basal activity was markedly diminished (Fig. 5, middle) relative to the full-length promoter. This degree of water inducibility was significantly less than that conferred by any of the other SRE/Ets-containing deletion mutants (constructs C, D, and E; Fig. 5, right). Consistent with these data, degree of induction by water in the Egr-1 minimal promoter (construct G) was similar to that of the SRE-deleted promoter (construct B). Reporter gene expression from the promoterless pXP2 vector alone (V) was unchanged after hypotonic treatment (Fig. 5, right). In summary, the absence of SRE/Ets motifs from an Egr-1 promoter mutant (Fig. 5, left) dramatically diminished basal reporter gene activity (Fig. 5, middle) and significantly diminished water inducibility (Fig. 5, right).
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Importance of Ets motifs, the Elk-1 binding sequence. Because the SREs appeared to be important for achieving maximal Egr-1 transcription after hypotonic treatment and because ERK and Elk-1 activation were shown to comprise part of the signaling repertoire in response to hypotonic stress, the ability of SREs and adjacent Ets motifs (which interact with activated Elk-1) to support hypotonic stress-inducible transcription was examined. The region in the immediate vicinity of SRE3 and SRE4 in the murine Egr-1 promoter [which were previously shown to confer a substantial portion of inducibility in other contexts (13, 32)] was PCR amplified to include or exclude each of the two adjacent clusters of Ets motifs (13). The resultant PCR fragments, named for the numbered primers used for their amplification (as well as the primers themselves), are depicted diagrammatically in Fig. 6, left. PCR fragments were subcloned upstream of the (heterologous) TK promoter in the enhancerless TK Luc vector [also called PT109 (40)]. In the absence of Egr-1 promoter elements (TK Luc), basal (unstimulated) reporter gene activity was relatively low, but a 2.7-fold induction by hypotonicity was observed (Fig. 6, right). In the presence of SREs alone (TK23), total reporter gene activity was substantially increased, and induction with hypotonic treatment rose to fourfold, although this did not achieve statistical significance with respect to TK Luc. When either of the two adjacent clusters of Ets motifs was included (TK13 or TK24), total reporter gene activity was again substantially increased, and induction with hypotonic treatment was significantly greater (between 5- and 6-fold). Finally, if both Ets clusters were included (TK14), basal reporter gene activity was increased further, and induction with hypotonic treatment was still greater (7-fold). In summary, the presence of adjacent clusters of Ets motifs significantly enhances water inducibility of an SRE-containing Egr-1 promoter fragment, in rough proportion to the number of clusters retained.
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Hypotonic stress activates other MAPK family members. To determine whether the ability of hypotonic stress to activate MAPKs was unique to members of the ERK family, detergent lysates prepared from control-treated and hypotonically stressed mIMCD3 cells were subjected to immunoblot analysis with antibodies specifically recognizing the phosphorylated (activated) forms of the p38 (Fig. 7, top) and SAPK/JNK (Fig. 7, bottom) stress-activated MAPKs. To validate the sensitivity of the antibodies, detergent lysates were prepared from mIMCD3 cells subjected to a treatment known to activate p38 and SAPK/JNK in this model [hypertonic stress (100 mM supplemental NaCl) (66)]. Hypertonic NaCl markedly activated p38 at 5 and 30 min of treatment (Fig. 7, top). Hypotonic stress, in contrast, modestly activated p38, and only at the earliest time point examined (5 min). In addition, hypertonic NaCl treatment resulted in the appearance of multiple molecular species recognized by the anti-phospho-SAPK/JNK antibody (Fig. 7, bottom). Three principal bands corresponding to (logarithmically interpolated) molecular masses of 41, 45, and 53 kDa were detected. The latter two bands appeared to correspond to JNK1 (46 kDa) and JNK2 (54 kDa), respectively, and this was confirmed with anti-JNK immunoblotting (data not shown). The 41-kDa band (and the faint 39.5-kDa band seen below it in several experiments) may represent cross-reactivity with phosphorylated p38, phosphorylated ERK, or another SAPK/JNK family member. No nonspecific bands were evident in control-treated cells (Fig. 7, bottom). Hypotonic stress activated at least three species, including JNK1 and those of ~39.5 and 41 kDa but exhibited no effect on the JNK2. In addition, as observed with ERK and p38, the effect was confined to the 5-min time point and, unlike NaCl treatment, was not observed thereafter.
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Hypotonic stress increases mIMCD3 cell volume. To examine a possible role for ERK-dependent pathways in cell volume regulation, the effect of PD-98059 on RVD after hypotonic stress was examined. Acute hypotonic stress induced by 50% medium dilution with water increased cell volume by ~100% relative to baseline at 10 min of treatment (data not shown). By 30 min of treatment, cell volume was ~50% greater than baseline. Therefore, the 30-min time point was examined to determine the effect of PD-98059 on the RVD occurring between 10 and 30 min. There was no significant difference between the effect of PD-98059 on cell volume in hypotonically stressed cells with respect to its effect on control-treated cells (data not shown). Hypotonic treatment increased cell volume by 56% at 30 min of treatment (Fig. 8A). Pretreatment with PD-98059 decreased basal cell volume by 10% and decreased cell volume in the presence of 30 min of hypotonic stress by 15% (Fig. 8A).
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Hypotonic stress decreases mIMCD3 cell viability.
Cell injury after different degrees and durations of hypotonic stress
was evaluated through determination of ATP content (2) and expressed
relative to total protein content. The data obtained through the use of
this assay paralleled those of the propidium iodide exclusion assay
previously employed (65); however, they were unaffected by the
potentially confounding fluorescence of inhibitory compounds and
vehicles. Medium dilution of 50% at 6 and 24 h of treatment failed to
appreciably alter cellular ATP content, whereas dilutions of 90%
resulted in marked loss of cell viability (data not shown). An
intermediate dilution of 75% was therefore examined at 24 h of
treatment to permit detection of protection from and greater
sensitivity to hypotonic stress in the presence of PD-98059. Hypotonic
stress (75% medium dilution) decreased ATP content by 56% (Fig.
8B); pretreatment with PD-98059 (100 µM for 30 min) failed to influence cellular ATP content under control
or hypotonic conditions.
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DISCUSSION |
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The present data indicate that, in cultured cells of the renal medulla, hypotonic stress activates the ERK MAPKs. ERK activation is further associated with activation of the ERK substrate and transcription factor Elk-1, as determined by reporter gene assay, and these events mediate, in part, the ability of hypotonic stress to activate transcription of a reporter construct driven by the promoter of the IEG Egr-1. Consistent with these data, hypotonic stress also increased Egr-1 mRNA abundance as well as Egr-1 protein abundance and trans-activating ability (65).
Evidence for the involvement of an ERK-independent signaling pathway in hypotonic stress-inducible Egr-1 transcription in this model includes 1) the (modest) effect of hypotonic treatment on enhancerless (minimal) promoters such as those of Egr-1 and TK, 2) the inability of PD-98059 to completely abolish the effect of hypotonicity on Egr-1 and Elk-1 activation, 3) the inability of gross deletions of SRE/Ets motifs to completely abolish hypotonicity-inducible transcription from the Egr-1 5'-flanking sequence, and 4) the inability of the absence of Ets motifs to completely abolish hypotonicity-inducible, SRE-mediated transcription. The nature of the signaling events responsible for this more generalizable transcriptional response is under investigation. It is conceivable that the change in intracellular and, therefore, intranuclear ionic strength engendered by hypotonic stress could directly alter protein-protein interactions among any of the host of basal transcription factors and thereby globally influence gene transcription. The presence of relative promoter selectivity (TK and Egr-1 > cytomegalovirus immediate early enhancer/promoter) suggests that the mechanism is not universal; a broader examination of minimal promoters is in progress.
Evidence for the involvement of an ERK-dependent pathway in hypotonic stress signaling to IEG transcription is provided by 1) the ability of hypotonic treatment to activate ERK and its substrate Elk-1, 2) the ability of the specific MEK inhibitor PD-98059 to partially and completely inhibit hypotonicity-inducible Egr-1 transcription and Elk-1 activation, respectively, 3) the diminution in hypotonicity responsiveness associated with deletion of SRE/Ets motifs from the native Egr-1 promoter, and 4) the diminution in hypotonicity responsiveness of isolated SREs in the absence of associated natively occurring adjacent Ets motifs, with which Elk-1 would be expected to interact.
A role for each of the three principal MAPK signaling pathways has been described in the setting of anisotonicity. In addition to being activated by hypotonic stress in the present study, the ERKs are activated by hypertonic stress in the renal epithelial Madin-Darby canine kidney cell line (25, 54) and by elevated urea concentrations in mIMCD3 cells (10). While the present data were in preparation, swelling-associated ERK activation was described in cells of gastrointestinal and cardiac origin (45, 47). The mammalian MAPK p38 is the higher eukaryotic homolog of the yeast osmoresponsive MAPK HOG1. Hypertonicity activates HOG1 in yeast (7) and p38 in mammalian cells (16, 23), including those derived from the renal medulla (6, 66). SAPK/JNK is also activated by hypertonic stress in mammalian cells in culture (19), including renal epithelial cells (6, 66). Interestingly, neither p38 nor SAPK/JNK is appreciably activated by hyperosmotic urea in renal medullary cells, as measured by immune-complex kinase assay (66). Because hyper- and hypotonicity activate ERKs, specificity must reside in modulation of downstream signaling events by additional signals that, in aggregate, result in acquisition of the dissimilar phenotypes of the hyper- and hypotonically stressed cell.
In the present model it was also observed that hypotonicity transiently activated the MAPKs p38 and SAPK/JNK. Consistent with these data, Tilly et al. (56) observed that swelling activated p38 in an intestinal cell line. In contrast, Sadoshima et al. (45) failed to detect p38 activation after hypotonic stress in cardiac myocytes; however, SAPK activation was demonstrable in this model. Whereas SAPK can activate Elk-1 (at least in vitro), the ability of p38 to activate Elk-1 remains unresolved. Activation of both of these kinases is insensitive to the MEK inhibitor PD-98059 in the present model; therefore, they likely play at most a secondary role in the ability of hypotonic stress to activate IEG transcription.
The relationship between ERK and Elk-1 phosphorylation/activation and previous observations regarding acute ion and osmolyte efflux pathways in response to hypotonicity remains to be elucidated. It is conceivable that ERK itself or another ERK-responsive or ERK-inducible kinase confers upregulation of one or more efflux pathways through posttranslational regulation (e.g., phosphorylation) of latent transport activity or through upregulation of transcription and translation of transport proteins. The former mechanism is favored in the case of inorganic ions and the osmolytes sorbitol (4, 14, 18, 22, 49, 63) and inositol (36, 38, 64), where extremely rapid efflux has been documented. Although ERK activation does not appear to play a major role in cell survival after acute hypotonic stress (Fig. 8B), a possible role for ERK activation in cell volume regulation is suggested by the decrement in cell volume (10-15%) observed in control and hypotonically stressed cells in the presence of PD-98059. ERK activation, however, is not required for the RVD observed at 30 min after acute hypotonic stress (Fig. 8B).
Phosphorylation events have previously been described in the context of
cell swelling in diverse models. In yeast, hypotonic stress activates a
MAPK cascade distinct from that activated by hypertonic stress.
Specifically, rapid tyrosine phosphorylation of the MAPK protein kinase
C (PKC1) follows exposure to hypotonicity and requires the presence of
upstream kinases (15). In a human intestinal cell line, Tilly and
colleagues (57) showed that hypotonic stress triggered a rapid increase
in tyrosine phosphorylation of multiple proteins, including a MAPK. A
potential physiological role for tyrosine phosphorylation events in
response to cell swelling was supported by experiments in which
specific inhibitors of tyrosine phosphorylation potentiated
osmosensitive ion efflux (55). Hypotonic shock of human peripheral
blood neutrophils resulted in tyrosine phosphorylation of multiple
proteins; the tyrosine kinase inhibitor genistein inhibited hypotonic
stress-inducible O2 generation in this
model (17). In skate and clam erythrocytes, swelling increased
phosphorylation of the anion exchanger band 3 (37) as well as other
proteins (41). In endothelial cells, however, inhibitor studies failed
to show a relationship between de novo phosphorylation and
Cl
conductance in response
to cell swelling (53).
The ERK/Elk-1 pathway can be activated by inducers of intracellular
Ca2+ release in a PKC- and
inositol trisphosphate-dependent fashion (reviewed in Ref. 58). Further
supporting a role for this pathway in the response to hypotonic stress,
Bagnasco et al. (3) and Mooren and Kinne (35) detected an increase in
intracellular Ca2+ within 1-5
min of hypotonic shock in renal epithelial Madin-Darby canine kidney
and IMCD cells, respectively; in the former example, inhibition of
intracellular Ca2+ release blocked
osmolyte efflux (3), underscoring its potential functional
significance. In contrast to its role in osmolyte efflux, intracellular
Ca2+ did not appear to mediate
Cl efflux in hypotonically
stressed IMCD cells (60). Hypotonicity-inducible intracellular
Ca2+ release was also observed in
permeabilized A7r5 embryonic vascular smooth muscle cells (34).
Surprisingly, Ca2+ release in this
model occurred independently of the inositol trisphosphate receptor.
Whether this observation is reflective of in vivo renal physiology or
is unique to the permeabilized state or this nonrenal cell line remains
to be established.
Speculation about other downstream physiological effectors of the ERK/Elk-1 pathway in hypotonic stress must include additional ERK substrates, as well as other trans-activation events inducible by activation of the ERK substrate Elk-1. Additional potential physiological ERK substrates include the transcription factor c-myc, as well as the enzymes phospholipase A2 and p90rsk. Phospholipase A2 cleaves membrane phospholipids into lipid-signaling intermediates, often in the context of cell stress (reviewed in Ref. 9). Activation of each of these substrates represents a potential downstream signaling event initiated by the upstream cascade described here. Genes responsive to Elk-1 regulation include those with composite SRE/Ets motifs within their genomic regulatory (5'-flanking) sequences, such as that encoding the transcription factor c-fos. Putative downstream genes of Egr-1 are legion (28, 52); their role in the cell response to hypotonic stress remains to be elucidated.
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ACKNOWLEDGEMENTS |
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We thank V. P. Sukhatme for the Egr-1 promoter, J. Epstein for HA3pCDNA3, and R. Maurer for the Elk-1/Gal4 chimera and 5XGAL4-Luc reporter vectors.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52494, the National Kidney Foundation, and the Medical Research Foundation of Oregon.
Address for reprint requests: D. M. Cohen, PP262, 3314 SW US Veterans Hospital Rd, Portland, OR 97201.
Received 12 November 1997; accepted in final form 6 July 1998.
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