Synthesis of the Na-K-ATPase -subunit is regulated at both the transcriptional and translational levels in IMCD3 cells
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《美国生理学杂志》
University of Colorado Health Sciences Center, Division of Renal Diseases and Hypertension, School of Medicine, Denver, Colorado
ABSTRACT
We previously reported that hypertonicity-mediated upregulation of the -subunit of Na-K-ATPase is dependent on both the JNK and the PI3 kinase pathways (Proc Natl Acad Sci USA 98: 13414, 2001). The present experiments were undertaken to explore the mechanisms whereby these pathways regulate the expression of the -subunit in inner medullary collecting duct cells (IMCD3). Inhibition of JNK with SP-600125 (20 μM), a concentration that causes an 95% inhibition of hypertonicity-stimulated JNK activation, markedly decreased the amount of the -subunit in response to 550 mosmol/kgH2O for 48 h. This was accompanied by a parallel decrease in the -subunit mRNA. The rate at which the -subunit mRNA decreased was unaffected by actinomycin D. In contrast, inhibition of PI3 kinase with LY-294002 results in a marked decrease in the amount of -subunit protein but without alteration in -subunit message. The rate at which the -subunit protein decreased was unaffected by cyclohexamide. Transfection of IMCD3 cells with a -subunit construct results in the expression of both -subunit message and protein. However, in cortical collecting duct cells (M1 cells) such transfection resulted in expression of only the message and not the protein. We conclude that JNK regulates the -subunit at the transcriptional level while PI3 kinase regulates -subunit expression at the translational level. There is also posttranscriptional cell specificity in the expression of the -subunit of Na-K-ATPase.
osmoregulation; hypertonicity
THE CELLS THAT INHABIT THE hypertonic environment of the inner medulla possess a number of adaptive mechanisms that allow them to survive this inhospitable environment. This survival is mediated initially by activation of ion transport systems (7) and thereafter by the cellular accumulation of a number of osmolytes (10). It has become increasingly evident that in addition to the proteins required for the cellular uptake and generation of these osmolytes, hypertonic stress brings about a coordinated response involving other proteins, many of which are critical to cell viability. Our laboratory has examined the role of the Na-K-ATPase with regard to the - and -subunits (6) and to the more recently described -subunit (5). We have done so both in cultured cells and in rodents at various states of hydration (5, 6). These studies strongly suggest that synthesis of the -subunit is important to cell survival when collecting duct cells are exposed to hypertonic stress (5). We have also demonstrated that -subunit synthesis is linked to signaling pathways that are known to be activated by hypertonicity. More specifically, the generation of the -subunits of Na-K-ATPase, but not the - and -subunits, is dependent on the activation of JNK (5). These experiments were performed with dominant-negative mutants of JNK that result in approximately a 50% decrement in JNK activation (27). Similarly, the pharmacological inhibition of PI3 kinase was found to significantly downregulate the expression of the -subunit but not the other two subunits of the protein (5). On this background, the present experiments were undertaken to define the molecular mechanisms that regulate the synthesis of the -subunit. More specifically, we examined the role of the aforementioned signaling pathways on the transcriptional and translational steps. Furthermore, we then determined whether the observed response is specific to the inner medullary collecting duct compared with the less osmotolerant renal cells that inhabit the renal cortex (6).
MATERIALS AND METHODS
Materials
Cell culture medium, serum, and antibiotics were obtained from Invitrogen (Carlsbad, CA). Antibody to the 1-subunit of Na-K-ATPase was purchased from Upstate Biotechnology (Lake Placid, NY). Antibody to the -subunit of Na-K-ATPase (a and b, splice variants) was generously provided by Dr. Steven Karlish (The Weizmann Institute of Science, Rehovot, Israel). Antibody for phospho-c-Jun was obtained from Cell Signaling (Beverly, MA). Cell signaling inhibitors (SP-600125, LY-294002) were purchased from Calbiochem (San Diego, CA). Osmolality was determined with an Advanced Instruments Micro-Osmometer (model 3300, Norwood, MA).
Cell Culture
The established murine inner medullary collecting duct cell line IMCD3 was previously provided by Dr. Steve Gullans (Boston, MA). Cell stocks were frozen in liquid N2 and propagated in 1:1 mixture of DMEM and Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. The mouse cortical collecting duct cell line M1 was obtained from Dr. Geza Fejes-Toth (22) and propagated in RPMI medium supplemented with FBS and antibiotics as described above. In experiments involving hypertonic stress, the media in culture dishes were exchanged for that with added NaCl to 550 or 600 mosmol/kgH2O depending on the experiment. At 550 mosmol/kgH2O, IMCD3 cells survive for at least 72 h; at 600 mosmol/kgH2O, survival is markedly decreased.
Treatment with Kinase Inhibitors
To minimize adsorption of the inhibitor, especially by albumin, confluent cell cultures were preincubated for 24 h in low-serum media (0.5% FBS) after which the kinase inhibitor was added. After 2 h, the osmolality was increased to 550 or 600 mosmol/kgH2O by the addition of sterile 5 M NaCl solution. After the required timed incubation, the cells were harvested as described below.
Inhibition of mRNA and Protein Synthesis
Nearly confluent IMCD3 cultures, growing under isotonic conditions in 100 x 20-mm tissue culture dishes, were first osmotically stressed at 550 mosmol/kgH2O for 24 h followed by osmotic stress at 550 mosmol/kgH2O in low-serum medium for 24 h. Cultures were pretreated for 2 h with either 10 μM LY-294002 (PI3 kinase inhibitor) or 20 μM SP-600125 (JNK inhibitor) and at time 0 5 μg/ml of actinomycin D or cyclohexamide added to interrupt further RNA or protein synthesis, respectively. RNA samples were obtained by harvesting treated cultures at 0, 1, 2, 4, and 6 h. Protein samples were obtained by harvesting treated cultures at 0, 4, 8, 12, and 24 h. RNA samples were analyzed by quantitative RT-PCR and protein samples by Western blot analysis.
Measurement of JNK Activity
JNK activity was assessed by Western blot analysis using (Ser 73) phospho-c-Jun-specific antibodies to determine the levels of c-Jun phosphorylation.
Western Blot Analysis
Cell lysates were prepared from confluent cell cultures in 100 x 20-mm tissue culture dishes as previously described (4, 5). Sample protein content was determined by BCA protein assay (Pierce, Rockford, IL). Depending on the experiment, from 25 to 150 μg of protein were loaded per lane for PAGE analysis. Gels were visualized using an alkaline phosphatase secondary antibody and Lumi-Phos reagent (Pierce) as described by the manufacturer. Chemiluminescence was recorded with an Image Station 440CF and results were analyzed with the 1D Image Software (Kodak Digital Science, Rochester, NY).
Plasmid Construction
The complete sequences of a and b mRNAs including both the 5'- and 3'-UTRs were obtained (4) using SMART RACE (BD Biosciences, Palo Alto, CA) and cloned in pGEM-T easy vector (Promega). To obtain mammalian expression constructs, -inserts were excised with EcoRI and ligated to an EcoRI-digested, shrimp alkaline phosphatase-treated pIRES vector (Clontech, Palo Alto, CA). On transfection in bacteria and cloning, constructs with the correct orientation were selected by digestion with StuI and confirmed by DNA sequencing (GenBank accession no. AY626243 and AY626244 for a and b, respectively).
Cell Transfection
Cultured cell lines were transfected using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Stable transfectants (clones) were selected from colonies in media prepared with 500 μg/ml of G-418 antibiotic. Clones were replated and a second colony selection was performed to provide clean clones for further analysis.
Northern Blot Analysis
Cytosolic RNA was isolated from cell cultures using the RNeasy kit (Qiagen, Valencia CA) separated by agarose gel electrophoresis and transferred to a nylon membrane as previously described (5). The 32P-labeled -specific oligonucleotide (5'-CATTGACCTGCCTATGTTTCTTACCGCC-3') was used as a probe.
Quantitative PCR
Prior to quantitative PCR (QPCR), RNA integrity was assessed by capillary electrophoresis using an Agilent Bioanalyzer [model 2100, Foster City, CA (using the 28 S to 18 S rRNA ratio)]. RNA was converted to cDNA using the Omniscript Reverse Transcriptase kit (Qiagen) as described by the manufacturer. Quantitative PCR primers (50 nM each) were designed using Primer Express 1.0 software (Applied Biosystems, Foster City, CA) targeting the conserved sequence (-forward 5'-CGACTATGAAACCGTCCGCAAAG-3' and -reverse 5'-TGCTGACTGTGCTGGGGTCTG-3'). QPCR was performed using the Quantitect SYBR Green PCR kit (Qiagen) on a Bio-Rad I-Cycler (Hercules, CA). QPCR runs were analyzed by agarose gel electrophoresis and melt curve to verify the correct amplicon was produced. To normalize the cDNA concentration in samples, QPCR assays for 18S rRNA were performed using an 18S rRNA Taqman kit (Applied Biosystems).
Statistics
Results were analyzed by ANOVA and a Tukey-Kramer multiple comparisons test using the InStat software package (GraphPad software). A value of P < 0.05 is considered significant.
RESULTS
Studies on the Mechanism Whereby Inhibition of JNK Dowregulates the Amount of -Subunit of Na-K-ATPase
Effect of the JNK inhibitor SP-600125 on hypertonicity-stimulated -subunit protein expression. We have previously shown that when cells transfected with a dominant-negative mutant of JNK are exposed to hypertonicity, the upregulation of the -subunit is significantly blunted. These mutants have only a 50% decrement in osmotic activation of the kinase (27). To achieve a fuller degree of inhibition of JNK, we employed a recently available specific inhibitor, SP-600125. This agent decreased hypertonicity-stimulated JNK activation in a dose-dependent manner (Fig. 1). At the dose used in the present experiments, 20 μM, an 95% inhibition was observed. Figure 2 depicts the results obtained when IMCD3 cells were exposed to isotonic media or to 550 mosmol/kgH2O for 48 h without or with the inhibitor SP-600125 at 20 μM for 2 h before hypertonic exposure. Such exposure to hypertonicity markedly increased the expression of the -subunit, as well as both splice variants of the -subunit of Na-K-ATPase in cells not exposed to the inhibitor. Pretreatment with the JNK inhibitor left the osmotic response of the -subunit unaltered; in contrast, 20 μM inhibitor prevented osmotic stress-dependent upregulation of both -subunit splice variants by >87% (P < 0.001).
Effect of the JNK inhibitor SP-600125 on hypertonicity-mediated increases in -subunit message. To assess whether this decrement in -subunit protein amount by the JNK inhibitor SP-600125 during osmotic shock occurred at the transcriptional or at the translational level, -subunit mRNA levels were assessed by quantitative PCR. The results shown in Fig. 3 depict that the robust increase in the -subunit message that accompanies exposure to hypertonicity and its reduction in cells pretreated with the JNK inhibitor. This inhibition is specific for the -subunit, as the increment in message for the - and -subunits of Na-K-ATPase was unaffected by such treatment.
Effect of the JNK inhibitor SP-600125 on the degradation (stability) of -subunit message. We undertook experiments to discern whether the JNK inhibition employed caused a decrease in message by affecting transcription (i.e., synthesis) or by enhancing the degradation. To this end, we employed the RNA synthesis inhibitor actinomycin D and examined by quantitative PCR the rate at which the message was degraded when its synthesis was halted in the presence and absence of JNK inhibition in cells exposed to hypertonicity for 48 h. Figure 4 depicts the levels of the -subunit mRNA in control cells and in those exposed to 20 μM SP-600125. Over the 6-h observation in four sets of cells, the rates of degradation were indistinguishable. These experiments strongly suggest that JNK inhibition per se does not enhance message degradation and that the observed decrease in the amount of -subunit message that is measured when cells are exposed to hypertonicity is due to decrements in message synthesis, reflecting an effect of this kinase on transcriptional regulation.
Studies of the Mechanism Whereby Inhibition of PI3 Kinase Downregulates the Amount of the -Subunit of Na-K-ATPase
Effect of the PI3 kinase inhibitor LY-294002 on -subunit message and protein. We previously showed that the pharmacological inhibition of PI3 kinase with LY-294002 produces a dose-dependent decrease in the amount of hypertonicity-stimulated -subunit protein (5). To ascertain whether this was a result of an effect on the transcription of the gene, we undertook Northern blot analysis and quantitative PCR in the presence of this agent and concomitantly measured protein synthesis. As is depicted in Fig. 5A, the increment in -subunit mRNA that accompanies exposure to 550 mosmol/kgH2O was not affected by doses of LY-294002 as high as 10 μM, a dose at which the amount of protein is markedly suppressed (Fig. 5B). This observation was confirmed with quantitative PCR measurements (data not shown).
Effect of the PI3 kinase inhibitor LY-294002 on degradation of the -subunit protein. The foregoing observation suggested an effect of PI3 kinase inhibition on the translation of the -subunit message. However, we could not exclude the possibility that this was a consequence of enhanced protein degradation. We thus undertook experiments in which IMCD3 cells exposed to hypertonicity for 48 h were studied in the presence and absence of PI3 kinase inhibition and the time course of the decrement in the amount of protein was studied for 24 h following inhibition of protein synthesis with cyclohexamide. Figure 6 depicts the results of these experiments. The baseline levels of protein were somewhat lower in cells already exposed to LY-294002 for 2 h. However, following addition of cyclohexamide, the rate of loss of protein was very similar in control and in LY-294002-treated cells. Thus over a 24-h period the level of -subunit protein decreased from 692 ± 71 to 289 ± 71 in LY-294002-treated cells and from 780 ± 73 to 386 ± 78 in control cells on the background of cyclohexamide treatment. These changes were not different from each other.
Effect of Cell Type on the Expression of the -Subunit: Comparison of M1 and IMCD3 Cells
We found that cortical collecting duct cells are more osmosensitive than IMCD3 cells (6). We examined the ability of the M1 cells to express the -subunit when transfected with constructs of either the a or b splice variant. The results of six such transfectants (4 in M1 cells and 2 in IMCD3 cells) are shown in Fig. 7. The neo controls were devoid of the message while the transfectants, as expected, robustly expressed mRNA. The expression of -subunit protein was different, however, as IMCD3 cells also expressed the protein that corresponded to the transfected splice variant whereas none of the M1 cells demonstrated expression of a or b protein, reflecting a posttranscriptional cell-specific regulation of protein expression.
DISCUSSION
The hypertonic environment of the inner medulla poses a survival challenge to the cells that inhabit this inhospitable milieu. The successful ability to meet this challenge is, in fact, critical to the normal operation of the renal concentrating mechanism. The cellular response to hypertonicity has been the subject of intense investigation in the last decade. Much of it has focused on the generation and cellular uptake of inert osmolytes (10, 26), preceded by the activation of various modules of the MAP kinase family (2, 15). It has also become evident that these signaling pathways impact the osmotic regulation of a number of proteins other than osmolyte transporters including COX2 (28) and the ANP receptor (8) as well as the -subunit of Na-K-ATPase (5) and heat shock proteins (16). The interest in our laboratory has been directed at understanding the role played in adaptation by this protein, a member of the FXYD family of small transmembrane proteins (23) that is exclusively expressed in the kidney (24). Within the kidney, the -subunit is expressed in the outer and inner stripe of the outer medulla (1), the ascending limb on Henle's loop (20), as well as in the inner medulla (19). In cultured renal cells the protein is clearly induced by hypertonicity as determined by protein expression (5) and immunocytochemistry (18) and dependent on both JNK and PI3 kinase (5). The present study further explores the mechanism whereby these signaling pathways are involved in the regulation of the -subunit.
The initial experiments we undertook extended the observations we previously reported employing a dominant-negative mutant of JNK2. Although such mutants provide a valuable tool for the study of this kinase, these cells only decrease hypertonicity-induced JNK activation by 50% (27). Also, such cells upregulate compensatory pathways (JNK1 for example) that could impact the observations. We thus confirmed our findings employing a highly specific JNK inhibitor at a dose that in intact cells suppressed hypertonicity-stimulated JNK activation by 95%. This agent specifically abrogates the upregulation of both splice variants of the -subunit, whereas the upregulation of the -subunit of Na-K-ATPase was unaltered. Because JNK impacts a number of downstream transcription factors (such as c-Jun), we examined whether the observed effect operates at the transcriptional step. Although the JNK inhibitor had no effect whatsoever on the transcription of the message for the - and -subunits of Na-K-ATPase, it caused a profound decrement in mRNA for the -subunit as assessed by quantitative PCR. These observations suggested an effect of JNK activation on the transcriptional step. However, we could not exclude the possibility that the agent accelerated the breakdown of the message. To investigate this possibility, we studied the time course of the decrement in -subunit when message synthesis was inhibited with actinomycin D. The rate at which this occurred was unaffected by the JNK inhibitor making it most likely that enhanced degradation does not account for the observed decrease in message. Rather our results suggest an effect of JNK in the regulation of message synthesis, i.e., at the transcriptional step. The mechanism whereby inhibition of PI3 kinase down regulated the expression of the -subunit was different. In contrast to the effect of JNK inhibition, the PI3 kinase inhibitor LY-294002 caused no decrement in the -subunit message levels. The decrement in -subunit protein levels that accompanies inhibition of PI3 kinase in the face of unaltered message levels suggested translational control. However, we could not exclude the possibility that the inhibition of the kinase enhances protein degradation. We thus studied the time course of the decrement in protein over a 24-h period in the background of inhibition of protein synthesis. The PI3 kinase inhibitor LY-294002 did not accelerate protein degradation suggesting that the observed decrement in protein levels is not mediated by this mechanism but is rather most likely a consequence of decreases in synthesis, i.e., a translational regulation. PI3 kinase is involved in a wide array of cellular functions (13) including cell growth, regulation of cell cycle, migration, and survival (3). Pathways activated by PI3 kinase appear to be involved in the regulation of effector molecules that impacts the translational process. Among these are mTor, PKC, and P70S6K (9, 17, 21). Future studies will be needed to define the precise level at which the inhibition of PI3 kinase impacts the translation of the -subunit. Careful analysis of -5'UTRs shows that both start with an oligopyrimidine motif, 5'-CCTCCCCTT-3' for a and 5'-CCCTCCTCCT-3' for b. In this regard, they belong to a class of mRNAs (5'TOP including those encoding ribosomal proteins and elongation factors) whose expression is controlled at the translational level (12, 14). Expression of these mRNAs has been shown to be blocked by inhibiting the activation (phosphorylation) of p70S6K with Rapamycin (11).
Our final experiments were designed to explore the cellular specificity of the regulation of -subunit expression. To this end, we compared the IMCD3 cells employed in the aforementioned experiments to that of cortical collecting duct cells. We transfected both cells with constructs of the a and b splice variants and the appropriate message was expressed in the transfected cells. However, although IMCD3 cells expressed the protein, this was not the case for M1 cells. These cells thus appear to lack the necessary signals to translate the presented message, underlying clearly the cell specificity involved in the transcriptional regulation of this protein.
Taken together, our experiments expand our understanding of the signaling pathways that regulate the expression of the -subunit of Na-K-ATPase in renal cells. They allow us to conclude that the JNK pathway regulation occurs at the transcriptional level by impacting the synthesis of the message. In contrast, the PI3 kinase pathway regulates -subunit expression at the translational level. Renal cells regulate the expression of the protein, at least in part, at a posttranscriptional step. Whether there is also a transcriptional regulation that also imparts cell specificity should be the subject of further investigation. It is perhaps surprising that a small protein such as the -subunit of Na-K-ATPase, whose sole function appears to be to modulate the activity of the enzyme (20, 25), is subject to such complex multilevel regulation.
GRANTS
This work was supported by National Institutes of Health Grant DK-19928.
ACKNOWLEDGMENTS
We thank J. Clegg for administrative support.
FOOTNOTES
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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ABSTRACT
We previously reported that hypertonicity-mediated upregulation of the -subunit of Na-K-ATPase is dependent on both the JNK and the PI3 kinase pathways (Proc Natl Acad Sci USA 98: 13414, 2001). The present experiments were undertaken to explore the mechanisms whereby these pathways regulate the expression of the -subunit in inner medullary collecting duct cells (IMCD3). Inhibition of JNK with SP-600125 (20 μM), a concentration that causes an 95% inhibition of hypertonicity-stimulated JNK activation, markedly decreased the amount of the -subunit in response to 550 mosmol/kgH2O for 48 h. This was accompanied by a parallel decrease in the -subunit mRNA. The rate at which the -subunit mRNA decreased was unaffected by actinomycin D. In contrast, inhibition of PI3 kinase with LY-294002 results in a marked decrease in the amount of -subunit protein but without alteration in -subunit message. The rate at which the -subunit protein decreased was unaffected by cyclohexamide. Transfection of IMCD3 cells with a -subunit construct results in the expression of both -subunit message and protein. However, in cortical collecting duct cells (M1 cells) such transfection resulted in expression of only the message and not the protein. We conclude that JNK regulates the -subunit at the transcriptional level while PI3 kinase regulates -subunit expression at the translational level. There is also posttranscriptional cell specificity in the expression of the -subunit of Na-K-ATPase.
osmoregulation; hypertonicity
THE CELLS THAT INHABIT THE hypertonic environment of the inner medulla possess a number of adaptive mechanisms that allow them to survive this inhospitable environment. This survival is mediated initially by activation of ion transport systems (7) and thereafter by the cellular accumulation of a number of osmolytes (10). It has become increasingly evident that in addition to the proteins required for the cellular uptake and generation of these osmolytes, hypertonic stress brings about a coordinated response involving other proteins, many of which are critical to cell viability. Our laboratory has examined the role of the Na-K-ATPase with regard to the - and -subunits (6) and to the more recently described -subunit (5). We have done so both in cultured cells and in rodents at various states of hydration (5, 6). These studies strongly suggest that synthesis of the -subunit is important to cell survival when collecting duct cells are exposed to hypertonic stress (5). We have also demonstrated that -subunit synthesis is linked to signaling pathways that are known to be activated by hypertonicity. More specifically, the generation of the -subunits of Na-K-ATPase, but not the - and -subunits, is dependent on the activation of JNK (5). These experiments were performed with dominant-negative mutants of JNK that result in approximately a 50% decrement in JNK activation (27). Similarly, the pharmacological inhibition of PI3 kinase was found to significantly downregulate the expression of the -subunit but not the other two subunits of the protein (5). On this background, the present experiments were undertaken to define the molecular mechanisms that regulate the synthesis of the -subunit. More specifically, we examined the role of the aforementioned signaling pathways on the transcriptional and translational steps. Furthermore, we then determined whether the observed response is specific to the inner medullary collecting duct compared with the less osmotolerant renal cells that inhabit the renal cortex (6).
MATERIALS AND METHODS
Materials
Cell culture medium, serum, and antibiotics were obtained from Invitrogen (Carlsbad, CA). Antibody to the 1-subunit of Na-K-ATPase was purchased from Upstate Biotechnology (Lake Placid, NY). Antibody to the -subunit of Na-K-ATPase (a and b, splice variants) was generously provided by Dr. Steven Karlish (The Weizmann Institute of Science, Rehovot, Israel). Antibody for phospho-c-Jun was obtained from Cell Signaling (Beverly, MA). Cell signaling inhibitors (SP-600125, LY-294002) were purchased from Calbiochem (San Diego, CA). Osmolality was determined with an Advanced Instruments Micro-Osmometer (model 3300, Norwood, MA).
Cell Culture
The established murine inner medullary collecting duct cell line IMCD3 was previously provided by Dr. Steve Gullans (Boston, MA). Cell stocks were frozen in liquid N2 and propagated in 1:1 mixture of DMEM and Ham's F-12 nutrient mixture supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin. The mouse cortical collecting duct cell line M1 was obtained from Dr. Geza Fejes-Toth (22) and propagated in RPMI medium supplemented with FBS and antibiotics as described above. In experiments involving hypertonic stress, the media in culture dishes were exchanged for that with added NaCl to 550 or 600 mosmol/kgH2O depending on the experiment. At 550 mosmol/kgH2O, IMCD3 cells survive for at least 72 h; at 600 mosmol/kgH2O, survival is markedly decreased.
Treatment with Kinase Inhibitors
To minimize adsorption of the inhibitor, especially by albumin, confluent cell cultures were preincubated for 24 h in low-serum media (0.5% FBS) after which the kinase inhibitor was added. After 2 h, the osmolality was increased to 550 or 600 mosmol/kgH2O by the addition of sterile 5 M NaCl solution. After the required timed incubation, the cells were harvested as described below.
Inhibition of mRNA and Protein Synthesis
Nearly confluent IMCD3 cultures, growing under isotonic conditions in 100 x 20-mm tissue culture dishes, were first osmotically stressed at 550 mosmol/kgH2O for 24 h followed by osmotic stress at 550 mosmol/kgH2O in low-serum medium for 24 h. Cultures were pretreated for 2 h with either 10 μM LY-294002 (PI3 kinase inhibitor) or 20 μM SP-600125 (JNK inhibitor) and at time 0 5 μg/ml of actinomycin D or cyclohexamide added to interrupt further RNA or protein synthesis, respectively. RNA samples were obtained by harvesting treated cultures at 0, 1, 2, 4, and 6 h. Protein samples were obtained by harvesting treated cultures at 0, 4, 8, 12, and 24 h. RNA samples were analyzed by quantitative RT-PCR and protein samples by Western blot analysis.
Measurement of JNK Activity
JNK activity was assessed by Western blot analysis using (Ser 73) phospho-c-Jun-specific antibodies to determine the levels of c-Jun phosphorylation.
Western Blot Analysis
Cell lysates were prepared from confluent cell cultures in 100 x 20-mm tissue culture dishes as previously described (4, 5). Sample protein content was determined by BCA protein assay (Pierce, Rockford, IL). Depending on the experiment, from 25 to 150 μg of protein were loaded per lane for PAGE analysis. Gels were visualized using an alkaline phosphatase secondary antibody and Lumi-Phos reagent (Pierce) as described by the manufacturer. Chemiluminescence was recorded with an Image Station 440CF and results were analyzed with the 1D Image Software (Kodak Digital Science, Rochester, NY).
Plasmid Construction
The complete sequences of a and b mRNAs including both the 5'- and 3'-UTRs were obtained (4) using SMART RACE (BD Biosciences, Palo Alto, CA) and cloned in pGEM-T easy vector (Promega). To obtain mammalian expression constructs, -inserts were excised with EcoRI and ligated to an EcoRI-digested, shrimp alkaline phosphatase-treated pIRES vector (Clontech, Palo Alto, CA). On transfection in bacteria and cloning, constructs with the correct orientation were selected by digestion with StuI and confirmed by DNA sequencing (GenBank accession no. AY626243 and AY626244 for a and b, respectively).
Cell Transfection
Cultured cell lines were transfected using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Stable transfectants (clones) were selected from colonies in media prepared with 500 μg/ml of G-418 antibiotic. Clones were replated and a second colony selection was performed to provide clean clones for further analysis.
Northern Blot Analysis
Cytosolic RNA was isolated from cell cultures using the RNeasy kit (Qiagen, Valencia CA) separated by agarose gel electrophoresis and transferred to a nylon membrane as previously described (5). The 32P-labeled -specific oligonucleotide (5'-CATTGACCTGCCTATGTTTCTTACCGCC-3') was used as a probe.
Quantitative PCR
Prior to quantitative PCR (QPCR), RNA integrity was assessed by capillary electrophoresis using an Agilent Bioanalyzer [model 2100, Foster City, CA (using the 28 S to 18 S rRNA ratio)]. RNA was converted to cDNA using the Omniscript Reverse Transcriptase kit (Qiagen) as described by the manufacturer. Quantitative PCR primers (50 nM each) were designed using Primer Express 1.0 software (Applied Biosystems, Foster City, CA) targeting the conserved sequence (-forward 5'-CGACTATGAAACCGTCCGCAAAG-3' and -reverse 5'-TGCTGACTGTGCTGGGGTCTG-3'). QPCR was performed using the Quantitect SYBR Green PCR kit (Qiagen) on a Bio-Rad I-Cycler (Hercules, CA). QPCR runs were analyzed by agarose gel electrophoresis and melt curve to verify the correct amplicon was produced. To normalize the cDNA concentration in samples, QPCR assays for 18S rRNA were performed using an 18S rRNA Taqman kit (Applied Biosystems).
Statistics
Results were analyzed by ANOVA and a Tukey-Kramer multiple comparisons test using the InStat software package (GraphPad software). A value of P < 0.05 is considered significant.
RESULTS
Studies on the Mechanism Whereby Inhibition of JNK Dowregulates the Amount of -Subunit of Na-K-ATPase
Effect of the JNK inhibitor SP-600125 on hypertonicity-stimulated -subunit protein expression. We have previously shown that when cells transfected with a dominant-negative mutant of JNK are exposed to hypertonicity, the upregulation of the -subunit is significantly blunted. These mutants have only a 50% decrement in osmotic activation of the kinase (27). To achieve a fuller degree of inhibition of JNK, we employed a recently available specific inhibitor, SP-600125. This agent decreased hypertonicity-stimulated JNK activation in a dose-dependent manner (Fig. 1). At the dose used in the present experiments, 20 μM, an 95% inhibition was observed. Figure 2 depicts the results obtained when IMCD3 cells were exposed to isotonic media or to 550 mosmol/kgH2O for 48 h without or with the inhibitor SP-600125 at 20 μM for 2 h before hypertonic exposure. Such exposure to hypertonicity markedly increased the expression of the -subunit, as well as both splice variants of the -subunit of Na-K-ATPase in cells not exposed to the inhibitor. Pretreatment with the JNK inhibitor left the osmotic response of the -subunit unaltered; in contrast, 20 μM inhibitor prevented osmotic stress-dependent upregulation of both -subunit splice variants by >87% (P < 0.001).
Effect of the JNK inhibitor SP-600125 on hypertonicity-mediated increases in -subunit message. To assess whether this decrement in -subunit protein amount by the JNK inhibitor SP-600125 during osmotic shock occurred at the transcriptional or at the translational level, -subunit mRNA levels were assessed by quantitative PCR. The results shown in Fig. 3 depict that the robust increase in the -subunit message that accompanies exposure to hypertonicity and its reduction in cells pretreated with the JNK inhibitor. This inhibition is specific for the -subunit, as the increment in message for the - and -subunits of Na-K-ATPase was unaffected by such treatment.
Effect of the JNK inhibitor SP-600125 on the degradation (stability) of -subunit message. We undertook experiments to discern whether the JNK inhibition employed caused a decrease in message by affecting transcription (i.e., synthesis) or by enhancing the degradation. To this end, we employed the RNA synthesis inhibitor actinomycin D and examined by quantitative PCR the rate at which the message was degraded when its synthesis was halted in the presence and absence of JNK inhibition in cells exposed to hypertonicity for 48 h. Figure 4 depicts the levels of the -subunit mRNA in control cells and in those exposed to 20 μM SP-600125. Over the 6-h observation in four sets of cells, the rates of degradation were indistinguishable. These experiments strongly suggest that JNK inhibition per se does not enhance message degradation and that the observed decrease in the amount of -subunit message that is measured when cells are exposed to hypertonicity is due to decrements in message synthesis, reflecting an effect of this kinase on transcriptional regulation.
Studies of the Mechanism Whereby Inhibition of PI3 Kinase Downregulates the Amount of the -Subunit of Na-K-ATPase
Effect of the PI3 kinase inhibitor LY-294002 on -subunit message and protein. We previously showed that the pharmacological inhibition of PI3 kinase with LY-294002 produces a dose-dependent decrease in the amount of hypertonicity-stimulated -subunit protein (5). To ascertain whether this was a result of an effect on the transcription of the gene, we undertook Northern blot analysis and quantitative PCR in the presence of this agent and concomitantly measured protein synthesis. As is depicted in Fig. 5A, the increment in -subunit mRNA that accompanies exposure to 550 mosmol/kgH2O was not affected by doses of LY-294002 as high as 10 μM, a dose at which the amount of protein is markedly suppressed (Fig. 5B). This observation was confirmed with quantitative PCR measurements (data not shown).
Effect of the PI3 kinase inhibitor LY-294002 on degradation of the -subunit protein. The foregoing observation suggested an effect of PI3 kinase inhibition on the translation of the -subunit message. However, we could not exclude the possibility that this was a consequence of enhanced protein degradation. We thus undertook experiments in which IMCD3 cells exposed to hypertonicity for 48 h were studied in the presence and absence of PI3 kinase inhibition and the time course of the decrement in the amount of protein was studied for 24 h following inhibition of protein synthesis with cyclohexamide. Figure 6 depicts the results of these experiments. The baseline levels of protein were somewhat lower in cells already exposed to LY-294002 for 2 h. However, following addition of cyclohexamide, the rate of loss of protein was very similar in control and in LY-294002-treated cells. Thus over a 24-h period the level of -subunit protein decreased from 692 ± 71 to 289 ± 71 in LY-294002-treated cells and from 780 ± 73 to 386 ± 78 in control cells on the background of cyclohexamide treatment. These changes were not different from each other.
Effect of Cell Type on the Expression of the -Subunit: Comparison of M1 and IMCD3 Cells
We found that cortical collecting duct cells are more osmosensitive than IMCD3 cells (6). We examined the ability of the M1 cells to express the -subunit when transfected with constructs of either the a or b splice variant. The results of six such transfectants (4 in M1 cells and 2 in IMCD3 cells) are shown in Fig. 7. The neo controls were devoid of the message while the transfectants, as expected, robustly expressed mRNA. The expression of -subunit protein was different, however, as IMCD3 cells also expressed the protein that corresponded to the transfected splice variant whereas none of the M1 cells demonstrated expression of a or b protein, reflecting a posttranscriptional cell-specific regulation of protein expression.
DISCUSSION
The hypertonic environment of the inner medulla poses a survival challenge to the cells that inhabit this inhospitable milieu. The successful ability to meet this challenge is, in fact, critical to the normal operation of the renal concentrating mechanism. The cellular response to hypertonicity has been the subject of intense investigation in the last decade. Much of it has focused on the generation and cellular uptake of inert osmolytes (10, 26), preceded by the activation of various modules of the MAP kinase family (2, 15). It has also become evident that these signaling pathways impact the osmotic regulation of a number of proteins other than osmolyte transporters including COX2 (28) and the ANP receptor (8) as well as the -subunit of Na-K-ATPase (5) and heat shock proteins (16). The interest in our laboratory has been directed at understanding the role played in adaptation by this protein, a member of the FXYD family of small transmembrane proteins (23) that is exclusively expressed in the kidney (24). Within the kidney, the -subunit is expressed in the outer and inner stripe of the outer medulla (1), the ascending limb on Henle's loop (20), as well as in the inner medulla (19). In cultured renal cells the protein is clearly induced by hypertonicity as determined by protein expression (5) and immunocytochemistry (18) and dependent on both JNK and PI3 kinase (5). The present study further explores the mechanism whereby these signaling pathways are involved in the regulation of the -subunit.
The initial experiments we undertook extended the observations we previously reported employing a dominant-negative mutant of JNK2. Although such mutants provide a valuable tool for the study of this kinase, these cells only decrease hypertonicity-induced JNK activation by 50% (27). Also, such cells upregulate compensatory pathways (JNK1 for example) that could impact the observations. We thus confirmed our findings employing a highly specific JNK inhibitor at a dose that in intact cells suppressed hypertonicity-stimulated JNK activation by 95%. This agent specifically abrogates the upregulation of both splice variants of the -subunit, whereas the upregulation of the -subunit of Na-K-ATPase was unaltered. Because JNK impacts a number of downstream transcription factors (such as c-Jun), we examined whether the observed effect operates at the transcriptional step. Although the JNK inhibitor had no effect whatsoever on the transcription of the message for the - and -subunits of Na-K-ATPase, it caused a profound decrement in mRNA for the -subunit as assessed by quantitative PCR. These observations suggested an effect of JNK activation on the transcriptional step. However, we could not exclude the possibility that the agent accelerated the breakdown of the message. To investigate this possibility, we studied the time course of the decrement in -subunit when message synthesis was inhibited with actinomycin D. The rate at which this occurred was unaffected by the JNK inhibitor making it most likely that enhanced degradation does not account for the observed decrease in message. Rather our results suggest an effect of JNK in the regulation of message synthesis, i.e., at the transcriptional step. The mechanism whereby inhibition of PI3 kinase down regulated the expression of the -subunit was different. In contrast to the effect of JNK inhibition, the PI3 kinase inhibitor LY-294002 caused no decrement in the -subunit message levels. The decrement in -subunit protein levels that accompanies inhibition of PI3 kinase in the face of unaltered message levels suggested translational control. However, we could not exclude the possibility that the inhibition of the kinase enhances protein degradation. We thus studied the time course of the decrement in protein over a 24-h period in the background of inhibition of protein synthesis. The PI3 kinase inhibitor LY-294002 did not accelerate protein degradation suggesting that the observed decrement in protein levels is not mediated by this mechanism but is rather most likely a consequence of decreases in synthesis, i.e., a translational regulation. PI3 kinase is involved in a wide array of cellular functions (13) including cell growth, regulation of cell cycle, migration, and survival (3). Pathways activated by PI3 kinase appear to be involved in the regulation of effector molecules that impacts the translational process. Among these are mTor, PKC, and P70S6K (9, 17, 21). Future studies will be needed to define the precise level at which the inhibition of PI3 kinase impacts the translation of the -subunit. Careful analysis of -5'UTRs shows that both start with an oligopyrimidine motif, 5'-CCTCCCCTT-3' for a and 5'-CCCTCCTCCT-3' for b. In this regard, they belong to a class of mRNAs (5'TOP including those encoding ribosomal proteins and elongation factors) whose expression is controlled at the translational level (12, 14). Expression of these mRNAs has been shown to be blocked by inhibiting the activation (phosphorylation) of p70S6K with Rapamycin (11).
Our final experiments were designed to explore the cellular specificity of the regulation of -subunit expression. To this end, we compared the IMCD3 cells employed in the aforementioned experiments to that of cortical collecting duct cells. We transfected both cells with constructs of the a and b splice variants and the appropriate message was expressed in the transfected cells. However, although IMCD3 cells expressed the protein, this was not the case for M1 cells. These cells thus appear to lack the necessary signals to translate the presented message, underlying clearly the cell specificity involved in the transcriptional regulation of this protein.
Taken together, our experiments expand our understanding of the signaling pathways that regulate the expression of the -subunit of Na-K-ATPase in renal cells. They allow us to conclude that the JNK pathway regulation occurs at the transcriptional level by impacting the synthesis of the message. In contrast, the PI3 kinase pathway regulates -subunit expression at the translational level. Renal cells regulate the expression of the protein, at least in part, at a posttranscriptional step. Whether there is also a transcriptional regulation that also imparts cell specificity should be the subject of further investigation. It is perhaps surprising that a small protein such as the -subunit of Na-K-ATPase, whose sole function appears to be to modulate the activity of the enzyme (20, 25), is subject to such complex multilevel regulation.
GRANTS
This work was supported by National Institutes of Health Grant DK-19928.
ACKNOWLEDGMENTS
We thank J. Clegg for administrative support.
FOOTNOTES
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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