p38 MAPK mediates acid-induced transcription of PEPCK in LLC-PK1-FBPase+ cells

Elisabeth Feifel1, Petra Obexer1, Manfred Andratsch1, Stephan Euler1, Lynn Taylor2, Aimin Tang2, Yu Wei2, Herbert Schramek1, Norman P. Curthoys2, and Gerhard Gstraunthaler1

1 Department of Physiology, University of Innsbruck, A-6010 Innsbruck, Austria; and 2 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523 - 1870


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LLC-PK1-FBPase+ cells are a gluconeogenic and pH-responsive renal proximal tubule-like cell line. On incubation with acidic medium (pH 6.9), LLC-PK1-FBPase+ cells exhibit an increased rate of ammonia production as well as increases in glutaminase and phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels and enzyme activities. The increase in PEPCK mRNA is due to an enhanced rate of transcription that is initiated in response to intracellular acidosis. The involvement of known MAPK activities (ERK1/2, SAPK/JNK, p38) in the associated signal transduction pathway was examined by determining the effects of specific MAPK activators and inhibitors on basal and acid-induced PEPCK mRNA levels. Transfer of LLC-PK1-FBPase+ cultures to acidic medium resulted in specific phosphorylation, and thus activation, of p38 and of activating transcription factor-2 (ATF-2), respectively. Anisomycin (AI), a strong p38 activator, increased PEPCK mRNA to levels comparable to those observed with acid stimulation. AI also induced a time-dependent phosphorylation of p38 and ATF-2. SB-203580, a specific p38 inhibitor, blocked both acid- and AI-induced PEPCK mRNA levels. Western blot analyses revealed that the SB-203580-sensitive p38alpha isoform is strongly expressed. The octanucleotide sequence of the cAMP-response element-1 site of the PEPCK promotor is a perfect match to the consensus element for binding ATF-2. The specificity of ATF-2 binding was proven by ELISA. We conclude that the SB-203580-sensitive p38alpha -ATF-2 signaling pathway is a likely mediator of the pH-responsive induction of PEPCK mRNA levels in renal LLC-PK1-FBPase+ cells.

metabolic acidosis; proximal tubule; ammoniagenesis; gluconeogenesis; mitogen-activated protein kinase; fructose 1,6-bisphosphatase; phosphoenolpyruvate carboxykinase


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SYSTEMIC METABOLIC ACIDOSIS initiates an array of adaptive responses in tubular cell metabolism and transport along the entire renal nephron (1, 9, 36, 37). The pH-induced changes in cell metabolism, however, are confined to the proximal convoluted tubule. In rats, renal proximal tubular cells respond with an increased extraction and catabolism of glutamine and enhanced rates of ammonium ion excretion and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> ion reabsorption, thereby partially restoring acid-base homeostasis (13, 19). This is accomplished, in part, by increases in phosphate-dependent glutaminase (PDG) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) activities, which result from increased levels of their respective mRNAs (19). However, the two genes are differentially regulated. Increased levels of PEPCK mRNA result from an enhanced rate of transcription, whereas PDG mRNA levels are increased by decreasing its rate of degradation (9, 19).

The differential regulation of the adaptive response of the two enzymes is reproduced in LLC-PK1-FBPase+ cells (13). These cells are a gluconeogenic strain of the renal epithelial LLC-PK1 cell line (12) that exhibit a number of proximal tubule-specific metabolic properties (17). On incubation with acidic medium (pH 6.9), LLC-PK1-FBPase+ cells respond with an increased rate of ammonia production and two- to threefold increases in PDG and PEPCK mRNA levels and enzyme activities. Furthermore, incubation of LLC-PK1-FBPase+ cells in low-potassium (0.7 mM)-containing media for 24-72 h elicits a decrease in intracellular pH while maintaining normal extracellular pH. The LLC-PK1-FBPase+ cells again respond with increased levels of PDG and PEPCK mRNAs, suggesting that an intracellular acidosis triggers the adaptive responses (13).

The mechanism of sensing cellular pH in renal proximal tubular cells and the associated signal transduction pathway for mediating the pH-responsive adaptations are unknown. In recent years, it has been well established that MAPK signaling cascades are activated by various extracellular stimuli, including hyperosmolarity (2, 21, 22, 39). In mammals, hypertonicity is unique to the renal medullary interstitium, and similarly to metabolic acidosis, it induces a plethora of cellular responses resulting from specific gene expression (reviewed in Refs. 4 and 14). The p38 stress-activated MAPK superfamily is activated by changes in extracellular osmolality and is likely to mediate the osmoadaptation of renal distal tubule and collecting duct cells (28, 35, 38). Other cellular stresses that are potent in vivo and in vitro activators of p38 MAPK include inflammatory cytokines, heat shock, and the protein synthesis inhibitor anisomycin (AI). Therefore, decreased intracellular pH might also activate an MAPK in LLC-PK1-FBPase+ cells.

The promoter region of the cytosolic PEPCK gene contains a cAMP-response element (CRE-1) (15, 16, 25, 32). The sequence of this element (TTACGTCA) was recently recognized as a perfect match to the consensus sequence reported for activating transcription factor-2 (ATF-2) homodimers (7), a downstream substrate of p38 MAPK. These findings prompted us to define the role of p38 kinase and other MAPK signaling cascades (ERK1/2, SAPK/JNK) in the pH-responsive induction of PEPCK mRNA transcription in LLC-PK1-FBPase+ cells (Fig. 1). Here, we show that incubation of LLC-PK1-FBPase+ cultures in acidic medium resulted in a biphasic phosphorylation, and thus activation, of p38 kinase and ATF-2. In addition, AI specifically increased PEPCK mRNA to levels similar to those observed in acidic cultures. SB-203580, a specific p38 kinase inhibitor, but not the MAPK ERK (MEK)1/2 inhibitor PD-098059 or the SAPK/JNK inhibitor curcumin, produced a dose-dependent inhibition of the acid- and AI-induced PEPCK mRNA levels. These results suggest that p38/ATF-2 signaling may mediate the pH-responsive induction of PEPCK mRNA levels in LLC-PK1-FBPase+ kidney cells.


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Fig. 1.   Schematic diagram of the signal transduction pathways explored in the present study. A decrease in intracellular pH (pHi), either elicited by extracellular acidosis or low-potassium culture media (13), results in increased mRNA levels of cytosolic phophoenolpyruvate carboxykinase (PEPCK) and mitochondrial phosphate-dependent glutaminase in LLC-PK1-FBPase+ cells. The MAPK cascades ERK1/2, JNK1/2, and p38, which might be involved in mediating this adaptive pH response, are depicted. Furthermore, upstream protein tyrosine kinases are shown. On the right, utilized activators and inhibitors of the signaling cascades are listed and their specific site(s) of action are detailed. Anisomycin (AI), a strong activator of the SAPKs, p38 and JNK, induced both PEPCK and glutaminase mRNA levels, as seen under acidic conditions. Preliminary results showed that TGF-beta specifically activates p38 and activating transcription factor (ATF)-2 in LLC-PK1-FBPase+ cells and increases cellular levels of PEPCK mRNA. Of the inhibitors listed, only the p38 inhibitors SB-203580 and SB-202190 produced a dose-dependent inhibition of acid- and AI-induced PEPCK mRNA levels. For further details see text. C/EBP, CCAAT/enhancer binding protein; CHOP, C/EBP homologous protein; MEK, MAPK/ERK kinase; MEKK, MEK kinase; MKK, MAPK kinase; PI, phosphatidylinositol; TAK1, TGF-beta -activated kinase 1; RK, reactivating kinase.


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Culture media and chemicals. Culture media were prepared from DMEM base (D-5030, Sigma) supplemented with 2.2 g/l (26.2 mM) NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and 1.0 g/l (17.8 mM) NaCl (to correct for medium osmolarity) (13). L-Glutamine was added from freshly prepared stock solutions (final concentration 2.0 mM). All media additives were of tissue culture grade and were obtained from Sigma-Aldrich (Vienna, Austria). Biochemicals and buffer chemicals were of the highest analytical grade available and were obtained from Boehringer Mannheim (Vienna, Austria) or from Sigma-Aldrich. MAPK inhibitors were purchased from Calbiochem. All tissue culture plasticware (Falcon Labware) was from Becton Dickinson (Heidelberg, Germany).

Cell culture, adaptation protocol, and cell harvest. Gluconeogenic LLC-PK1-FBPase+ cells (12, 13, 17) were cultured in DMEM with 5.5 mM D-glucose, 2 mM L-glutamine, and 26 mM NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (pH 7.4), supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Acidic medium (pH 6.9) contained 9 mM NaHCO3 and was supplemented with the appropriate amount of NaCl to maintain equivalent osmolarity. Cultures were incubated at 37°C in a 5% CO2-95% air atmosphere. Routinely, cultures were fed three times a week. Experimental cultures were always fed 24 h before adaptation. Confluent monolayers were subcultured (split ratio 1:3) with 0.25% trypsin and 0.02% EDTA in Ca2+- and Mg2+-free buffered saline. Experimental cultures were grown for 10-12 days to produce confluent monolayers in 10-cm plastic tissue culture dishes (Falcon Optilux 3003) by using 10 ml of culture medium or in six-well plates (Falcon 3046) with 2 ml of medium. Thereafter, cultures were adapted to acidic media (pH 6.9) for the indicated times.

For Western blotting, monolayer cultures were harvested in freshly prepared ice-cold lysis buffer [(in mM) 50 Tris · HCl, pH 7.5, 150 sodium chloride, 5 EDTA, 40 beta -glycerophosphate, 50 sodium fluoride, and 0.1 phenylmethylsulfonyl fluoride, as well as 1% (vol/vol) Nonidet P-40 (Amresco), 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, 200 µM sodium orthovanadate, 1 µg/ml leupeptin, and 1 µM pepstatin A] (34). Cells were lysed on ice for 20 min, followed by the forcing of the lysate through a 21-gauge needle with a syringe. The lysates were then transferred to microcentrifuge tubes and centrifuged at 14,000 g for 15 min at 4°C. Aliquots of supernatants were stored at -80°C. For Northern blot analysis, total RNA was isolated by using the acid guanidinium thiocyanate method as described previously (13, 17).

Western blotting and antibodies. SDS-PAGE was performed under standard denaturing conditions using 16 × 16-cm slab gels (SE-400, Hoefer). Equal amounts of protein were loaded onto each lane of 10% polyacrylamide gels. Rainbow Marker (RPN 756, Amersham) was used as a molecular weight standard. Electrophoresis was performed overnight at a constant 80 V. Gels were blotted immediately after electrophoresis onto polyvinylidene fluoride membrane (Immobilon-P, Millipore). Gels were rinsed briefly in transfer buffer [25 mM Tris, 200 mM glycine, pH 8.3, 0.1% (wt/vol) SDS, 20% (vol/vol) methanol], and transfer was carried at a constant current of 400 mA for 3 h at 4°C. For immunodetection, membranes were blocked overnight with 5% (wt/vol) dry milk and 0.1% (vol/vol) Tween 20 in Tris-buffered saline (pH 7.6) at 4°C and processed according to the instructions of the manufacturers of the antibodies. The following antibodies were used: PhosphoPlus Antibody Kits (New England Biolabs) for the detection of phosphorylated forms of ERK1/2 (p44/42 MAP Kinase Thr202/Tyr204 Antibody Kit 9100), SAPK/JNK (Thr183/Tyr185 Antibody Kit 9250), and p38 (Thr180/Tyr182 Antibody Kit 9210), respectively. The state of phosphorylation of transcription factor ATF-2 was determined by using the PhosphoPlus ATF-2 (Thr71) Antibody Kit (model 9220, New England Biolabs). The anti-MAPK kinase (anti-MKK) antibodies (anti-MEK-3, sc-959; anti-MEK-4, sc-964; and anti-MEK-6, sc-1991) and the antibodies against the p38 isoforms (anti-p38, sc-535-G; anti-p38beta , sc-6176; anti-SAPK4, sc-7585; and anti-ERK 6, sc-2020) were obtained from Santa Cruz Biotechnology. Visualization of blots was carried out with enhanced chemiluminescence by using either the Western Star System (Tropix) for the PhosphoPlus antibodies or the ECL System (Amersham) for all other antibodies. All blots were exposed to Hyperfilm ECL (Amersham).

Northern blot analysis and cDNA probes. Formaldehyde-agarose gel electrophoresis of total RNA samples, transfer to GeneScreen Plus membranes (New England Nuclear), and hybridization and posthybridization washings of blots were carried out as described previously (13, 17-19). Blots were exposed to autoradiographic film (Kodak BioMax MS). Quantitation of mRNA levels was accomplished by using a Personal Densitometer SI-Scanner (Molecular Dynamics). Sample integrity and equal loading of 20 µg RNA/lane were monitored by staining with ethidium bromide after electrophoresis. For probing PEPCK mRNA, a 1.6-kb BglII fragment of pPCK-10 (13, 17), which encodes the rat cytosolic PEPCK, was used. The pPCK-10 plasmid was kindly provided by Dr. R. Hanson (Case Western Reserve) (15, 16).

Isolation of nuclear extract and electrophoretic mobility shift assays. Nuclear extracts of LLC-PK1-FBPase+ cells were prepared as described recently (25). The CRE-1 probe was synthesized by Macromolecular Resources (Ft. Collins, CO) as complementary oligonucleotides that contained bases -99 to -77 of the PEPCK promoter. The sequence of the sense strand is 5'-GATCCGGCCCCTTACGTCAGAGGCGAG-3', in which the nucleotides derived from the PEPCK promoter are underlined and the CRE-1 element is in bold. The additional sequence was included to create 5' BamHI overhangs. The oligonucleotides were annealed in 50 mM NaCl, 66 mM Tris · HCl, and 6.6 mM MgCl2, pH 7.5, by heating to 85°C and cooling to 25°C.

The double-stranded oligonucleotide was 5'-end labeled with [gamma -32P]ATP by using T4 polynucleotide kinase (33). The indicated amount of nuclear extract was incubated for 20 min on ice with 200 ng poly[dI-dC], 200 ng pUC19, and the labeled oligonucleotide (20 fmol, 20,000-60,000 counts/min). For the supershift analysis, the indicated amount of antibody was preincubated with the nonspecific competitors and the nuclear extract for 45 min on ice. The labeled probe was then added, and the complete sample was incubated for another 20 min on ice. The electrophoresis was performed at 170 V for 1.5 h at 4°C by using a 4% polyacrylamide gel.

Specificity of ATF-2 binding to the CRE-1 element was further assessed with the newly developed Mercury TransFactor Kit (model K2062-1, Clontech Laboratories). In this ELISA-based assay, oligonucleotides containing the CRE-1 consensus binding sequence are immobilized in 96-well plates. When nuclear extracts containing the transcription factors are incubated in the wells, ATF-2 and CRE binding protein (CREB)-1 bind to the consensus sequence. Transcription factor binding is then detected by a specific primary antibody and is quantitated after horseradish peroxidase-conjugated secondary antibody binding and measurement of enzymatic color reaction in a standard microplate reader. Nuclear extracts of control (pH 7.4) and acid-adapted LLC-PK1-FBPase+ cells (15 h at pH 6.9) were prepared as described above (25), except that 40 mM beta -glycerophosphate, 50 mM sodium fluoride, and 200 µM sodium orthovanadate were added as phosphatase inhibitors (34). The state of phosphorylation of ATF-2 in nuclear extract preparations was proven by Western blotting.

Statistical analysis of results was performed using an unpaired Student's t-test.


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Effects of specific MAPK activators and inhibitors on PEPCK mRNA levels in LLC-PK1-FBPase+ cells. Incubation of LLC-PK1-FBPase+ cells in acidic media (pH 6.9) results in an increased level of the cytosolic PEPCK mRNA (13, 17), which is due to a pH-responsive induction of transcription (18, 19). The time course of this response is depicted in Fig. 2. Within 6 h after transfer of cells to acidic media, the 2.7-kb cytosolic PEPCK mRNA is increased approximately threefold. The induced level is then sustained for at least 24 h.


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Fig. 2.   Time course of the pH-responsive increase in cytosolic PEPCK mRNA levels in LLC-PK1-FBPase+ cells. Confluent LLC-PK1-FBPase+ cultures were treated with acidic medium containing 9 mM NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (pH 6.9). At the indicated time points, cells were harvested for RNA isolation. Total RNA samples (20 µg) were separated by agarose gel electrophoresis and blotted onto GeneScreen Plus membranes. The membranes were hybridized with the cytosolic PEPCK mRNA probe generated from pPCK-10 (13, 17, 19). A: representative Northern blot of the 2.7-kb cytosolic PEPCK mRNA. B: quantitation of independent sets of blots by densitometry. Values are means ± SE; n are in parentheses.

To determine a possible involvement of MAPK signaling in the acid-mediated induction of PEPCK mRNA transcription, the effects of specific MAPK activators and inhibitors on PEPCK mRNA levels in LLC-PK1-FBPase+ cells were examined. A detailed pathways diagram is displayed in Fig. 1.

A representative Northern blot is shown in Fig. 3A, and the series of independent experiments are summarized in Fig. 3B. Stimulation of cells with 5 µM AI at pH 7.4 for 2 h increased PEPCK mRNA to levels comparable to acid stimulation. The stimulatory effects of AI and acidic conditions (pH 6.9) were additive, suggesting the activation of different targets upstream of MAPKs. Cycloheximide (100 µM), a second eukaryotic protein synthesis inhibitor, had no significant effect on PEPCK mRNA levels (Fig. 3A). The p38 inhibitor SB-203580 (23, 30) inhibited the acid-induced increase in PEPCK mRNA levels without affecting basal mRNA expression at pH 7.4 (Fig. 3, A and B). SB-203580 produced a dose-dependent inhibition of the acid-stimulated increase in PEPCK mRNA levels (Fig. 4). Complete inhibition was achieved with 10 µM SB-203580 (Fig. 4B). The use of SB-202190, another pyridinyl imidazole inhibitor (23), revealed identical results (data not shown). AI-dependent stimulation of PEPCK mRNA levels under normal (pH 7.4) or acidic conditions (pH 6.9) was also blocked by SB-203580 (Fig. 3, A and B). These data strongly indicate an involvement of the p38 MAPK cascade in mediating the AI-stimulated and the pH-responsive induction of PEPCK mRNA transcription.


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Fig. 3.   Effects of AI, cycloheximide (CHX), and SB-203580 (SB) on control and pH-induced levels of PEPCK mRNA in LLC-PK1-FBPase+ cells. Total RNA was isolated from control cells (CO; pH 7.4) and from cells adapted to acidic medium (pH 6.9) for 18 h. Where indicated, 10 µM SB-203580 was added throughout the adaptation period; 5 µM AI or 100 µM cycloheximide was added 2 h before isolation of RNA. A: representative Northern blots. B: summary of data quantitated by densitometry. Values are means ± SE (n = 7). ** P < 0.01 compared with control (pH 7.4).



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Fig. 4.   SB-203580 produces a dose-dependent inhibition of the pH-responsive induction of PEPCK mRNA levels in LLC-PK1-FBPase+ cells. Cells were adapted to pH 6.9 for 18 h in the absence or presence of the indicated concentrations of SB-203580. Total RNA was then isolated and subjected to Northern blot analysis as described in Fig. 2. A: representative Northern blot of the 2.7-kb cytosolic PEPCK mRNA. B: densitometric quantitation of blots of independent series of experiments. Values are means ± SE; n are in parentheses.

The MEK1/2 inhibitor PD-098059 (11, 34) had no effect on basal or acid-induced PEPCK mRNA levels (Fig. 5A). Experiments with U0126, another MEK1/2 inhibitor (11), produced identical results (data not shown). The effectiveness of PD-098059 in LLC-PK1-FBPase+ cells at the applied concentrations was established by phospho-specific Western blot analysis, as shown in Fig. 5B. LLC-PK1-FBPase+ cultures were serum depleted for 24 h and then stimulated by addition of 10% (vol/vol) fetal bovine serum for 10 min. Preincubation of serum-depleted cultures with 50 µM PD-098059 completely inhibited serum-stimulated ERK1/2 phosphorylation. Curcumin (6) was used to inhibit the SAPK/JNK cascade. As depicted in Fig. 6A, curcumin did not affect acid- and AI-induced PEPCK mRNA levels at a concentration of up to 50 µM. However, AI-stimulated phosphorylation, and thus activation, of JNK1/2 was completely abolished at 20 µM curcumin (Fig. 6B).


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Fig. 5.   Effect of PD-098059 on cytosolic PEPCK mRNA levels and on ERK1/2 phosphorylation in LLC-PK1-FBPase+ cells. A: Northern blot analysis of the 2.7-kb PEPCK mRNA contained in total RNA isolated from control and acid-adapted (PD) LLC-PK1-FBPase+ cells that were treated for 18 h in the absence or presence of the indicated concentrations of PD-098059. B: Western blot analysis of ERK1/2 phosphorylation in whole cell lysates of LLC-PK1-FBPase+ cells. Confluent cultures were serum depleted for 24 h and then stimulated for 10 min by the addition of 10% (vol/vol) fetal bovine serum. Parallel cultures were preincubated with 50 µM PD-098059 for 2 h before serum stimulation. Immunoblots were stained with ERK1/2 antibody to assess equal loading and ERK1/2 expression (top) and with phospho-specific ERK1/2 antibody to assess the extent of phosphorylation (bottom). FCS, fetal calf serum.



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Fig. 6.   Effect of the SAPK/JNK inhibitor curcumin on cytosolic PEPCK mRNA levels and on JNK1/2 phosphorylation in LLC-PK1-FBPase+ cells. A: representative Northern blot of the 2.7-kb PEPCK mRNA contained in total RNA isolated from control and acid-adapted (AI) LLC-PK1-FBPase+ cells that were treated for 18 h in the absence or presence of the indicated concentrations of curcumin. For stimulation with AI, cultures were preincubated for 2 h with 20 µM curcumin and thereafter AI stimulated for 3 h. B: Western blot analysis of JNK1/2 phosphorylation in whole cell lysates of LLC-PK1-FBPase+ cells. Confluent cultures were stimulated with AI for 5 and 30 min. Parallel cultures were preincubated with 20 µM curcumin for 2 h before AI stimulation. Immunoblots were stained with a phospho-specific JNK1/2 antibody to assess the extent of phosphorylation, and thus activation, of the JNK.

Acidic incubation of LLC-PK1-FBPase+ cells induces p38 MAPK and ATF-2 activation. On the basis of the preceding results, further experiments focused specifically on the p38 MAPK pathway. Initial experiments tested whether incubation of LLC-PK1-FBPase+ cells in acidic media (pH 6.9) per se is sufficient to activate the p38 cascade. As depicted in Fig. 7, this treatment resulted in a biphasic phosphorylation, and thus activation, of p38 MAPK with peaks occurring at 0.5-1 and 9 h. A major downstream target of p38 MAPK is the transcription factor ATF-2 (7, 8, 22). An acid-induced phosphorylation of ATF-2 was also evident and occurred with a slight lag. Phosphorylation of ATF-2 peaked at 3 and 9-15 h after transfer of cells to acidic medium.


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Fig. 7.   Acid-mediated phosphorylation of p38 MAPK and of transcription factor ATF-2. Confluent cultures of LLC-PK1-FBPase+ cells were serum starved for 24 h to reduce the potential effects of serum factors. Cells were then switched to serum-free acidic medium (9 mM NaHCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, pH 6.9), and whole cell extracts were isolated at the indicated times. Immunoblot analysis was performed with phospho-specific p38 MAPK and phopsho-specific ATF-2 antibodies (New England Biolabs). Quantitation of six independent phospho-p38 blots was performed by densitometry. Arbitrary units are given as means ± SE. * P < 0.05 and ** P < 0.01 compared with control (pH 7.4).

The direct activation of ATF-2 by the p38 MAPK was further tested by examining the time courses of phosphorylation of p38 and ATF-2 after stimulation of LLC-PK1-FBPase+ cells with AI or hyperosmotic sorbitol (39). The results of a representative series of experiments are summarized in Fig. 8. AI caused a time-dependent increase in phosphorylation of p38, with a peak at 30-60 min after onset of stimulation (Fig. 8A). The phosphorylation of ATF-2 again occurred with a lag compared with p38, with a peak in activation at 60-120 min (Fig. 8B). The AI-induced phosphorylation and activation of ATF-2 was specifically blocked by the p38 inhibitor SB-203580 (Fig. 8C), indicative of signaling through an SB-203580-sensitive isoform of p38 MAPK (20, 23, 30). Hyperosmotic sorbitol showed a slower time course in activating p38 MAPK and ATF-2 compared with AI. Cycloheximide again did not activate either p38 MAPK or ATF-2 (Fig. 8, A and B), which may explain the lack of an effect on PEPCK mRNA levels shown by Northern blot analysis in Fig. 3A.


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Fig. 8.   The effects of AI, cycloheximide, sorbitol (SORB), and SB-203580 on the kinetics of phosphorylation of p38 MAPK and ATF-2. Confluent LLC-PK1-FBPase+ cultures were treated with 5 µM AI, 100 µM cycloheximide, or 250 mosmol/kgH2O sorbitol for the indicated times. Immunoblots of whole cell lysates were separated by SDS-PAGE and stained with anti-phospho p38 MAPK antibody (A) and anti-phospho ATF-2 antibody (B). C: cells were stimulated with 5 µM AI in the absence (CO) or presence of 10 µM SB-203580 (+SB), and cell lysates were probed for ATF-2 phosphorylation. The presented data are representative of experiments performed in duplicate.

ATF-2 from LLC-PK1-FBPase+ cells binds to the CRE-1 element of the PEPCK promoter. Incubation of nuclear extracts from LLC-PK1-FBPase+ cells with a labeled oligonucleotide containing the CRE-1 element of the PEPCK promoter results in the formation of specific complexes that can be resolved on a nondenaturing polyacrylamide gel (25). As shown in Fig. 9, preincubation of the nuclear extract with increasing amounts of antibodies specific for ATF-2 results in the disappearance of the top band and the appearance of a supershifted band. Quantification of the radioactivity contained in each of the shifted bands indicated that a maximum of ~20% of the complexed oligonucleotide was supershifted with the AFT-2-specific antibodies. The percentage supershifted was not increased when nuclear extracts of acid-adapted LLC-PK1-FBPase+ cells were used (data not shown). Thus ATF-2 is one of the proteins contained in LLC-PK1-FBPase+ cells that bind to the CRE-1 element of the PEPCK promoter.


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Fig. 9.   Gel-shift analysis of ATF-2 binding to the CRE-1 element of the PEPCK promoter. Nuclear extracts were prepared from confluent cultures of LLC-PK1-FBPase+ cells. Samples containing 1.6 µg of nuclear protein were preincubated for 45 min with the indicated µl of AFT-2-specific antibody (New England Biolabs). Then 1-µl aliquots of [32P]-labeled CRE-1 oligonucleotide (20 fmol) were added to each sample to yield a final volume of 20 µl. The samples were incubated for an additional 20 min and then separated by nondenaturing PAGE. The presented data are representative of experiments performed in duplicate. Dashed arrow, supershifted bands; solid arrows, bound protein complexes.

The specificity of ATF-2 binding to the CRE-1 element was further confirmed using an ELISA-based binding assay (Mercury TransFactor Kit, Clontech Laboratories). As depicted in Fig. 10, a low, but specific, binding of ATF-2 was obtained with nuclear extracts of control (pH 7.4) LLC-PK1-FBPase+ cells. In a parallel series of experiments, nuclear extracts of LLC-PK1-FBPase+ cells that were adapted to acidic pH (6.9) for 15 h were used. The 15 h of acidic adaptation were chosen because at that time the increase in PEPCK mRNA levels was completed (Fig. 2) and ATF-2 phosphorylation was at its second peak (Fig. 7). A marked phosphorylation of ATF-2 was observed in nuclear extracts of 15-h acid-adapted (pH 6.9) LLC-PK1-FBPase+ cells (Fig. 10, inset), but no increase in ATF-2 binding could be detected. Thus these data are consistent with the results of the gel-shift assays described above. Therefore, in vitro, ATF-2 and phosphorylated ATF-2 bind with equal affinity to the CRE-1 site (3). The TransFactor assay for CREB-1 binding revealed no signals (data not shown), indicating that transcription factor CREB is not present in LLC-PK1-FBPase+ nuclear extracts. Nuclear extracts of PC-12 cells served as positive controls for CREB binding.


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Fig. 10.   Detection of specific binding of ATF-2 to the CRE-1 element of the PEPCK promoter by Mercury TransFactor Kit (Clontech Laboratories). Microtiter 96-well plates coated with oligonucleotides containing the CRE-1 consensus sequence were incubated with nuclear extracts (protein content 2 µg/µl) of control (pH 7.4) and acid-adapted LLC-PK1-FBPase+ cells (pH 6.9 for 15 h). Specificity of ATF-2 binding was determined by incubating nuclear extracts with competitor oligonucleotides (500 ng/µl) of the same nucleotide sequence as the test oligonucleotides coated in the 96-well plate. Nuclear extracts of Jurkat cells that were provided with the assay kit served as positive controls. Horseradish peroxidase-catalyzed color reaction was measured in a microplate reader at 655 nm. Values are means of 2 independent series of experiments. Inset: Western blot of nuclear extracts used in the TransFactor assay, showing maximal phosphorylation of ATF-2 after 15 h of acid adaptation at pH 6.9 (for a complete time course of pH-induced ATF-2 phosphorylation, see Fig. 7). OD, optical density.

LLC-PK1-FBPase+ cells express the alpha -isoform of p38 MAPK and the upstream MKKs. At present, four distinct isoforms of p38 MAPK have been identified: p38alpha (SAPK2a), p38beta (SAPK2b), p38gamma (ERK6 or SAPK3), and p38delta (SAPK4) (20, 22, 30). Thus extracts of LLC-PK1-FBPase+ cells were screened for expression of the four p38 homologs by Western blot analysis using isoform-specific antibodies (Fig. 11A). LLC-PK1-FBPase+ cells strongly express the alpha -isoform of p38 MAPK, whereas the other isoforms are barely detectable. p38delta was present in very low levels, but p38beta and p38gamma were barely detectable even when crude cell homogenates were initially immunoprecipitated with isoform-specific antibodies (data not shown). LLC-PK1 wild-type cells showed an identical pattern of p38 kinase protein expression. HepG2 cells, which have been shown to express all four p38 isoforms (22), served as a positive control. The p38 isoforms are functionally divided into two subgroups: the p38alpha and p38beta isoforms, which are inhibited by the pyridinyl imidazole inhibitor SB-203580, and p38gamma and p38delta isoforms, which are resistant to this inhibitor (23, 30). Because SB-203580 inhibited the pH-mediated and AI-stimulated increases in PEPCK mRNA levels (Figs. 3 and 4) and ATF-2 phosphorylation (Fig. 8C), the above-mentioned Western blot analysis suggests that the alpha -isoform of p38 MAPK mediates the observed responses in LLC-PK1-FBPase+ cells. Further Western blot analysis indicates that all three MKKs upstream of the p38 pathway, MKK3, MKK6, and MKK4, are also present in LLC-PK1-FBPase+ cells (Fig. 11B).


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Fig. 11.   Basal expression of p38 MAPK isoforms and of MAPK kinases (MKK) in LLC-PK1-FBPase+ cells. Whole cell lysates of confluent LLC-PK1-FBPase+ (PK1-F+) cultures grown under standard conditions were separated by SDS-PAGE and immunoblotted. Blots were stained with antibodies (A) against the p38alpha and p38delta isoforms and against upstream MKKs, MKK3, MKK6, and MKK4 (Santa Cruz Biotechnology; B). A: cell lysates from HepG2 cells served as positive controls, and lysates from LLC-PK1 wild-type cells (PK1) were used for comparison. The presented blots are representative of 5-6 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The pH-responsive and gluconeogenic LLC-PK1-FBPase+ cell line (12, 13, 17) provides an effective model system to identify the molecular mechanism by which the onset of metabolic acidosis leads to the cell-specific induction of the mitochondrial glutaminase (PDG) and cytosolic PEPCK activities in renal proximal convoluted tubular cells. Recent studies indicated that the pH-responsive increases in PDG and PEPCK mRNAs in LLC-PK1-FBPase+ cells (13) occur by means of the separate mechanisms previously characterized to occur in rat kidney cortex (19). Furthermore, a decrease in intracellular pH was recently identified as the stimulus that initiates both responses (13). However, the associated signal transduction mechanism that mediates the pH-responsive activation of PEPCK mRNA transcription had not been previously characterized.

Specific gene expression, either in a temporal or tissue-specific manner, or in response to extracellular or environmental stimuli, requires the coordinate activation of specific transcription factors. In recent years, the MAPK family has been shown to play a pivotal role in the control of cellular responses to external stimuli (22, 39). Many important substrates for MAPKs are transcription factors; however, the biochemical links among environmental signals, MAPKs, and transcriptional regulation are largely unknown.

The best understood MAPK pathway is the ERK1/2 cascade, which appears to be involved in growth factor-induced mitogenesis, differentiation, and cellular transformation (34). The SAPKs, JNK and p38 MAPK, represent two independent and parallel MAPK pathways that are activated in response to a variety of extracellular stimuli and cellular stresses, including inflammatory cytokines, heat shock, high osmolarity, ceramides, and TGF-beta (Fig. 1 and Refs. 22, 30, 38, and 39).

The p38 MAPK has been identified as the mammalian homolog of the Saccharomyces cerevisiae osmosensing gene HOG1 (defined as high osmolarity glycerol response 1), which is part of the yeast adaptive response to hyperosmotic stress (22). Similar to HOG1, p38 is activated in response to increases in extracellular osmolarity, suggesting an essential role in the osmoregulation of mammalian cells. Indeed, the p38 pathway is activated in renal cells exposed to extracellular hypertonicity (2, 28, 38). In addition, hypertonicity-mediated induction of mRNAs for betaine transporter in Madin-Darby canine kidney cells (35) and of aldose reductase in HepG2 cells (28) is specifically blocked by the p38 inhibitor SB-203580. In a more recent study (29), in which hypertonicity-induced genes were screened in mouse inner medullary collecting duct-3 cells by subtractive hybridization and cDNA microarray analysis, the induction of 11 of the 12 genes found was also inhibited by SB-203580.

In the present study, the effects of specific activators and inhibitors of the MAPK pathways on basal and acid-induced PEPCK mRNA levels were investigated. The resulting data revealed both activation of the p38 MAPK/ATF-2 cascade when LLC-PK1-FBPase+ cells are incubated in acidic culture media (Fig. 7) and inhibition of the acid-mediated increase in PEPCK mRNA by SB-203580 (Figs. 3 and 4). In addition, the protein synthesis inhibitor AI activated p38 MAPK and ATF-2 and increased PEPCK mRNA to levels similar to those observed during acidosis (Figs. 3 and 8). These effects of AI were also blocked by SB-203580 (Figs. 3B and 8C). Interestingly, cycloheximide, a second inhibitor of eukaryotic protein synthesis, was unable to activate p38 and ATF-2 or to induce PEPCK mRNA transcription (Figs. 3A and 8). Thus the effects of AI are unrelated to its ability to inhibit protein synthesis and are specific to the p38/ATF-2/PEPCK cascade. Finally, gel-shift analysis (Fig. 9) and ELISA-based binding assays (Fig. 10) indicated that ATF-2 is at least one of the transcription factors contained in nuclear extracts of LLC-PK1-FBPase+ cells that binds to the CRE-1 element of the PEPCK promoter. In total, the reported data strongly support the hypothesis that the pH-responsive induction of PEPCK mRNA transcription is mediated by phosphorylation of p38alpha MAPK that, in turn, phosphorylates and activates ATF-2.

A Western blot survey revealed that p38alpha is the major isoform expressed in LLC-PK1-FBPase+ cells (Fig. 11A). The upstream MKKs of p38, MMK3 and MKK6 (22), were also detectable in LLC-PK1-FBPase+ cells. In addition, MKK4 (39) was expressed in these cells (Fig. 11B). The finding that all three MAP/SAP kinases, ERK1/2, JNK, and p38 (39), can be phosphorylated in LLC-PK1-FBPase+ cells (Figs. 5-8) indicates that all of the essential components of each MAPK pathway are present in these cells.

Recent reports showed that incubation of MCT cells, an SV40-transformed mouse proximal tubule cell line, in acidic medium induced a twofold increase in c-Src activity, a soluble tyrosine kinase, which was paralleled by an increased phosphotyrosine content of cytosolic proteins (1, 40). These studies suggest that a decrease in extracellular pH may lead to activation of c-Src or a related tyrosine kinase, causing increased expression of Fos and Jun, which in turn activate transcription of pH-responsive genes (40). However, the tyrosine kinase inhibitors, herbimycin A, genistein, or tyrphostin (Fig. 1), had no effect on PEPCK mRNA levels in LLC-PK1-FBPase+ cells (data not shown), confirming earlier findings (18). Taken together, these results indicate that ERK1/2 (Fig. 5), SAPK/JNK (Fig. 6), and tyrosine phosphorylation by c-Src (40) do not play a significant role in pH-induced transcription of cytosolic PEPCK mRNA in renal LLC-PK1-FBPase+ cells.

The transcriptional regulation of the gene that encodes the cytosolic isoform of PEPCK has been studied primarily in liver tissue (reviewed in Refs. 8, 15, 16). The sequence of the promoter for the PEPCK gene from mice, rats, and humans has been remarkably conserved (>95% sequence identity). Thus mechanisms of transcriptional regulation deduced from studies with the rodent PEPCK promoter are likely to be characteristic of the control in most mammalian species. The initial 490 bp of the rat PEPCK gene are very complex. They contain at least 12 separate elements that mediate the hormonal and dietary control of PEPCK gene expression in the liver. However, transcription of PEPCK mRNA in the liver and kidney are differentially regulated. Hepatic gluconeogenesis is primarily involved in the maintenance of blood glucose homeostasis, whereas renal gluconeogenesis is linked to ammoniagenesis and the maintenance of acid-base balance. Thus pH-responsive regulation of PEPCK gene expression occurs only in the kidney (13, 17-19, 36).

Previous studies have shown that binding of hepatocyte nuclear factor-1 to the P2 element (-200 to -164) of the PEPCK promoter is essential for basal expression of PEPCK in the kidneys of transgenic mice (5, 15). In addition, mutation or deletion of the P2 element in PEPCK-reporter gene constructs significantly decreased expression in LLC-PK1-FBPase+ cells (18). Furthermore, studies by Cassuto et al. (5) showed that the P2 site may also be required for a full induction of PEPCK activity in response to acidic pH. Similar studies have also implicated the CRE-1 and the P3(II) region as elements that contribute to the pH-responsive induction of the PEPCK gene (18).

The P3(II) region contains a 7-bp sequence, TTAGTCA, that binds activator protein-1 (AP-1; Jun/Fos heterodimers). This sequence differs from an AP-1 consensus (TGAGTCA) sequence by a single G-to-T substitution. The CRE-1 or cAMP response element within the PEPCK promoter is located (-91 to -84) ~60 bp 5' from the TATA box (-29 to -23) (15, 16). CREs are highly conserved regulatory elements found in numerous cellular genes that are induced by cAMP. They typically consist of an 8-bp palindromic consensus sequence (TGACGTCA) located within 100 nucleotides of the TATA box. Within the promoter of the cytosolic PEPCK gene, the sequence of the CRE-1 element (TTACGTCA) again diverges from the consensus sequence by a single G-to-T substitution (8, 15, 16, 32). Several members of the leucine zipper-containing transcription factors have been shown to bind to the CRE-1 element, including CREB, CCAAT/enhancer binding protein (C/EBP)alpha , C/EBPbeta , AP-1, and Jun/Jun homodimers (8, 15, 25).

Previous studies using dominant-negative forms of CREB and of C/EBP (24, 31) and more recent experiments using GAL4-chimeric constructs (25) revealed that binding of C/EBPbeta , and not CREB, to CRE-1 mediates the cAMP-dependent activation of PEPCK mRNA transcription in subconfluent LLC-PK1-FBPase+ cells (see Fig. 1). In an earlier gel-shift analysis, the shift produced by incubating the CRE-1 element with a nuclear extract of LLC-PK1-FBPase+ cells was supershifted with an antibody against C/EBPbeta but not with an antibody against CREB (24). This is in line with data from an ELISA-based binding assay used in the present study, in which no CREB-1 binding signals could be detected, indicating that CREB is not present in LLC-PK1-FBPase+ nuclear extracts.

LLC-PK1-FBPase+ cells differ significantly in their responses to either cAMP or an acidic environment, depending on the state of confluence of the cultures and thus on the state of differentiation. The cAMP-responsive induction of PEPCK gene transcription, mediated by C/EBPbeta , is maximal in subconfluent cells (24, 25), whereas the pH response, triggered by SB-203580-sensitive p38 MAPK signaling, is maximal in confluent, fully differentiated LLC-PK1-FBPase+ monolayer cultures or in LLC-PK1-FBPase+ epithelia grown on permeable culture supports (13, 17, 18). The time point in culture duration or state of confluency, respectively, of this transition as well as the molecular mechanisms are unknown at present.

Recently, it was recognized that the PEPCK CRE-1 sequence TTACGTCA is identical to the consensus sequence required for binding of ATF-2 homodimers (7). This study demonstrated that in Fao hepatoma cells, a sodium arsenite-induced activation of PEPCK mRNA transcription was mediated by p38 MAPK transactivation of ATF-2. ATF-2 is a basic-leucine zipper transcription factor that exhibits transcriptional activation after dual phosphorylation on Thr69 and Thr71. Activated ATF-2 forms a homodimer or heterodimer with c-jun, binds to CREs, and stimulates CRE-dependent transcription of genes (3). These observations provide the basis on which to interpret the data obtained in the present study. They support the conclusion that phosphorylation of ATF-2 by the SB-203580-sensitive alpha -isoform of p38 MAPK may mediate the increased transcription of cytosolic PEPCK mRNA during metabolic acidosis.

On the basis of the existing data, the following model (Fig. 12) was developed as a hypothesis for the mechanism by which PEPCK mRNA transcription is induced in the renal proximal convoluted tubule during metabolic acidosis. During normal acid-base balance, C/EBPbeta and ATF-2 are constitutively bound to the CRE-1 site. In addition, the P2 and possibly the P3(II) regions of the PEPCK promoter are occupied with bound transcription factors. Phosphorylation of C/EBPbeta by protein kinase A leads to nuclear import (27), and phosphorylation of ATF-2 by p38 MAPK exposes its transcriptional activation domain (26). These observations may explain how C/EBPbeta and ATF-2 could act through the same element to mediate different responses. Thus a decrease in intracellular pH leads to activation of the alpha -isoform of p38 MAPK that in turn phosphorylates and activates ATF-2. The activated ATF-2 then recruits the "auxiliary" factors and/or coactivators that are necessary for transcriptional activation of the PEPCK gene. Similar studies of tumor necrosis factor receptor 1 signaling (3) demonstrated that autoregulation of TNF-alpha gene expression is also mediated through the p38-dependent phosphorylation of ATF-2/Jun heterodimers that are bound to the TNF-alpha CRE promoter element. The associated gel-shift analysis using nuclear extracts from unstimulated or rhTNF-alpha -stimulated L929 cells again demonstrated no differences in ATF-2 binding. Thus the binding of ATF-2 to a CRE element is not affected by phosphorylation.


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Fig. 12.   Model of the pH-responsive induction of PEPCK mRNA transcription during metabolic acidosis. At pH 7.4, basal transcription of the PEPCK gene is mediated by binding of hepatocyte nuclear factor-1 (HNF-1) and possibly c-Fos/c-Jun to the P2 and P3(II) elements, respectively. The CRE-1 site of the PEPCK promoter is occupied with nonphosphorylated ATF-2 and/or C/EBPbeta . A decrease in intracellular pH leads to phosphorylation of p38alpha MAPK, which in turn phosphorylates ATF-2 at Thr69 and Thr71. Subsequently, the activated ATF-2 recruits or interacts with one or more coactivator. The resulting complex facilitates the recruitment of RNA polymerase II (Pol II) or modifies the associated nucleosomes, leading to activated transcription of the PEPCK mRNA.

Recent experiments suggest that additional cis-acting elements either upstream (10) or downstream (9) of the proximal promoter of the PEPCK gene may be necessary for the pH-responsive induction of PEPCK mRNA transcription. Thus additional experiments will be required to identify and characterize the additional elements and to further test this hypothesis.


    ACKNOWLEDGEMENTS

This work was supported by Austrian Science Fund Grants P12705 and P14981 (to G. Gstraunthaler) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43704 (to N. P. Curthoys). Parts of this study were presented at the Annual Meetings of the American Society of Nephrology in Philadelphia, PA, 1998 (J Am Soc Nephrol 9: 52A, 1998), Miami Beach, FL, 1999 (J Am Soc Nephrol 10: 52A, 1999), and San Francisco, CA, 2001 (J Am Soc Nephrol 12: 48A, 2001).


    FOOTNOTES

Address for reprint requests and other correspondence: G. Gstraunthaler, Dept. of Physiology, Univ. of Innsbruck, Fritz-Pregl-Str. 3, A-6010 Innsbruck, Austria (E-mail: gerhard.gstraunthaler{at}uibk.ac.at).

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.

10.1152/ajprenal.00097.2002

Received 12 March 2002; accepted in final form 6 May 2002.


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DISCUSSION
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