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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ABSTRACT |
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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 p38
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
p38
-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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INTRODUCTION |
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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
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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MATERIALS AND METHODS |
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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
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
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-p38,
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.
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RESULTS |
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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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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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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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LLC-PK1-FBPase+ cells
express the -isoform of p38 MAPK and the upstream MKKs.
At present, four distinct isoforms of p38 MAPK have been
identified: p38
(SAPK2a), p38
(SAPK2b), p38
(ERK6 or SAPK3),
and p38
(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
-isoform of p38 MAPK, whereas the other isoforms are barely
detectable. p38
was present in very low levels, but p38
and
p38
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 p38
and p38
isoforms, which are inhibited by the
pyridinyl imidazole inhibitor SB-203580, and p38
and p38
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
-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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DISCUSSION |
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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- (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 p38 MAPK that, in
turn, phosphorylates and activates ATF-2.
A Western blot survey revealed that p38 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)
, C/EBP
, 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/EBP, 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/EBP
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/EBP, 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 -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/EBP 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/EBP
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/EBP
and ATF-2 could act through the same element to
mediate different responses. Thus a decrease in intracellular pH leads
to activation of the
-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-
gene expression is also mediated through the
p38-dependent phosphorylation of ATF-2/Jun heterodimers that are bound
to the TNF-
CRE promoter element. The associated gel-shift analysis
using nuclear extracts from unstimulated or rhTNF-
-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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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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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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