1 Renal Division, Emory University School of Medicine, Atlanta, Georgia 30322; 2 Division of Human Genetics, Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201, and 3 Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287
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ABSTRACT |
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Acidification or
glucocorticoids increase the maximal activity and subunit mRNA levels
of branched chain -ketoacid dehydrogenase (BCKAD) in various cell
types. We examined whether these stimuli increase transcription of
BCKAD subunit genes by transfecting BCKAD subunit promoter-luciferase
plasmids containing the mouse E2 or human E1
-subunit promoter into
LLC-PK1 cells, which do not express glucocorticoid
receptors, or LLC-PK1-GR101 cells, which we have engineered
to constitutively express the glucocorticoid receptor gene.
Dexamethasone or acidification increased luciferase activity in
LLC-PK1-GR101 cells transfected with the E2 or
E1
-minigenes; acidification augmented luciferase activity in
LLC-PK1 cells transfected with these minigenes but
dexamethasone did not. A pH-responsive element in the E2 subunit
promoter was mapped to a region >4.0 kb upstream of the transcription
start site. Dexamethasone concurrently stimulated E2 subunit promoter
activity and reduced the binding of nuclear factor-
B (NF-
B) to a
site in the E2 promoter. Thus acidification and glucocorticoids
independently enhance BCKAD subunit gene expression, and the
glucocorticoid response in the E2 subunit involves interference with
NF-
B, which may act as a transrepressor.
acidosis; branched-chain amino acids; gene expression; branched-chain ketoacid dehydrogenase
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INTRODUCTION |
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BRANCHED-CHAIN AMINO
ACIDS (BCAA) are the most common essential amino acids in
protein, and they can influence metabolic pathways in muscle and other
tissues. For example, leucine, its ketoacid -ketoisocaproate, or
3-methylvalerate, the irreversibly decarboxylated product of
-ketoisocaproate, suppresses muscle protein degradation. [Valine,
isoleucine, and their metabolites do not influence muscle protein
degradation in isolated rat muscles (41).] Amino acids including leucine stimulate protein synthesis in muscle (30, 41,
51) and adipocytes (18, 34) by enhancing
translation initiation. Jefferson and colleagues (18, 30, 34,
51) found that leucine stimulates translation initiation by
enhancing the activity of eukaryotic initiation factor-4E (eIF-4E)
through mammalian target of rapamycin (mTOR)-dependent
mechanisms. Leucine also stimulates insulin production, and
insulin modulates protein turnover and other metabolic processes
(21, 42, 44). Thus it is not surprising that BCAA
degradation is tightly controlled in normal adults and in patients with
kidney disease (35, 36, 50).
Branched-chain -ketoacid dehydrogenase (BCKAD) regulates BCAA
disposal by catalyzing their irreversible oxidative decarboxylation. The BCKAD complex is located in mitochondria and is composed of unique
E1
, E1
, and E2 subunits and an E3 subunit that is also present in
pyruvate dehydrogenase. BCKAD activity can be regulated by BCKAD
kinase, which phosphorylates the E1
subunit, inhibiting BCKAD
activity. In muscle, BCKAD is primarily in the inactive, phosphorylated
state (2-5% of enzyme is active), whereas its activity state (the
proportion of dephosphorylated BCKAD) is much higher in kidney
(60-70% in the active state) and other tissues (23). We and others have documented concurrent changes in total BCKAD activity (a measure of enzyme content), BCKAD subunit proteins, and the
mRNAs encoding BCKAD subunits (i.e., E1
, E2) in response to acidosis
and other physiological stimuli (e.g., glucocorticoids, insulin)
(7, 8, 10, 17, 45, 52). Because acidosis results in higher
glucocorticoid production, it is difficult to separate the two signals
in vivo (3, 17, 37).
To separate the influences of acidification from those of
glucocorticoids, we studied how these signals change BCKAD activity in
LLC-PK1 pig kidney cells because: 1) the kidney
is a major organ for BCAA degradation in humans (49), and
proximal tubules constitute the bulk of the kidney mass; and
2) LLC-PK1 cells do not respond to
glucocorticoids (25, 52). By generating
glucocorticoid-responsive LLC-PK1 cells through stable
transfection of LLC-PK1 cells with an expression vector encoding the
rat glucocorticoid receptor (LLC-PK1-GR101 cells)
(52), we were able to study the effects of acidosis and
glucocorticoids separately in the same general cell type. We found that
either a low extracellular pH or dexamethasone can independently
stimulate BCKAD activity and increase the amount of enzyme subunit
proteins in these kidney cells. We have extended our findings in kidney
cells by showing that a low extracellular pH and/or glucocorticoids
stimulate the promoters of the BCKAD E1 and E2 subunit genes.
Recently, we showed that nuclear factor-
B (NF-
B) participates in
the transcriptional regulation of the proteasome C3 subunit by
glucocorticoids in L6 muscle cells (15), and we noted that
there are NF-
B-like sites in the mouse BCKAD E2 subunit promoter.
Therefore, we evaluated whether NF-
B is involved in the
glucocorticoid-dependent regulation of BCKAD E2 subunit transcription.
Our results provide evidence of genetic mechanisms that act to regulate
BCKAD function in response to catabolic stimuli.
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METHODS |
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BCKAD subunit promoter-luciferase minigenes.
Promoter-luciferase chimeric minigenes containing the 5'-flanking
region [7,000 to 140 base pairs (bp)] of the murine BCKAD E2 subunit
gene have been described (11). To evaluate whether NF-B
is involved in the regulation of BCKAD subunit transcription by
glucocorticoids, we changed the 10-base NF-
B-like motif in pE2-0.14
to 5'-TAGTTTAGAT-3' using substituted-primer PCR. A more conservative
alteration was also made by changing the first three bases to
5'-AAATCTTTCC-3' in pE2-0.14. [This should not disrupt the NF-
B p65 subunit interaction site (31)].
These sequence changes were verified by DNA sequencing.
Cell culture and transfections.
LLC-PK1 and LLC-PK1-GR101 cells were maintained
and transfected as described (52). Cells were transfected
with 3 µg of the E2 subunit minigene plasmids or pE1-0.8 kb plus
6.5 µg of salmon testis DNA using calcium phosphate; 0.5 µg of a
control plasmid DNA (pSV
encoding
-galactosidase or pRL-TK
encoding renilla luciferase) was cotransfected to correct for
differences in transfection efficiency (52). Control cells
were cotransfected with pGL2 Basic without a promoter element and a
control reporter plasmid. Subsequently, cells were maintained
in DMEM with 10% fetal bovine serum (FBS; without hygromycin) for
24 h followed by DMEM with 10% charcoal-treated FBS for 48 h
(52). Some cells were incubated with 50 nM
dexamethasone and/or acidified (pH 7.0) media for 24 h. The
biological potency of this concentration of dexamethasone is ~15
times higher than the circulating level of cortisol in an adult
(46). To ensure that dexamethasone responses were mediated through the glucocorticoid receptor, cells were incubated with dexamethasone and an equimolar concentration of the steroid receptor antagonist RU-486 (added 1 h before dexamethasone) (28,
52). The media pH was stable during the 24-h treatment period
(52).
Luciferase activity.
Firefly luciferase activity was measured as described by Brasier et al.
(4). Briefly, cells were harvested in lysis buffer [25 mM
Tris, pH 7.8, 4 mM EGTA, 1% Triton X-100, 10% glycerol, and 2 mM
dithiothreitol (DTT)]. Samples were diluted (1:8) with assay buffer
(25 mM Tris, pH 7.8, 4 mM EGTA, 20 mM MgSO4, 2 mM ATP, and
1 mM DTT), and luminescence was measured with a Turner model TD-20/20
luminometer after the addition of D-luciferin. We assessed
transfection efficiencies by either 1) dividing the firefly
luciferase activity by the respective -galactosidase activities
after normalizing each activity for protein content (33)
or 2) measuring the firefly and renilla luciferase
activities in the same sample with the Dual Luciferase Reporter Assay
system (Promega).
Electrophoretic mobility shift assays.
Nuclear protein extracts were prepared from LLC-PK1-GR101
cells according to Dignam et al. (14). The sense strand of
the DNA probes used in the binding assays was
5'-GAGGAGGCGTCTTTCCCAGCTG-3' for the normal NF-B motif
or 5'-GAGGAGATCAAATCTACAGCTG-3' for the mutant NF-
B
site. Binding reactions were performed as described (15).
Reaction products were separated in 4% polyacrylamide, 2.5% glycerol
gels with 0.5× TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) and
visualized with a Molecular Dynamics Storm imaging system.
Statistical analyses.
Results (means ± SE) between treatment groups were compared with
an unpaired Student's t-test, but for three or more groups, significance was evaluated by one-way ANOVA followed by Tukey's t-test. In all cases, a value of P 0.05 was
considered significant.
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RESULTS |
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Glucocorticoids induce BCKAD E1 and E2 transcription.
To evaluate the influence of glucocorticoids on BCKAD subunit
promoters, LLC-PK1-GR101 cells were transfected with a
BCKAD E1
or E2 subunit promoter-luciferase reporter plasmid. [These cells respond to dexamethasone (52).] Basal luciferase
activity (without dexamethasone) in cells transfected with either the
pE2-7.0 or pE1
-0.8 kb subunit-promoter minigenes was higher than in
cells transfected with the control pGL2 Basic luciferase expression plasmid with no promoter (P < 0.05; Fig.
1A). After dexamethasone (50 nM) was added for 24 h, firefly luciferase activity in cells transfected with pE2-7.0 was increased by 127% (P < 0.05 vs. no dexamethasone). Dexamethasone stimulated pE1
-0.8
minigene luciferase activity 259% (P < 0.01 vs. no
dexamethasone; Fig. 1B). When cells transfected with either
pE2-7.0 or pE1
-0.8 were incubated with equimolar concentrations of
dexamethasone plus RU-486, luciferase activity was not increased (Fig.
1B). In a preliminary study, RU-486 alone did not change
luciferase activity in cells transfected with pE2-7.0 (data not shown),
whereas renilla luciferase activity in cells transfected with pRL-TK
(controlled by the thymidine kinase promoter) was not increased by
dexamethasone (37.3 ± 5.4 light U/mg without dexamethasone vs.
36.8 ± 3.8 with dexamethasone; P, not
significant). Thus glucocorticoids specifically stimulate the
E1
and E2 subunit promoters by mechanisms requiring the
glucocorticoid receptor.
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The E2 subunit glucocorticoid-responsive element is an NF-B
binding site.
E2 promoter-luciferase reporter gene plasmids with progressively
shorter fragments of the proximal 5'-flanking region (Fig. 2A) were transfected into
LLC-PK1-GR101 cells to localize the region of the promoter
that responds to glucocorticoids. Minigenes containing
140 bp of E2
promoter were stimulated by dexamethasone but to a variable degree
(i.e., between a 2- and 4-fold increase; Fig. 2B). Maximal
activation by dexamethasone was measured in cells transfected with the
E2 minigene containing ~900 bases of 5'-flanking region (pE2-0.9 kb).
With additional distal promoter sequence, induction of luciferase
activity by dexamethasone was less effective (Fig. 2B).
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Acidification stimulates transcription of BCKAD E2 and
E1-subunits.
To determine if extracellular acidification activates the BCKAD E2 or
E1
-promoters, we transfected steroid-receptor null LLC-PK1 cells with the BCKAD subunit promoter-luciferase
minigenes (25, 52). These cells make it possible to
distinguish responses to a reduced extracellular pH from those of the
glucocorticoid receptor (25, 52). Acidification (24 h)
increased luciferase activity from pE2-7.0 kb by 87%
(P < 0.05 vs. pH 7.4) but not in cells transfected
with pE2-0.3 kb (Fig. 4A).
Likewise, acidification increased luciferase activity from pE1
-0.8
kb by 60% (P < 0.05 vs. pH 7.4; Fig. 4A).
Dexamethasone did not increase luciferase activity in any of the
transfected cells. Similar responses were obtained when
LLC-PK1-GR101 cells were transfected with the pE2-7.0 or
pE1
-0.8 kb minigenes; however, acidification did not increase luciferase activity in cells transfected with E2 minigenes containing ~4,000 bp or less of promoter sequence (Fig. 4B). Thus
acidification acts independently of glucocorticoids to stimulate E2 and
E1
gene expression.
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Acidification and glucocorticoids act cooperatively to stimulate
BCKAD subunit transcription.
To evaluate if extracellular acidification and glucocorticoids can act
cooperatively to increase E2 or E1 transcription, LLC-PK1-GR101 cells were transfected with pE2-7.0 or
pE1
-0.8 kb and then were subjected to one or both stimuli together.
With either subunit minigene, dexamethasone plus acidification
increased luciferase activity more than in cells incubated with either
dexamethasone or a low pH alone (Fig. 5).
In cells transfected with the pE2-0.3-kb minigene, dexamethasone plus
acidification increased luciferase activity to a level similar to that
measured with glucocorticoids alone (Fig. 5).
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DISCUSSION |
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In patients or rats with uremia or metabolic acidosis, the plasma
levels of BCAA are reduced (2, 19, 22, 37, 39). In humans,
the tissues that contribute the most to whole body leucine oxidation
are skeletal muscle and kidney (49). In muscle, catabolic
stimuli (e.g., acidosis or glucocorticoids) increase the steady-state
levels of subunit mRNAs and total BCKAD activity concurrently (7,
8, 10, 17, 20, 45, 48). In intact rats, it is difficult to
separate the effects of acidosis from those of glucocorticoids because
glucocorticoid production is stimulated by acidosis (38).
Therefore, we studied the regulation of BCKAD activity in
LLC-PK1 and LLC-PK1-GR101, because these cells
allowed us to determine the separate influences of acidosis and
glucocorticoids in the same general cell type. We found that acidification and glucocorticoids independently increase BCAA catabolism and the amounts of mitochondrial BCKAD subunit proteins (52). Our current findings demonstrate that acidosis or
glucocorticoids stimulate the transcription of the E2 and E1 BCKAD
subunit genes through unique cis-acting elements under
conditions that also increase BCKAD activity.
We were surprised to find that stimulation of the mouse E2 subunit
promoter by glucocorticoids involves a binding site for the
transcription factor NF-B. First, we found that
dexamethasone-induced stimulation of the pE2-0.14 kb reporter gene was
prevented by changing the base sequence of the NF-
B binding motif at
position
28 to
19. When the first three bases of the NF-
B site
were changed, basal luciferase activity increased 12-fold, suggesting that NF-
B suppresses E2 subunit promoter activity. Second, an abundant nuclear protein in untreated LLC-PK1-GR101 cells
binds to the NF-
B binding site in the E2 subunit promoter, and
incubating cells with dexamethasone reduced the binding of this nuclear
protein. Formation of the protein-DNA complex was blocked by polyclonal antibodies against NF-
B p65, suggesting that protein epitopes recognized by the p65 antibodies are in close proximity to the DNA
binding domain of the protein. Thus, glucocorticoids stimulate E2
subunit transcription by reducing the binding of NF-
B and, hence,
its suppressor action on the E2 subunit promoter in LLC-PK1-GR101 cells. This is a different mechanism of glucocorticoid stimulation than
occurs in cultured hepatic cells (12). In H4-II-E-C3
hepatoma cells, E2 subunit transcription is increased by dexamethasone, but the response involved the
140 to
70 region of the E2 subunit promoter, which is upstream of the NF-
B site that is important for
the response to glucocorticoids in LLC-PK1-GR101 cells.
Notably, we found that dexamethasone increased the transcription of the proteasome C3 subunit gene in L6 muscle cells by a mechanism that is
similar to the one we identified for the glucocorticoid-mediated induction of the BCKAD E2 subunit promoter (15). In both
cases, dexamethasone interferes with the binding of NF-
B, which acts as a transcriptional suppressor for both promoters. The results of our
studies with the BCKAD E1
-subunit promoter suggest that glucocorticoids stimulate this promoter by a NF-
B-independent mechanism, because a NF-
B binding site was not present in the segment of the human E1
promoter that we used in the transfection experiments.
We did not determine how glucocorticoids interfere with NF-B binding
to the E2 subunit promoter, but activated glucocorticoid receptors can
block NF-
B-induced transcription of inflammatory genes (e.g.,
interleukin-6, intercellular adhesion molecule-1) by directly
interfering with their binding to DNA response elements and, hence,
transactivation (13, 32, 40). In contrast to the these
reports, NF-
B appeared to be a transrepressor of the E2 subunit gene
in our studies. Other potential mechanisms include a
glucocorticoid-induced increase in the amount of inhibitory protein of
NF-
B (I
B) leading to sequestration of NF-
B in the cytosol
(15, 47).
Acidification has been shown to increase transcription of a few specific genes, including renal glutaminase, sodium-hydrogen exchanger-3, and most notably, phosphoenolpyruvate carboxykinase (PEPCK) in rat kidney and proximal tubule cells (5, 6, 24, 26). In the kidney, PEPCK expression is stimulated by either acidosis or glucocorticoids, but the location in the PEPCK promoter where acidification exerts its effects and the identity of the acidification-induced transactivating factor(s) binding to the PEPCK promoter have not been conclusively identified (6, 24, 43). Cassuto et al. (6) reported that a HNF-1 (P2) recognition motif is required for pH sensitivity, whereas Holcomb et al. (24) identified other promoter regions that appear to be crucial. In both cases, the upstream elements that were reported to be responsive to acidification were located close to the transcription initiation site.
How could acidification stimulate E2 or E1 promoters? The
intracellular pH in cultured muscle and LLC-PK1 cells
decreased when the extracellular pH was reduced (16, 29).
In acidemic rats, including those with chronic uremia, the
intracellular pH in muscle was not different from that of control rats.
In rats that were treated identically, BCKAD activity and the levels of BCKAD subunit mRNAs were increased (1, 22). Thus it is
plausible that extracellular acidification stimulates an intracellular
signaling pathway with effectors that can regulate the transcription of genes including the BCKAD E1
and E2 subunits. In one case, acute acidification increased the transcription of a number of immediate early genes (e.g., Egr-1, c-fos,
c-jun) in vivo in rat renal cortical cells and in simian
virus 40-transformed, mouse proximal tubule cells (MCT cells)
(53). Our current results indicate that there is
an acidification-responsive element in the E1
and E2 BCKAD subunit
promoter regions. In the E2 subunit gene, this cis-acting element does not require a position close to the transcription initiation site as it is >4,000 bp upstream. In contrast, the acidification-response element in the E1
gene is located in a position more proximal (<710 bp) to the transcription start site. If
the acidification-responsive elements in the regions upstream of the
E1
and E2 genes are identical, we conclude that they do not require
a specific location in the gene (e.g., adjacent to the transcription
start site). Alternatively, the response elements responding to
acidification in the E1
and E2 genes may be different.
In summary, we have shown that two BCKAD subunit promoters are activated by dexamethasone or acidified media. The stimuli also increased BCKAD subunit protein and total enzyme activity in LLC-PK1-GR101 cells (52). The concurrent nature of these responses suggests that increased BCKAD subunit transcription could be a mechanism for the increase in BCKAD subunit proteins in these renal proximal tubular cells. Such a mechanism, coupled with activation/inactivation of the BCKAD enzyme by a kinase-phosphatase system, would allow cells to precisely regulate the activity of BCKAD, the key enzyme responsible for the degradation of the essential BCAA in different catabolic conditions.
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ACKNOWLEDGEMENTS |
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We thank Drs. D. Danner and W. Mitch for helpful discussions and encouragement.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50740 (S. R. Price) and the Life and Health Insurance Medical Research Fund (J. M. Chinsky).
Address for reprint requests and other correspondence: S. R. Price, Renal Div., Rm. 338 WMB, 1639 Pierce Dr., Emory Univ., Atlanta, GA 30322 (E-mail: medrp{at}emory.edu).
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.
Received 3 August 2000; accepted in final form 18 December 2000.
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