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3
Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA;
*
Department of Surgery, Pennsylvania State University, Hershey School of Medicine, Hershey, PA; and
Department of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, Albuquerque, NM
3To whom correspondence should be addressed. E-mail: sabcouwer{at}salud.unm.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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KEY WORDS: • glutamine • glutamine synthetase • 26S proteosome
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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GLN catabolism is initiated by the deamidation of the molecule to form
glutamate. This can proceed via a number of cytosolic transamidase
enzymes that use the 
-amido nitrogen of GLN in a variety of
metabolic syntheses (Zalkin and Smith 1998
). However,
the rate at which these reactions utilize GLN depends ultimately upon
the metabolic demand for the reaction products and is, therefore, not
appropriate for control of GLN homeostasis. On the other hand, the
mitochondrial enzyme GA catalyzes the hydrolysis of the -amido group
of GLN to form glutamate and ammonia. This ammonia can be used to form
carbamoyl phosphate or can diffuse from the mitochondria and the cell
itself. Glutamate can be further deaminated to form 
-ketoglutarate
and thus enter the citric acid cycle. Thus, the catabolism of GLN
through GA can be increased without the production of excessive amounts
of specific metabolites.
In contrast to the many enzymes that utilize GLN as a substrate, only one enzyme, GS, is responsible for de novo synthesis of GLN. This ligase catalyzes the formation of GLN from glutamate and ammonia in the cytoplasm. Because both of these substrates are relatively abundant and therefore not limiting, the rate of GLN formation is highly dependent upon the activity of GS. To date, no one has found that the specific activity of the mammalian GS is affected by post-translational modifications (e.g., adenylation, phosphorylation). Thus, the intracellular content of GS protein and, therefore, the GS expression level, is a major determining factor of GLN production rate.
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Mechanisms by which GA expression is regulated |
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TOPABSTRACTINTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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, Watford et al. 1984 and 1985
). In all of
these states, there is a heightened catabolism of amino acids, whether
derived from muscle stores or from dietary sources. The purposes of
this up-regulation of GA expression and increased GLN catabolism
are partly to support increased gluconeogenesis, which requires
precursors derived from the carbon skeleton of GLN (Nurjhan et al. 1995
, Stumvoll et al. 1998
), and to deliver
additional nitrogen to the urea cycle for disposal (Nissim et al. 1992
, Watford 1993
). Watford and colleagues
demonstrated that, at least in the case of high protein diets and
starvation, up-regulation of GA activity was due to elevated GA
mRNA expression via increased transcription of the hepatic GA gene
(Watford and Smith 1989
, Watford et al. 1994
). Hepatic GA transcription is probably up-regulated by
a cAMP-dependent mechanism (Watford and Smith 1989
).
It has been established that hepatic GA expression is responsive to
glucagon and drugs that increase cAMP (Brosnan et al. 1994
, Häussinger et al. 1983
, Lacey et al. 1981
, McGivan et al. 1991
). Thus,
transcription of hepatic GA and expression of GA activity in liver is
increased under conditions of low insulin and/or high glucagon levels.
Indeed, our laboratory has shown that injection of insulin or glucose
feeding of food-deprived rats rapidly returns hepatic GA mRNA
expression to normal levels (Elgadi et al. 1997
). Recent
cloning and analysis of the rat hepatic GA promoter revealed a
cAMP-responsive element (CRE) in this gene (Chung-Bok et al. 1997
). The presence of this promoter element would seem to
explain the gene’s responsiveness to glucagon and insulin. However, in
the hepatoblastoma model system employed to analyze the promoter, the
transcriptional activity of the promoter was not increased by
8-bromo-cAMP (Chung-Bok et al. 1997
). Surprisingly, the
promoter was responsive to glucocorticoid hormone when a functional
glucocorticoid receptor was overexpressed. This is an unexpected
finding because published data do not suggest that glucocorticoid
hormones control GA expression in the liver (McGivan et al. 1991
).
In a rat model of chronic metabolic acidosis, ammonia excretion and the
expression of kidney GA activity increase appreciably in cells of the
proximal convoluted tubule (Curthoys and Lowry 1973
,
Davies and Yudkin 1952
, Leonard and Orloff 1955
). One purpose of up-regulating kidney GA expression is
to increase the production of ammonia in the kidney, so that it may
sequester hydronium ions and allow their excretion in ammonium. This
results in the disposal of protons and amelioration of acidosis.
Increased GA activity in the kidney coincides with increased GA protein
and with up-regulation of kidney-type GA mRNA expression
(Curthoys et al. 1976
, Hwang and Curthoys 1991
, Tong et al. 1986 and 1987
). However, in
this case, the heightened mRNA levels are not due to an increase in
kidney GA gene transcription; rather, they arise from a
post-transcriptional regulatory mechanism (Hwang and Curthoys 1991
, Hwang et al. 1991
). Using a cell
culture model, Curthoys and colleagues demonstrated that expression of
GA mRNA increases under acidic culture conditions due to stabilization
of this mRNA and a reduced rate of mRNA decay (Hwang et al. 1991
). The rates at which specific mRNAs are degraded in the
cytoplasm are not random; rather, they are controlled by various RNA
binding proteins (RBP) that recognize and bind to specific sequence
elements within mRNAs and thus exert stabilizing or destabilizing
effects (Derrigo et al. 2000
). As discovered by Curthoys
and colleagues, there exist sequence elements in the 3'-untranslated
region (3'-UTR) of rat kidney-type GA mRNA that allow for its
stabilization under acidotic conditions (Laterza and Curthoys 2000b, Laterza et al. 1997
). This sequence, called a
pH-response element (pHRE) consists of a direct repeat of 8-base
adenosine and uridine-rich sequences. Curthoys and colleagues
demonstrated that these elements are bound by a pH-response-element
binding protein, recently identified as the protein zeta-crystallin
(Curthoys 2001
, Laterza and Curthoys 2000a
). Zeta-crystallin is a NADPH:quinone oxidoreductase
that is abundant in the lens of some mammals (Rao et al. 1992
). Although this protein binds single-stranded DNA
(Gagna et al. 1998
), its role as an RBP is novel.
Our laboratory recently discovered that alternative mRNA splicing is an
additional post-transcriptional control mechanism influencing GA
expression (Elgadi et al. 1999
). While cloning the human
kidney-type GA cDNA, three unique cDNAs were identified. One of
these cDNA, termed hKGA, coincided closely with the kidney-type GA
cDNAs that had been previously cloned from rats and pigs (Porter et al. 1995
, Shapiro et al. 1991
). This cDNA
also matches hKGA cDNAs recently cloned from human brain cDNA libraries
(Holcomb et al. 2000
, Nagase et al. 1998
). To our surprise, we found a novel GA cDNA that we termed
hGAC. This cDNA contains a protein-coding sequence with
amino-terminal amino acid sequence matching hKGA but with a
different carboxy-terminus. At amino acid 550, the amino acid
sequence of hGAC diverges from hKGA and exhibits a 48 amino acid–long
C-terminus that has no homology or similarity to the 113 amino
acid–long C-terminus of hKGA. While these studies were proceeding,
Imbert et al. (1996)
also isolated a human GA cDNA,
which was one of several clones containing CAG trinucleotide repeats
isolated from a spinocerebellar ataxia patient lymphoblastoid cell line
library. (The rat KGA cDNA as well as all human GA cDNAs have variable
CAG trinucleotide repeats in their 5' ends; the significance of this is
unknown.) When fully analyzed, we noted that this cDNA, termed hGAM,
represents yet another novel GA cDNA (Elgadi et al.1999
). hGAM contains an open reading frame that encodes a
169-amino acid peptide that is identical to hKGA and hGAC up to amino
acid 161 and then encodes a unique C-terminus.
Because the 5' ends of all three human GA cDNAs contain identical
sequences and on the basis of preliminary analysis of human GA genomic
BAC clones, we concluded that all three hGA cDNAs were derived by
alternative splicing of a common primary transcript from a single GA
gene locus. The limited tissue distribution of these GA mRNA
spliceforms was determined by Northern blot analysis (Elgadi et al. 1999
). These results suggested that the three nonhepatic
hGA isoforms are expressed in unique tissue-specific patterns,
i.e., hKGA is expressed predominantly in brain and kidney; hGAC is
expressed in cardiac muscle, pancreas, placenta, kidney and lung; and
of the tissue tested, hGAM is expressed exclusively in cardiac and
skeletal muscle. None of these isoforms was detected in liver.
It is noteworthy that only hGAC was found to contain sequences
resembling the pH-response elements found in the 3'-UTR of rat
kidney-type GA by Curthoys and colleagues. Neither hKGA nor hGAM
contains these elements. The 3'-UTR of hGAC contains two 8-base
sequences (UUUAAAUA) that match the first half of this element and two
separate 8-base sequences (UUAAAAUA) that match the second half of this
element. Results from Curthoys’ laboratory suggest that an 8-base
half-element is sufficient for pH-responsive function
(Laterza and Curthoys 2000b
). Therefore, the presence of
these pH-response element half-sites in hGAC and the absence of
similar sequences in hKGA suggest that only the former mRNA may be
up-regulated in the human kidney during acidosis.
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Mechanisms regulating GS expression |
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TOPABSTRACTINTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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During physiologic stress, GS activity is markedly up-regulated,
thereby allowing the lung to become an appreciable net producer of GLN
(Ardawi 1990
, Ardawi and Majzoub 1991
).
Sepsis and glucocorticoid administration elevate rat lung GS mRNA
levels on the order of 10-fold (Abcouwer et al. 1995
,
Lukaszewicz et al. 1997
). Studies in a rat
pulmonary-derived epithelial cell line (L2) have shown that
glucocorticoids regulate GS transcription in this model through a
glucocorticoid receptor–dependent mechanism (Abcouwer et al. 1996
). The transcriptional response of the rat GS gene to
glucocorticoid has been characterized and shown to be attributable to
two genetic regions as follows (Chandrasekhar et al. 1999
): one region lies nearly 6 kb upstream of the
transcription initiation site, and the other lies within the first
intron of the gene. Both of these regions contain
glucocorticoid-response elements. The intron region contains a
canonical glucocorticoid response-element, whereas the
far-upstream region contains three separate glucocorticoid
response-element half-sites. Each of these regions imparts large
glucocorticoid induction of transcription to the GS promoter as well as
a heterologous promoter in a glucocorticoid-dependent fashion.
Despite a large transcriptional response, GS protein levels do not
always increase with GS mRNA levels, which suggests that
post-transcriptional control mechanism(s) also regulate GS
expression. We believe that the ability of GLN to promote GS protein
turnover is the basis of this irregularity and a mechanism by which GLN
production is governed by GLN demand. To test this premise, we
conducted a study in rats that measured the acute effects of
dexamethasone (DEX) and GLN-depletion produced by GLN-free diet
and treatment with methionine sulfoximine (MSO). The data showed that
DEX increased lung GS protein levels by twofold, and GLN-free diet
with MSO treatment increased GS protein levels by fourfold, whereas the
combination of the two treatment raised GS protein levels 12-fold over
control rats (Labow et al. 1998
). These studies
indicated that GS expression in lung could be regulated by both
transcriptional and post-transcriptional mechanisms.
Approximately 25 years ago, it was shown that GS activity in hepatoma
and Chinese hamster cells was diminished by GLN due to accelerated
degradation of the protein (Arad et al. 1976
,
Arad and Kulka 1978
, Milman et al. 1975
).
Subsequent reports demonstrated similar results in astrocytes and
neuroblastoma cells, as well as a skeletal muscle cell line
(Feng et al. 1990
, Lacoste et al. 1982
,
Patel et al. 1986
). Collectively, these studies
demonstrated that the presence of GLN regulates GS expression via a
post-transcriptional mechanism in which the rate of GS protein
degradation is altered. Although it is known that the effect of GLN on
GS can be blocked by inhibitors of protein synthesis (e.g.,
cycloheximide) (Crook and Tomkins 1978
) and requires ATP
(Milman et al. 1975
), the exact nature of this
regulatory mechanism remains unknown.
In addition to GS, many key regulatory proteins are degraded in a rapid
and highly selective manner (Rechsteiner 1987
). Some of
these proteins are degraded by the 26S proteosome, a multisubunit
complex with a highly selective protease activity that comprises the
major nonlysosomal pathway for intracellular proteolysis
(Ciechanover 1994
, Goldberg 1995
). One
such protein is ornithine decarboxylase, an enzyme in the polyamine
biosynthesis pathway that shares many biochemical features with GS
(Murakami et al. 1996
). Both enzymes are regulated by
glucocorticoids and GLN, and both can be degraded rapidly and
selectively (Patel et al. 1986
).
We hypothesized, therefore, that GLN regulates GS degradation by
facilitating its degradation by the 26S proteosome. To test this
hypothesis, we used a pulmonary-derived cell line (L2) to study the
mechanism by which GLN regulates GS expression and to test the effects
of lactacystin (LAC). LAC is a compound derived from bacteria that
specifically inhibits the 26S proteosome by covalently binding to its
active threonine residue in the amino-terminus of the catalytic ß
subunit (Fenteany et al. 1995
). As expected, GLN
starvation up-regulated GS expression in L2 cells via a
post-transcriptional mechanism, and the combined effects of
glucocorticoid stimulation by DEX and GLN starvation produced a
synergistic increase in GS protein levels. The addition of GLN to
culture media produced a rapid decline in GS protein levels, and the
effect was almost completely abrogated by inhibition of the 26S
proteosome by LAC. We concluded that the regulation of GS by its
product GLN occurs in cells of pulmonary origin and that this
regulation is mediated through the 26S proteosome.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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All chemicals were purchased from Sigma Chemical (St. Louis, MO) and Fisher Biotech (Pittsburgh, PA). [32P]dCTP was purchased from Amersham (Arlington Heights, IL). DEX was prepared as a 1.25 mmol/L stock solution in absolute ethanol, and LAC was purchased from Kamiya Biomedical (Seattle, WA) and prepared as a 2 mmol/L stock solution in dimethylsulfoxide (DMSO). These stock solutions were filter-sterilized using a 0.2-µm Sterile Acrodisc (Gelman Sciences; Pittsburgh, PA) before use.
Tissue culture methods and protocols.
L2 cells (CCL-149) were originally purchased from American Type Culture
Collection (Rockville, MD). L2 cells were derived through clonal
isolation from a rat alveolar type II pneumocyte primary culture
(Kain and Douglas 1973
) and exhibited phosphatidate
phosphohydrolase activity and immunohistochemical reactivity patterns
similar to type II alveolar cells (Aiba et al. 1988
,
Douglas et al. 1983
). All cells were grown in
Dulbecco’s modified Eagle medium (DMEM; high glucose and without
GLN or sodium pyruvate; Gibco BRL, Grand Island, NY)
supplemented with 10% heat-inactivated and dialyzed fetal bovine
serums (FBS; Gibco), 50 µg/mL gentamicin (Gibco) and 4
mmol/L GLN. Cells were routinely passaged twice weekly by trypsinizing
and seeding at 1:4 dilutions in T-150 flasks (Falcon; Piscataway, NJ).
For all experiments, cells were seeded with supplemented DMEM (as
above) into 10- or 15-cm sterile plates and grown to confluence (3–4
d). Cells were provided with fresh media 24 h before initiating
each experiment.
To measure the effects of GLN on GS expression, L2 cells were grown to
confluence, and before the initiation of each experiment
(t = 0) all plates were washed with DMEM containing
no GLN and then fed with supplemented DMEM media containing between 0
and 2 mmol/L GLN. After 24 h, cells were harvested for Northern
and Western blot analysis. The combined effects of GLN concentration
and DEX on GS expression were measured in confluent L2 cells fed with
supplemented DMEM media containing either 0 or 4 mmol/L GLN and either
1 µmol/L DEX or an equal volume of EtOH vehicle. This
concentration of DEX was selected because it had previously been shown
to produce a maximal induction of GS mRNA and protein expression in L2
cells between 18 and 24 h after exposure (Abcouwer et al. 1996
). After 24 h, cells were harvested for analysis.
To examine the effect of GLN and the proteosome inhibitor LAC on GS protein decay over time, all cells were fed supplemented DMEM media containing 1 µmol/L DEX 24 h before initiating the experiment. Then, 4.5 h before initiating the time course, cells were washed twice and refed with supplemented DMEM media containing no DEX and no GLN (the use of dialyzed FBS ensured that no GLN and a minimum of glucocorticoid hormone were contributed by this serums component). Four hours later, LAC (or an equal volume of DMSO vehicle) was added to the media to a final concentration of 20 µmol/L; after a 30-min incubation, GLN (or an equal volume of PBS vehicle) was added to a final concentration of 4 mmol/L. Cells were harvested 1, 2, 4 and 8 h after the addition of GLN for analysis.
Northern blotting.
Total RNA was isolated from cells by the one-step acid-phenol
guanidinium procedure (Chomczynski and Sacchi 1987
) with
RNA-Stat 60 (TelTest; Friendswood, TX) and Northern blotting was
performed as previously described in detail (Abcouwer et al. 1996
). Northern blots were hybridized sequentially with probes
derived from an 800-bp segment of rat GS cDNA containing primarily
coding sequence and a cDNA of rat glyceraldehyde 3-phosphate
dehydrogenase (GAPDH). Hybridization, autoradiography, and laser
densometric analysis and quantification were performed as previously
described (Abcouwer et al. 1996
).
Western blotting.
L2 cell monolayers were rinsed with PBS and covered with PBS containing
2 mmol/L EDTA for 5–10 min, at room temperature. After cells had
rounded, they were detached from tissue culture plates with a cell
lifter (Fisher, Biotech), placed in 15-mL conical vials (Fisher,
Biotech), and sedimented by centrifugation at 200 x g for 10 min, at 4°C. After the supernatant had been
removed, the cells were stored at -80°C until processed. Cytoplasmic
lysates were obtained, analyzed by Western blotting using a murine
anti-sheep brain glutamine synthetase antibody that
cross-reacts with rat GS protein (Transduction Laboratories;
Lexington, KY) and quantified by laser densitometry as described in
detail previously (Abcouwer et al.1996
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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To determine whether GLN concentrations in a physiologic range affect
GS protein expression of L2 cells, confluent cultures were fed with
media containing between 0 and 2 mmol/L GLN and were harvested after
24 h. GS protein content was measured using Western blot analysis.
A single major 45-kDa band was visualized in each lane using a murine
anti-sheep brain GS monoclonal antibody that cross-reacts with
the rat GS protein (data not shown). This analysis demonstrated that
decreasing ambient GLN concentration increased GS protein levels in L2
cells in a dose-dependent manner (Fig. 1
). ;F1>GS protein levels rose twofold between GLN concentrations
of 2 and 0.5 mmol/L, and then exhibited a substantial increase to
10-fold basal level as GLN concentrations fell to zero. Thus, GLN
did affect L2 GS protein expression in a physiologically relevant range
of concentrations, but this effect was most dramatic during severe GLN
starvation.
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To determine whether GLN concentration exerts a
post-transcriptional effect on GS protein expression in L2 cells,
the combined effects of GLN deprivation and glucocorticoid stimulation
on GS expression were measured in confluent cultures that were fed
media containing either 0 or 4 mmol/L GLN and either 1
µmol/L DEX or an equal volume of ethanol (EtOH) carrier.
Northern blots hybridized with a rat GS cDNA probe revealed a 2.8- and
1.4-kb transcript in both DEX-stimulated and control cells at both
0 and 4 mmol/L GLN (data not shown). GS mRNA levels were equivalent in
L2 cells incubated with EtOH vehicle and either 0 mmol/L or 4 mmol/L
GLN (Fig. 2
A). ;F2A>DEX induced GS mRNA expression 14-fold in cells incubated
with GLN-free media, and 15-fold in cells incubated with media
containing 4 mmol/L GLN (Fig. 1)
. Thus, ambient GLN concentration did
not influence GS mRNA levels nor did it affect the DEX-mediated
increase in GS transcription. For cells incubated under identical
conditions, GS protein content was measured using Western blot
analysis, and again a single major 45-kDa band was visualized in each
lane (data not shown). In contrast, the level of GS protein within L2
cells was highly dependent upon media GLN (Fig. 2
B).
;F2B>GLN deprivation alone led to a fivefold increase in
EtOH-treated controls and a fourfold increase in GS protein in
cells simultaneously incubated with DEX. DEX alone raised GS protein
levels sixfold in cells incubated in GLN-free media and sevenfold
in those cultured with 4 mmol/L GLN. The combined effects of DEX and
GLN starvation raised GS protein levels 27-fold over EtOH-treated
cells grown in 4 mmol/L GLN. Therefore, through distinct mechanisms,
the combined influences of glucocorticoid and GLN depletion acted
synergistically to augment L2 GS expression.
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To determine whether GLN affects the turnover rate of GS protein in L2
cells and whether proteosome activity plays a role in GS protein
degradation, previously DEX-stimulated L2 cells were incubated in
low and high GLN conditions and in the presence of the proteosome
inhibitor LAC or DMSO vehicle. DEX-stimulated confluent cultures
were rinsed and incubated in glucocorticoid-free media to allow the
decay of GS protein to basal levels to be observed in the absence of
drugs that inhibit translation. [We previously confirmed that
GLN-mediated degradation of GS was almost completely eliminated by
cycloheximide (data not shown).] GS protein content was measured using
Western blot analysis and again a single major 45-kDa band was
visualized in each lane (data not shown). The analysis demonstrated
that the addition of GLN to tissue culture media produced a rapid
decline in total GS protein levels in cells exposed only to DMSO
vehicle (Fig. 3
). The half-life of GS protein in the presence of 4 mmol/L GLN and
DMSO was 2.7 h. When L2 cells were incubated in media containing
no GLN and DMSO, the half-life of the GS protein increased to
12 h. However, when LAC was present in the culture media (20
µmol/L), the half-life of the GS protein was 9.3 and
10.2 h in cells incubated with media containing 4 and 0 mmol/L
GLN, respectively. Thus, GLN greatly increased the rate of GS protein
decay in L2 cells and selective inhibition of the 26S proteosome by LAC
effectively eliminated the effect of GLN.
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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). It should also be noted that in the present study,
GS protein levels increased most dramatically when the GLN
concentrations fell below 0.5 mmol/L. The normal concentration of GLN
in both rat (Brosnan et al. 1983
, Herbert et al. 1966
, Labow et al. 1999
) and human plasma
(Bergstrom et al. 1974
, Parry-Billings et al. 1992
, van der Hulst et al. 1996
) is 0.6
mmol/L. However, during critical illness, plasma GLN concentration may
fall by as much as 50% (Askanazi et al. 1980
,
Greig et al. 1996
, Palmer et al. 1996
).
Thus, the effect of GLN on GS expression occurred over physiologically
meaningful GLN concentrations, with the greatest effect occurring at
concentrations that may be experienced by severely ill patients.
Previously published studies from our laboratory have shown that
L2 cells stimulated with DEX, increase GS mRNA and protein levels to a
maximal and sustained value by 12 and 18 h after exposure,
respectively (Abcouwer et al. 1996
). In the present
study, GS mRNA levels measured after 24 h of treatment were
increased by DEX treatment, but not affected by GLN starvation. In
contrast, GS protein levels were significantly affected by both DEX and
GLN concentrations. These two effects seem to act synergistically to
induce GS expression. In contrast, glucocorticoids and GLN starvation
have been previously shown to work additively in cerebellar astrocytes
(Patel et al. 1986
). However, our previous in vivo
studies measuring the acute effects of DEX and GLN-depletion on GS
expression in the lungs of rats provide support for a multiplicative
effect of these two stimuli (Labow et al. 1998
). A
synergistic effect would be expected if DEX and GLN starvation had
independent effects upon transcription and post-transcriptional
steps in gene expression, respectively.
Although the ability of GLN to accelerate GS protein degradation has
been known for many years, the mechanism by which GLN acts to do this
remains unknown. In the present study, the presence of GLN increased
the rate of GS protein degradation over fourfold. Selective inhibition
of the 26S proteosome by LAC increased the half-life of the GS
protein in the presence of 4 mmol/L GLN more than threefold, so that
GLN had very little effect on GS protein turnover in the presence of
LAC. Although it has previously been suggested that the GS protein is
degraded within lysozomes (Freikopf-Cassel and Kulka 1981
), the present study suggests that the
proteosome-pathway is utilized for GS proteolysis in response to
GLN. The proteosome pathway is also utilized in the degradation of a
related enzyme, ornithine carboxylase. Like GS, ornithine decarboxylase
is degraded in a rapid and selective manner that requires ATP, and is
inhibited by cycloheximide (Murakami et al. 1996
).
Moreover, of the known proteins that are regulated through the
proteosome pathway, ornithine decarboxylase is somewhat unique because
its degradation in vitro is ubiquitin-independent (Murakami et al. 1992
). In fact, degradation of ornithine decarboxlyase
requires the synthesis and binding of an antizyme, which accounts for the effect of cycloheximide (Murakami et al. 1996
). By analogy, GS may share this unique mode of enzyme
regulation. However, Osada et al. (1999)
recently showed
that a previously unidentified ubiquinated protein that is present at
abnormally high levels in hepatocellular carcinomas is, in fact, GS.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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, Sevdalian et al. 1980
,
Szondy and Newsholme 1991
). In times of infection, stress or trauma, glucocorticoids cause an increase in GS transcription, particularly in lung and muscle tissue. The increased GS mRNA level presumably increases the rate of translation of GS mRNA to protein by a comparable amount. However, because intracellular glutamate and ammonia concentrations are not limiting, an accumulation of GS protein causes increased GLN production. If this extra GLN is not disposed of, a feedback loop analogous to alosteric regulation also increases the degradation rate of GS protein. Therefore, the ultimate GS protein level increases appreciably only if GLN disposal keeps pace with or outpaces the increased GLN production. The reason for including a transcriptional component in this regulatory mechanism could be to increase the robustness of the response of GS activity to GLN-demanding catabolic states. Without an increase in the GS mRNA level and, therefore, in translation of GS protein, GS activity levels could respond to increased GLN demand by virtue of increased protein stability alone. However, this response would be relatively slow and would come at the expense of appreciable depletion of intracellular GLN stores. During physiologic stress, when stress hormone and GLN levels may vary widely, this dual regulatory mechanism could allow the lung and muscle to finely tune GLN production to meet systemic demands.
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FOOTNOTES |
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2 Supported by the Edward D. Churchill Fellowship,
MGH Department of Surgery (B.I.L.), National Institutes of Health Grant
R01 HL44986 (W.W.S.) and National Institutes of Health Grant R29
CA72772 (S.F.A.).
4 Abbreviations used: CRE, cAMP-response
element; DEX, dexamethasone; DMEM, Dulbecco’s modified Eagle medium;
DMSO, dimethylsulfoxide; EtOH, ethanol; FBS, fetal bovine sera; GA,
glutaminase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GLN,
L-glutamine; GS, glutamine synthetase; LAC, lactacystin;
MSO, methionine sulfoximine; pHRE, pH-response element; RBP, RNA
binding protein; 3'-UTR, 3'-untranslated region.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMechanisms by which GA...Mechanisms regulating GS...MATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSLITERATURE CITED |
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1. Abcouwer S. F., Bode B. P. & Souba W. W. (1995) Glucocorticoids regulate rat glutamine synthetase expression in a tissue-specific manner. J. Surg. Res. 59:59-65.[Medline]
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