REVIEW ARTICLE
Control of mammalian gene expression by amino acids,
especially glutamine
Carole Brasse-Lagnel, Alain Lavoinne and Annie Husson
Appareil Digestif, Environnement et Nutrition, EA 4311, Universite
´
de Rouen, France
A growing number of reports clearly demonstrate that
amino acids are able to control physiological functions
at different levels, including the initiation of protein
translation, mRNA stabilization and gene transcrip-
tion [1–3]. Although the molecular mechanisms
involved in the control of gene expression by amino
Keywords
AARE; amino acids; ATF; gene transcription;
glutamine; mammalian cells; NF-jB; NSRE;
signalling pathways; transcription factors
Correspondence
A. Lavoinne, Groupe ADEN, Faculte
´
de
Me
´
decine-Pharmacie de Rouen, 22
Boulevard Gambetta, Rouen Cedex, France
Fax: +33 2 35 14 82 26
Tel: +33 2 35 14 82 40
E-mail:
(Received 12 November 2008, revised 9
January 2009, accepted 21 January 2009)
doi:10.1111/j.1742-4658.2009.06920.x

+
-
dependent transport system; ASNS, asparagine synthetase; ASS, argininosuccinate synthetase; ATF, activating transcription factor; C/EBP,
CCAAT/enhancer-binding protein; CHOP, C/EBP homology binding protein; ERK, extracellular signal-related kinase; FXR, farnesoid X
receptor; HIF, hypoxia-inducible factor; HNF, hepatocyte nuclear factor; HSF, heat shock factor; IL, interleukin; IjB, inhibitor of kappa B; JNK,
c-Jun N-terminal kinase; LPS, lipopolysaccharide; NF-jB, nuclear factor kappa B; NSRE, nutrient-sensing response elements; PPAR,
peroxysome proliferator-activated receptor; RXR, retinoid X receptor; TNF, tumour necrosis factor.
1826 FEBS Journal 276 (2009) 1826–1844 Journal compilation ª 2009 FEBS. No claim to original French government works.
acid availability have been extensively studied in lower
eukaryotes such as yeasts [4], the control of transcrip-
tional events including signalling pathways, transcrip-
tion factors and their corresponding cis-acting DNA
sequences is still unclear in mammalian cells. Never-
theless, some in vitro experiments have shown that
under specific conditions such as amino acid depriva-
tion, the expression of individual genes is changed via
the activation of specific transcription factors and reg-
ulatory sequences. The first studies, performed about
20 years ago, concerned stimulation of ASS gene tran-
scription by arginine deprivation in human cell lines
[5]. A small region (149 bp) of the ASS gene promoter
was proposed to be involved in arginine sensitivity,
suggesting the existence of an arginine responsive ele-
ment, but the specific cis element within this region
and the involved transcription factor(s) were not iden-
tified [6,7]. Further extensive studies on the ASNS
[8,9] and CHOP genes [10,11] allowed characterization
of specific responsive sequences in their promoter,
which were named either nutrient-sensing response ele-
ments (NSRE) or amino acid responsive elements

Tables 1 and 2 summarize the molecular data obtained
on the transcriptional effects of different amino acids
(except glutamine), together with the identified tran-
scription factors and the responsive elements involved.
Most of the data concern the inhibitory effect of
amino acids. Initial studies were performed to explore
the molecular mechanisms involved in the inhibitory
effect of asparagine and histidine on the expression of
ASNS and that of leucine on CHOP (also known as
GADD 153) gene expression (Table 1). Indeed, the first
identification of a sequence responsive to amino acid
(AARE) was performed by Guerrini et al. [8], while
studying the functionality of the ASNS gene promoter
in asparagine- or leucine-deprived ts11 and HeLa cells.
Further studies by Kilberg’s group on the inhibiting
effect of histidine on the human ASNS gene in HepG2
Table 1. AARE-NSRE sequences and the inhibiting effect of amino acids on gene transcription.
Cell
model
Amino
acid(s)
deprivation
Target
gene
a
Transcription
factor(s)
involved
Localization of
the responsive

glucose addition. It was subsequently referred to as
NSRE-1, a composite site which could be recognized
in vitro by two factors, namely the CCAAT/enhancer-
binding protein-b (C/EBP-b) and activating transcrip-
tion factor-4 (ATF4) [13,16]. An additional sequence,
named NSRE-2, located 11 nucleotides downstream
of NSRE-1, was found to amplify NSRE-1 activity
in response to amino acid starvation. Accessory
sequences such as GC boxes were also required for
maximal activation of the ASNS gene [9,17,18]. In
addition to the involvement of ATF4 and C/EBP-b,an
additional regulatory role of ATF3 on transcription of
the ASNS gene was also recognized following histidine
deprivation in HepG2 cells [19]. Further studies dem-
onstrated that stimulation of ASNS gene transcription
following ATF4 binding to NSRE-1 might involve
acetylation of histones H3 and H4, and the subsequent
binding of general transcription factors [20]. In para-
llel, extensive studies from Fafournoux’s group demon-
strated that transcription of the human CHOP gene is
stimulated by leucine deprivation in HeLa cells via a
specific AARE in the promoter. This element was able
to bind ATF2 and ATF4 in vitro [12,21]. Furthermore,
it was shown that binding of ATF4 and phosphoryla-
tion of ATF2 bound to CHOP AARE were essential
for the acetylation of histones H4 and H2B within the
AARE sequence leading to the response to leucine
starvation [22]. This result was recently supported by
the observation that the p300/CBP-associated factor, a
transcriptional co-activator with intrinsic histone ace-

CAT-1 gene [28,29], the xCT gene encoding a compo-
nent of the cystine/glutamate transport system (system
x
À
c
)
, [30] and the SNAT2 gene encoding an isoform of
the system A amino acid transporter [31,32]. Similarly,
such sequences were also found in genes encoding
transcription factors, such as ATF3 [33] and C/EBP-b
[34]. Again, evidence was obtained for increased
Table 2. Other proposed sequences involved in the effect of amino acids on gene transcription.
Cell model
Amino acid(s)
manipulation Target gene
Transcription
factor
involved
Localization of
the responsive
sequence
Proposed
responsive
sequence
a
Reference
Rat liver
in vivo
Protein-free diet IGFBP-1
stimulation

5¢-TTACGTCA-3¢
[39]
Mouse
macrophages
Homocysteine
addition
Gcl stimulation Nrf2 activation 5¢-Flanking region
(-6.5 kb/-3.8 kb)
ARE sequence [40]
Mouse
cerebellum
Glutamate
addition
Glast inhibition c-jun activation 5¢-Flanking region
(-135/-129)
AP-1 site [43]
a
Accessory sites and additional factors are not cited.
Glutamine and transcription factors C. Brasse-Lagnel et al.
1828 FEBS Journal 276 (2009) 1826–1844 Journal compilation ª 2009 FEBS. No claim to original French government works.
binding of ATF4 and C/EBP-b to these sequences
following amino acid deprivation, emphasizing the
major role played by ATF and C/EBP factors in the
inhibiting effect of amino acids on gene expression.
Concerning the stimulation of gene expression by the
presence of amino acids, only one gene, Pept 1, encod-
ing a peptide transporter, was shown to contain an
AARE-like sequence activated by phenylalanine addi-
tion but the functionality of the sequence in the pro-
moter has not been further specified [35].

summarizes the different genes regulated by amino
acids with the identified transcription factors and
responsive sequences. It is beyond the scope of this
review to detail the case of glutamate, a major excit-
atory neurotransmitter, regulating the transcription of
numerous genes in the central nervous system [41,42].
Indeed, glutamate acts through its binding to specific
membrane receptors, which is not the case for the
other amino acids. In this context, glutamate-respon-
sive elements were recently identified as a functional
AP-1 site in the 5¢-flanking sequence of some genes
in mammalian neurons and glial cells, such as the
glast gene in mouse cerebellum [43]. However, it
should be pointed out that glutamate may also exert
Amino acids
Deprivation Deprivation or addition
Cultured cells
Cytoplasm
Nucleus
C/EBPs, ATFs
USFs, AP-1, HNF-1,
ATF2, Nrf2
AAR pathway
?
NSRE
Target
genes
Corresponding
sequences
Target

level of their expression (mRNA or protein). In the lat-
ter case, the specific responsive sequences in the target
genes were not characterized further. It can be noted
Table 3. Transcription factors involved in the action of amino acids on gene expression.
Amino acid(s) manipulations Factor(s) implied Experimental model Reference
Inhibiting effect resulting from the presence of amino acids
Pooled amino acids deprivation Increased c-myc mRNA stabilisation Cultured rat hepatocytes [46]
Dietary protein restriction Increased HNF-3, HNF-1, C/EBP,
Sp1 binding and HNF-1 mRNA level
Rat liver in vivo [14]
Leu + Ile + Cys + Trp
deprivation
Increased CHOP mRNA and protein levels Mouse fibroblasts [48]
Protein-free diet Increased HNF-3c mRNA level Rat liver in vivo [49]
Increased Id2 mRNA level [50]
Increased FoxO4 mRNA level [51]
Methionine
Deprivation Increased c-jun,c-myc and jun-B mRNA levels CHO cells [52]
Addition Decreased p53 mRNA and protein levels Human breast cancer cells [53]
Homocysteine addition Decreased NF-jB binding TNF-stimulated HUVEC [54,55]
Decreased AP-1 binding NIH/3T3 cells [56]
Decreased PPAR a, c mRNA and protein levels Human monocytes [57]
Leucine deprivation Increased NF-jB binding Mouse embryo fibroblasts [58]
Histidine
Deprivation Increased ATF3 mRNA stabilisation HepG2 cells [47]
Addition Inhibited NF-jB activation TNF-induced Caco-2 cells [59]
Arginine
Deprivation Increased NF-jB binding Murine keratinocytes [60]
Addition Inhibited PPAR-c binding Post-ischaemic rat jejunum [61]
Leu or Ile or Val

1830 FEBS Journal 276 (2009) 1826–1844 Journal compilation ª 2009 FEBS. No claim to original French government works.
that the stabilization of specific mRNAs encoding
transcription factors can contribute to the stimulation
of gene expression following amino acid deprivation,
as demonstrated for c-myc and ATF3 [46,47]. The
observations reported in Table 3 underline the diver-
sity of the mechanisms by which amino acids modu-
lates gene expression. It can be seen that some amino
acids act by inhibiting several transcription factors
[14,46–63], whereas others act through a stimulatory
effect [14,64–80]. Interestingly, two amino acids,
namely homocysteine [54,70–74] and arginine [60,79],
are able to inhibit or stimulate nuclear factor kappa B
(NF-jB), depending on the physiological conditions
and cell types studied. This underlines the need to
understand the molecular mechanism by which these
amino acids act. Because increased circulating concen-
trations of homocysteine have been reported to be
associated with a variety of diseases [81], the molecular
mechanisms involved in the effects of the amino acid
were extensively studied, revealing multiple regulated
transcription factors (Tables 2 and 3).
Thus, as assessed by these studies, the regulation of
transcription by amino acids relies on different mecha-
nisms involving various transcription factors, but their
corresponding cis elements are not yet completely
characterized.
Complexity in the action of glutamine
on gene transcription
Because glutamine is the most abundant amino acid in

Addition Postischaemic rat intestine PPAR-c Increased DNA binding [61]
Deprivation Human fetal intestinal cell line
(H4) and Caco-2 cells
NF-jB Decreased IjBa level; increased p65 binding
and nuclear protein level
[92]
Addition Irradiated rat ileum in vivo NF-jB Decreased protein amount [94]
Addition Rat colon (and pancreas) in vivo
(experimental colitis)
NF-jB Decreased protein amount [95]
Addition Human intestinal (HTC-8) cells NF-jB Increased IjBa level [93]
Addition human intestinal (Caco-2) cells NF-jB Decreased DNA binding and nuclear p65 amount [44]
Addition Rat colon in vivo (experimental colitis) NF-jB Increased IkB Protein and decreased p65 protein [96]
Addition Rat intestine in vivo (brain trauma injury) NF-jB Decreased DNA binding and p65 protein level [98]
Addition Rat colon in vivo (experimental colitis) NF-jB and
STATs
Decreased nuclear p50 and p65 levels and
phosphorylated STAT1 and STAT5
[97]
Addition Adipose tissue in high fat diet rat NF-jB Decreased IKKb and decreased p65 binding [99]
Addition Rat lung in vivo NF-jB Increased IjBa expression and
decreased p65 binding
[100]
Addition Mouse lung in vivo (LPS-treatment) NF-jB Decreased LPS-induced DNA binding [101]
Addition LPS-treated rat alveolar epithelial cells NF-jB Decreased LPS-induced DNA binding [102]
Addition Septic mouse lung in vivo NF-jB Decreased DNA binding activation [113]
Addition Septic mouse lung in vivo HSF-1 and Sp1 Increased O-glycosylation and phosphorylation [111]
Addition Mouse embryonic fibroblasts (HSF)/)) HSF-1 Activation [112]
C. Brasse-Lagnel et al. Glutamine and transcription factors
FEBS Journal 276 (2009) 1826–1844 Journal compilation ª 2009 FEBS. No claim to original French government works. 1831

GCACGTAGC
Caco-2 cells
Fig. 2. Schematic representation of the
influence of glutamine on the transcription
of genes involved in intermediary
metabolism.
Table 5. Influence of glutamine on transcription factors involved in cell proliferation, apoptosis and survival.
Glutamine Experimental model
Transcription
factor(s)
involved Effect and mechanism involved Reference
Addition Porcine enterocyte line c-jun Increased mRNA level [123]
Addition Rat and pig intestinal cell lines AP-1 (c-jun)
and c-myc
Increased mRNA levels and increased
c-jun activity
[124]
Addition Induced rat mammary tumours p53 and c-myc Increased p53 phosphorylation and
decreased c-myc mRNA level
[139]
Deprivation Murine hybridoma cells p53 Decreased mRNA level [131]
Addition Exercised rat neutrophils p53 Decreased exercise-induced mRNA level [151]
Addition Pig renal epithelial cell line CHOP Decreased mRNA level [86]
Deprivation Human breast cells CHOP and Gadd 45 Increased mRNA stabilization [134]
Deprivation CHO cells CHOP Increased mRNA level [132]
Addition Murine hybridoma cells CHOP Decreased mRNA and protein levels [135]
Deprivation Human lung carcinoma cells HIF-1a/2a, Gadd 34
and CHOP
Decreased HIF-1/2 a protein, increased
Gadd 34 and CHOP mRNA levels

inhibitor of kappa B (IjB) because, in lipopolysac-
charide (LPS)-treated Caco-2 cells, glutamine depri-
vation decreased the level of IjB-a leading to an
increase in NF-jB within the nucleus [92]. In line
with this, addition of glutamine to HTC-8 cells was
shown to increase the IjBa content by limiting its
ubiquitination [93]. In addition, glutamine might also
act via a decrease in NF-jB synthesis or an increase
in its degradation because administration of the
amino acid decreased the immunoreactive NF-jB
protein in the intestine of injured rats [94,95]. More
recently, we demonstrated that glutamine addition
was able to decrease the nuclear content of p65 NF-
jB within 2 h, in control or cytokine-stimulated
Caco-2 cells [44]. Finally, in an experimental model
of colitis in the rat, glutamine administration not
only prevented the decrease in IjBa and the subse-
quent increase in nuclear p65, but also prevented the
increase in IjB kinases (IKKa and IKKb), thereby
reducing the production of pro-inflammatory media-
tors [96,97]. This was also reported in rat intestine
following brain trauma injury [98] and adipose tissue
following high fat diet [99]. Such studies were also
performed in septic rat lung in vivo, where glutamine
inhibited IjB-a degradation resulting in the attenua-
tion in tumour necrosis factor (TNF)-a and IL-6
production. In this condition, the amino acid was
shown to interfere with the NF-jB pathway through
the inhibition of p38 MAPK and ERK phosphory-
lation [100]. In septic mouse lung, glutamine

mesangial cells [109].
In addition to its influence on NF-jB and consis-
tent with its role as an anti-inflammatory molecule,
a protective effect of glutamine in injured intestine
was also observed via the inhibition of the DNA-
binding activity of AP-1 [78]. This was mediated by
the stimulation of peroxisome proliferator-activated
receptor c (PPAR-c) [61,110] and also through a
decrease in the phosphorylated form of STAT1 and
STAT5 [97]. Also contributing to its anti-inflamma-
tory action, the amino acid could induce the heat
shock protein response involving the O-glycosylation
and phosphorylation of the heat shock factor-1
(HSF-1) [111]. Notably, glutamine addition could
attenuate cytokine-induced NO production only in
HSF-1
+/+
mouse embryonic fibroblasts, the effect
being lost in HSF-1
)/)
cells [112]. In this regard, the
attenuation of NF-jB activation, the inhibition of
proinflammatory cytokine production and the subse-
quent decrease in lung injury following glutamine
treatment were lost in Hsp70()/)) mice [113].
Collectively, these data show that glutamine exerts
anti-inflammatory effects through several pathways, at
least in part through the inhibition (NF-jB, AP-1 and
STAT) or activation (PPAR-c and HSF-1) of specific
transcription factors. Moreover, the anti-inflammatory

two classes of MAP kinase, the ERKs and the c-Jun
N-terminal kinase (JNK) [125]. Through ERK signal-
ling, glutamine was shown to specifically stimulate
MEK-1, the upstream kinase that activates ERK-1
and ERK-2, leading to subsequent phosphorylation of
transcription factor Elk-1 involved in cellular
differentiation. Through JNK signalling, the increased
expression of c-jun gene by glutamine led to the
subsequent activation of factor AP-1 involved in cell
proliferation. The metabolism of glutamine was
required to activate the requested regulatory protein
kinases but the underlying mechanism remains uniden-
tified [125]. In parallel, glutamine could also stimulate
specific cell differentiation as shown by microarray
analysis in a pancreatic b-cell line revealing multiple
gene changes with a particular stimulation of the Pdx1
that is essential for pancreatic b-cell differentiation and
function [126].
By contrast, glutamine addition downregulated some
genes encoding factors involved in the inhibition of
proliferation or in protein degradation and apoptosis
[127,128]. Indeed, its inhibiting effect on specific cas-
pase activity protects against DNA breakage in various
tissues, but the underlying molecular mechanisms are
not yet fully understood [121,122,129–131]. Neverthe-
less, the inhibiting effect of glutamine on transcription
factors involved in the cessation of growth, such as
CHOP, was clearly demonstrated in a number of stud-
ies. For example, glutamine addition partly suppressed
the expression of CHOP mRNA in pig renal epithelial

addition could protect cells from apoptosis induced by
c-myc overexpression, as reported in human hepatoma
cell line [137] and inversely, glutamine deficiency could
induce apoptosis through an increase in the MYC pro-
tein level in different human cell lines [138]. In rat
mammary tumours, the dietary amino acid also coun-
teracted the proliferative effect of c-myc by reducing
its phosphorylation and mRNA level and by stimulat-
ing phosphorylation of p53, leading to tumour reduc-
tion [139]. Thus, in experimental breast cancer, dietary
glutamine could paradoxically promote the process of
apoptosis. This was reported to be the result of gluta-
thione downregulation [140,141]. These results illus-
trate the complex regulation exerted by glutamine on
transcription factor such as c-myc, i.e. the activation
of its gene expression in enterocyte lines in favour
of proliferation, as pointed out above [124], and its
inhibition in some tumours and other cell lines limiting
proliferation.
Glutamine and transcription factors C. Brasse-Lagnel et al.
1834 FEBS Journal 276 (2009) 1826–1844 Journal compilation ª 2009 FEBS. No claim to original French government works.
In the context of heat shock, an anti-apoptotic effect
of glutamine was also exerted via the stimulation of
Hsp protein production, both at transcriptional and
post-transcriptional levels [103,142]. Concerning its
transcriptional effect, activation of nuclear factor
HSF-1 and binding to a heat shock element (HSE)
resulting in the transcription of Hsp genes was
reported in rat intestinal cells and mouse fibroblasts
[143–146]. In particular, heat stress injury was

exercised rat neutrophils, depending on the cell type
and physiological condition.
Taken together, the data show that glutamine is able
to promote cell growth, attenuate the pathological
stress response and modulate apoptosis, at least partly
through the activation of specific transcription factors.
These observations have led to proposals that the
amino acid is a ‘survival factor’. However, glutamine
was also reported to act in the context of hypoxia, a
situation known to stimulate transcription factor
hypoxia-inducible factor-1 (HIF-1). HIF-1 is involved
in the maintenance of oxygen homeostasis, angiogene-
sis and hence, in tumour progression [152]. Indeed,
studies performed on human carcinoma cells showed
that glutamine deprivation decreased HIF-1a and
HIF-2a with an impaired release of vascular endo-
thelial growth factor (VEGF-A, a prominent mediator
of angiogenesis), limiting tumour oxygenation and
favouring cancer cell death [133]. Furthermore, gluta-
mine deprivation was also able to inhibit the hypoxia-
induced HIF-1a protein at the translational level in
human pancreatic and prostatic cancer cells [153].
Transcription factors involved in the regulatory
role of glutamine on intermediary metabolism
In parallel to its role as a metabolic substrate, gluta-
mine also stimulates a number of metabolic pathways,
namely hepatic lipid formation and glycogen synthesis
[154], hepatic and renal gluconeogenesis [155], and
muscle protein synthesis [156]. About 12 years ago, the
expression of some genes encoding enzymes directly or

regulated by growth factors and nutritional status, par-
ticularly amino acid availability [164]. Thus, the
ADSS1 response to protein kinase A and mammalian
target of rapamycin signalling subsequently involved
phosphorylation of the cAMP response element modi-
fier and its binding to a cAMP response element in the
promoter region of the ADSS1 gene [163]. A third
study performed by our group showed that glutamine
addition increased ASS gene transcription in human
C. Brasse-Lagnel et al. Glutamine and transcription factors
FEBS Journal 276 (2009) 1826–1844 Journal compilation ª 2009 FEBS. No claim to original French government works. 1835
enterocytes [165] but, by contrast to the results
obtained in hepatocytes [159], cell swelling was not
involved in the effect of the amino acid. Indeed, we
demonstrated that glutamine metabolism was involved
in Sp1 O-glycosylation via the hexosamine pathway.
This post-translational event induced the subsequent
nuclear translocation of Sp1 and its binding to GC
boxes in the promoter of the ASS gene [165]. More-
over, via another pathway, namely glutamate produc-
tion, glutamine was able to mask the stimulating effect
of IL-1b on ASS gene expression via a decrease in the
nuclear amount of NF-jB [44]. This illustrates that
glutamine may regulate expression of the same gene
via different pathways as a function of cell type and
pathophysiological conditions. In addition to its effect
on the ASS gene, glycosylation of Sp1 can also stimu-
late the ClC-2 gene expression, as observed after gluta-
mine addition to rat lung cell lines [166]. In addition,
increased expression of phosphorylated Sp1 after the

AARE/NSRE sequences and ATF factors involved in
the effects of amino acids on gene transcription, a
number of studies have reported that a variety of tran-
scription factors, much larger than initially thought,
can be modulated by amino acids with major func-
tional implications. This is particularly illustrated by
glutamine, which has received increased attention in
recent years and turned out to be an important regula-
tor of gene expression without any evidence for a ‘glu-
tamine-responsive element’. Microarray techniques
[126,170,171] and proteomic studies [172–174] are now
identifying the extent of the genetic programme con-
trolled by glutamine and the underlying molecular
mechanisms are being extensively deciphered. The
emerging data show that cells have developed various
molecular mechanisms to respond to changes in extra-
cellular glutamine concentrations. Indeed, through the
activation of different signalling pathways (ERK,
JNK, PKA and mTOR pathways) and a variety of
transcription factors including bZIP proteins (ATFs,
C/EBP), helix–turn–helix proteins (HSF-1), zinc fingers
proteins (Sp1) and nuclear receptors (PPAR, FXR/
RXR), glutamine significantly contributes to the regu-
lation of genes involved in major cellular processes,
namely the inflammatory response, proliferation, sur-
vival and metabolism. Moreover, glutamine modulates
the activity of transcription factors at multiple levels,
i.e. synthesis or degradation, posttranslational modifi-
cations or modulation of their activators or inhibitors.
The amino acid appears to be a valuable tool to study

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