Báo cáo khoa học: Control of mammalian gene expression by amino acids, especially glutamine potx

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

Molecular data rapidly accumulating on the regulation of gene expression
by amino acids in mammalian cells highlight the large variety of mecha-
nisms that are involved. Transcription factors, such as the basic-leucine
zipper factors, activating transcription factors and CCAAT/enhancer-bind-
ing protein, as well as specific regulatory sequences, such as amino acid
response element and nutrient-sensing response element, have been shown
to mediate the inhibitory effect of some amino acids. Moreover, amino
acids exert a wide range of effects via the activation of different signalling
pathways and various transcription factors, and a number of cis elements
distinct from amino acid response element/nutrient-sensing response
element sequences were shown to respond to changes in amino acid con-
centration. Particular attention has been paid to the effects of glutamine,
the most abundant amino acid, which at appropriate concentrations
enhances a great number of cell functions via the activation of various
transcription factors. The glutamine-responsive genes and the transcription
factors involved correspond tightly to the specific effects of the amino acid
in the inflammatory response, cell proliferation, differentiation and sur-
vival, and metabolic functions. Indeed, in addition to the major role played
by nuclear factor-jB in the anti-inflammatory action of glutamine, the
stimulatory role of activating protein-1 and the inhibitory role of C/EBP
homology binding protein in growth-promotion, and the role of c-myc in
cell survival, many other transcription factors are also involved in the
action of glutamine to regulate apoptosis and intermediary metabolism in
different cell types and tissues. The signalling pathways leading to the acti-
vation of transcription factors suggest that several kinases are involved,
particularly mitogen-activated protein kinases. In most cases, however, the
precise pathways from the entrance of the amino acid into the cell to the
activation of gene transcription remain elusive.
Abbreviations
AAR, amino acid response; AARE, amino acid response elements; ADSS1, adenylosuccinate synthetase; AP, activating protein; ASCT2, Na

+
-
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

(AARE). Specific transcription factors involved in the
amino acid response pathway (AAR) were also identi-
fied, and are members of the basic region/leucine zip-
per superfamily of transcription factors [12,13]. In
parallel, some amino acids involved in many cellular
functions, particularly glutamine, were shown to exert
a wide range of effects via the activation of different
signalling pathways and transcription factors. In this
case, a number of cis elements distinct from AARE/
NSRE were shown to respond to changes in amino
acid concentration. Although the molecular details of
these effects are not completely known, the heteroge-
neity of the involved factors might suggest multiple
AAR pathways depending on the amino acid studied,
the cell type used and the gene promoter configura-
tion. Moreover, this complexity is enhanced by the
fact that some target genes encode transcription fac-
tors which may in turn act on many subordinated
genes [14]. Among the amino acids, glutamine has the
ability to regulate gene expression in a number of
physiological processes, as reported in a recent review
illustrating the vast panel of regulated genes [15].
Thus, in this review, we intend to summarize recent
data obtained on the molecular mechanisms involved
in the effects of amino acids on gene expression, focus-
ing on the transcription factors responsive to gluta-
mine.
The importance of AARE sequences
and ATF/C/EBP transcription factors in
the AAR pathway

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

sequence(s)
Responsive
sequence(s)
b
Reference
HeLa Asparagine ASNS (ns) 5¢-Flanking region (-70/-64) AARE 5¢-CATGATG-3¢ [8]
HepG2 Histidine ASNS C/EBPb, ATF4 5¢-Flanking region (-68/-60) NSRE 1 5¢-TGATGAAAC-3¢ [13,16,18]
HeLa Leucine CHOP ATF2, ATF4 5¢-Flanking region (-310/-302) AARE 5¢-ATTGCATCA-3¢ [12,21]
NIH/T3T Cystine xCT ATF4 5¢-Flanking region (-94/-86
and -76/-68)
AARE 5¢-TGATGCAAA-3¢
and 5¢-TTTGCATCA-3¢
[30]
HepG2 Histidine C/EBPb ATF4 3¢-UTR region (+1554/+1646) multiple sites [34]
HepG2 Histidine SNAT2 ATF4,
C/EBPa, b, d
First intron (+712/+724) AARE 5¢-TGATGCAAT-3¢ [31,32]
HepG2 Histidine ATF3 ATF3, ATF4, C/EBPb 5¢-Flanking region (-23/-15) 5¢-TGATGCAAC-3¢ [33]
Rat C6
glioma
All amino
acids
CAT-1 ATF4, C/EBPb, ATF3 First exon (+45/+53) AARE 5¢-TGATGAAAC-3¢ [28,29]
a
Transcription factors studied as regulated target genes are given in bold.
b
Accessory sites are not specified.
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. 1827
cells specified that this element also responds to

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-

tyltransferase activity, could interact with ATF4 to
enhance CHOP transcription following leucine depri-
vation [23]. Although the CHOP AARE and ASNS
NSRE-1 sequences shared structural and functional
similarities, the CHOP AARE sequence is able to
function alone and is more sensitive to amino acid
deprivation than NSRE-1 alone [24]. These data show
that ATF factors might largely contribute to promote
the changes in the chromatin structure required to
enhance transcription of amino acid-regulated genes.
The mechanism(s) of detection of amino acid limita-
tion by the ARR pathway relies on free tRNA accu-
mulation which activates a stress kinase called the
GCN2 kinase. This kinase, in turn, phosphorylates the
eIF2a, thereby inhibiting general protein synthesis
[25,26], as shown previously in yeasts. Paradoxically,
in this condition, the specific synthesis of some tran-
scription factors from pre-existing mRNAs such as
ATF4 was observed with the subsequent activation of
target genes, namely those containing an AARE.
Signalling pathways involved in these effects were
recently studied in human hepatoma cells revealing the
activation of specific mitogen-activated protein kinase
cascades, such as the mitogen-activated protein kinase
kinase/extracellular signal-related kinase (ERK) path-
way [27].
Table 1 also shows that, in addition to original
AARE and NSRE, similar functional sequences were
identified in various regions of other amino acid-regu-
lated genes involved in amino acid transport such the

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

USF1-USF2
activation
5¢-Flanking region
(AARU: -112/-77)
E box : -88/-83
(5¢-CACGGG-3¢)
[36]
Human
endothelial
Homocysteine
addition
Endothelin-1
inhibition
AP-1 inhibition 5¢-Flanking region
(-109/-102)
AP-1 site
(5¢-GTGACTAA-3¢)
[37]
HepG2 Phenylalanine
deprivation
Albumin inhibition HNF1a inhibition 5¢-Flanking region
(-60/-46)
HNF-1 site [38]
HUVEC Homocysteine
addition
ATF3 stimulation ATF2 and c-jun
activation
5¢-Flanking region
(-92/-84)
ATF/CRE sequence

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].

Transcription factors other than
ATF/C/EBP are involved in the effects
of amino acids
In addition to ATF/C/EBP factors and specific AARE
sequences, a few other transcription factors and their
corresponding cis elements are modulated by amino
acids (Table 2). Thus, increased DNA-binding activity
of the upstream stimulatory factors USF1 and USF2
on the E box of the IGFBP-1 gene promoter was
observed in the liver of rats fed a protein-free diet [36].
Similarly, decreased binding of the activating protein 1
(AP-1) on the promoter of the endothelin-1 gene was
observed following homocysteine addition in endothe-
lial cells, resulting in inhibition of endothelin-1 expres-
sion [37]. In these two cases, the presence of amino
acid(s) resulted in inhibition of the DNA binding of
the involved transcription factors. By contrast, the
presence of amino acid may also result in stimulation
of the DNA binding of the involved transcription fac-
tor. Thus, in phenylalanine-deprived hepatoma cells,
the transcriptional activity of the hepatocyte nuclear
factor-1 (HNF-1) decreased, limiting expression of the
albumin gene [38]. Another example is brought about
by the activation of ATF2 and c-jun in homocysteine-
treated endothelial cells, stimulating ATF3 gene
transcription [39]. This was also observed in homocy-
steine-treated macrophages in which activation of
nuclear factor-E2-related factor 2 (Nrf2) stimulated the
gcl gene via an antioxidant response element, a path-
way involving the MEK/ERK1/2 kinases [40]. Figure 1

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

genes
or AARE
ASNS,CHOP,
xCT,C/EBP,
SNAT2,ATF3,
CAT-1
IGFBP-1,Endothelin-1,
Albumin,ATF3,Glast,
Gcl
Specific mRNAs
Cultured cells or
in vivo study
Fig. 1. Schematic representation of the
influence of amino acids on gene expression
in mammals. The figure is limited to the
transcription factors involving AARE and
NSRE, as well as the other known transcrip-
tion factors where responsive sequences
were identified in the gene. Details of the
responsive sequences are given in Tables 1
and 2.
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. 1829
transcriptional regulation via its production from the
intracellular metabolism of glutamine, as we [44] and
others [45] have recently reported in intestinal cells.
Finally, Table 3 summarizes studies reporting the
influence of amino acid removal or addition on some
transcription factors, either at the level of their activa-
tion (mainly by their ability to bind DNA) or at the

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

Addition Inhibited SREBP-2 mRNA level Human intestinal cell line [62]
Cysteine
Deprivation Increased ATF3, C/EBPb, C/EBPc, FoxO3A
and Gadd45 mRNA levels
Human hepatoma cells [63]
Stimulating effect resulting from the presence of amino acids
Dietary protein restriction Inhibited HNF-4 and NF1 binding Rat liver in vivo [14]
Mixed amino acids addition Increased phosphorylated STAT3 Perfused rat heart [64]
Tryptophan addition Increased AP-1 binding Human fibroblasts [65]
Homocysteine addition Increased CHOP, Gadd45, ATF4, Id-1, SREBP
and YY1 mRNA levels
HUVEC [66]
Increased c-Fos mRNA level Murine macrophages [67]
Increased ATF4 protein level Human retinal cell line [68]
Increased ATF4 mRNA level HUVEC [69]
Increased NF-jB binding Rat aortic muscle cells [70]
Human VSMCs [71]
Rat VSMCs [72]
HUVEC [73]
Rat kidney mesangial cells [74]
Activated IjB kinases and increased
NF-jB binding
Human endothelial cells [75]
Increased CREB binding HepG2 cells [76]
Increased AP-1 binding Rat hepatocytes [77]
Arginine addition Increased AP-1(c-jun) binding Rat jejunum in vivo [78]
Increased NF-jB protein Diabetic rat pancreas [79]
Glycine addition Increased PPAR-c mRNA level Mouse adipocytes [80]
Glutamine and transcription factors
C. Brasse-Lagnel et al.

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

plasma and human skeletal muscle, a number of stud-
ies recently explored its mode of action on gene expres-
sion, revealing the existence of a large variety of target
genes involved in major functions in the organism
[15,82,83]. Tables 4 and 5 and Fig. 2 illustrate both the
diversity of the studies into the effect of glutamine and
the variety of transcription factors involved in its
action. Although glutamine deprivation was also able
to stimulate the expression of ASNS [84,85] and
CHOP [86] genes in different kinds of mammalian
cells, the involvement of the NSRE and AARE
sequences in these effects was not studied. Moreover,
none of these responsive elements were identified in
the other target genes studied. The only putative
AARE identified in a glutamine-responsive gene was
found in the promoter of the glutamine transporter
ASCT2 gene, but its involvement in the regulation by
Table 4. Influence of glutamine on transcription factors involved in inflammation.
Glutamine Experimental model
Transcription
factor(s)
involved
Effect and
mechanism
involved Reference
Deprivation Human breast cancer cells NF-jB and AP-1 Increased DNA binding [90]
Deprivation Human intestinal (Caco-2) cells STAT-4 Increased DNA binding and
nuclear protein level
[91]
Addition Rat jejunum in vivo AP-1 (c-jun) Decreased DNA binding [78]

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

the amino acid in HepG2 cells was not demonstrated
[87]. Interestingly, studies on the glutamine-responsive
genes and the involved transcription factors revealed
some functional categorization corresponding to
specific effects of the amino acid in: (a) the inflamma-
tory response; (b) cell proliferation, differentiation and
survival; and (c) metabolic functions. We therefore
attempted to delineate the contribution of the gluta-
Sp1 glycosylation
Sp1
Glutamine
HepG2 cells
Rat cardiomyocytes

HepG2 cells
GAPDH
C/EBP α, β
–126 –118
PKA mTOR
CREM
ADSS1
CRE
Hexosamine pathway
GC boxes
ASS
RXR/FXR
ASCT2
AGGTGAATGACTT
FXR
–586 –574

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

[133]
Deprivation Human pancreatic and prostatic
cancer cells
HIF-1a Decreased protein level [153]
Addition Rat heat-shocked intestinal cell line HSF-1 Increased DNA binding [143]
Addition Mouse embryonic fibroblasts HSF-1 Increased phosphorylated nuclear HSF-1
and DNA binding
[144]
Addition Rat intestine in vivo HSF-1 Increased protein level [146]
Addition Mouse fibroblasts (HSF)/)) HSF-1 Activation [145]
Deprivation Human carcinoma cells ATF5 Increased mRNA stabilization [136]
Addition Pancreatic b-cell line Pdx1 Increased mRNA level and DNA binding [126]
Glutamine and transcription factors C. Brasse-Lagnel et al.
1832 FEBS Journal 276 (2009) 1826–1844 Journal compilation ª 2009 FEBS. No claim to original French government works.
mine-modulated transcription factors within each
category.
NF-jB and the effect of glutamine in the
inflammatory response (Table 4)
It is well known that glutamine is able to exert local
and systemic immunoregulatory activity [88,89]. In
particular, the anti-inflammatory role of glutamine
has been extensively studied both in vivo and in vitro,
and data obtained on the regulation of cytokine pro-
duction by the amino acid led to demonstration of
the involvement of specific transcription factors,
mainly NF-jB. Indeed, in glutamine-deprived human
breast cancer cells, activation of NF-jB DNA-bind-
ing may account in part for increased expression of
the IL-8 gene [90]. In addition to STAT [91], the
amino acid was shown to act at the level of the

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

administration before LPS injection also decreased
NF-jB activation and subsequent TNF- a production
[101]. This was recently demonstrated in vitro in
LPS-stimulated rat alveolar cells in which addition of
glutamine increased the glutathione level, prevented
NF-jB activation and attenuated TNF-a release
[102].
Taken together, these results highlight the physio-
logical importance of glutamine which, by counter-
acting activation of the NF-jB pathway, contributes
to the attenuation of local inflammation in the gut
and lung. The pathway by which glutamine attenu-
ates NF-jB activation is not yet clear although it
may involve enhanced intracellular glutathione in
turn inhibiting NF-jB activation [103] or an increase
in the O-linked N-acetylglucosamine protein levels
[104]. In line with these observations, glucosamine, a
metabolite of glutamine, was also shown to exert
anti-inflammatory properties through the inhibition
of the IL-1b-induced activation of NF-jB in cultured
rat or human chondrocytes [105,106] and in TNF-a-
stimulated human retinal cells [107]. Furthermore,
glucosamine was recently reported to suppress the
LPS-induced production of NO via a decrease in the
expression of iNOS by inhibiting NF-jB activation
and phosphorylation of p38 MAP kinase in mouse
macrophages [108]. However, this effect might be tis-
sue-specific because glucosamine remained without
any effect on the IL-1b-induced NF-jB pathway in
Caco-2 cells [44] and could even activate NF-jBin

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

effects of glutamine are tightly linked to the mecha-
nisms of cell survival, as discussed below.
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. 1833
Transcription factors involved in the regulatory
role of glutamine on cell proliferation, apoptosis
and survival
Different effects on cellular processes may contribute
to the trophic role of glutamine, namely an increase in
protein and nucleotide synthesis [114,115], a decrease
in proteolysis [116], reinforcement of the mitogenic
action of growth factors like epidermal growth factor
or growth hormone [117–120] and inhibition of apop-
tosis [121,122] (Table 5). Some of these actions were
shown to be exerted partly through the synthesis or/
and activation of specific transcription factors in vari-
ous kinds of cells. For example, in a porcine jejunal
cell line, glutamine addition was followed by rapid
stimulation of the immediate early gene c-jun expres-
sion, followed by an increase in mRNA and protein
levels of ornithine decarboxylase leading to subsequent
induction of the polyamine synthesis [123]. This was
also reported in rat and pig intestinal cell lines in
which expression of factors c-myc and c-jun, both
involved in cellular proliferation and differentiation,
was stimulated by glutamine addition, accounting for
the important contribution of the amino acid to cellu-
lar growth [124]. Concerning the signalling pathways
involved in the proliferative effect of glutamine on
enterocytes, the amino acid was shown to activate

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

cells lowering growth-cessation signals [86]. Con-
versely, depletion of the amino acid induced activation
of CHOP gene expression in Chinese hamster ovary
cells increasing cell death [132] and induced a parallel
increase in CHOP and GADD 34 mRNA levels in he-
patocarcinoma cells in favour of cancer cell death
[133]. Such a stimulation of CHOP and GADD 45,
another growth-inhibiting gene, was obtained follow-
ing glutamine depletion in human breast cell lines,
decreasing their growth and viability, an effect occur-
ring mainly at a post-transcriptional level [134]. Two
different lines of approach using murine hybridoma
cells showed that glutamine has an anti-apoptotic
effect. One study demonstrated that addition of gluta-
mine to the culture medium limited cell death via a
negative control on CHOP gene expression [135],
whereas the other study showed that its removal
increased cell death through the regulation of several
genes, namely a decrease in the tumour suppressor p53
mRNA level and a parallel stimulation in the expres-
sion of receptor FAS [131]. In parallel, glutamine
could also had an anti-apoptotic role in HeLaS3 cells
through the destabilization of ATF5 mRNA, a tran-
scription factor involved in cellular differentiation and
apoptosis [136]. Glutamine was also able to counteract
the effects of c-myc, a transcription factor involved in
proliferation and apoptosis, conducting paradoxically
either to a reduction or to a stimulation of the apopto-
sis process, depending mainly on the level of c-myc
expression and on the cell type. Indeed, glutamine

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

improved by glutamine treatment in wild-type mouse
embryonic fibroblasts (HSF-1
+/+
) although in knock-
out cells (HSF-1
)/)
), the beneficial effect of glutamine
on survival was lost [112]. Activation of HSF-1 by glu-
tamine was also demonstrated in rat intestine in vivo
improving survival after hyperthermia [146]. Lastly,
HSF-1 was also proposed to be involved in the gluta-
mine-induced expression of Hsp72 in the liver of rat
submitted to heat shock [147].
Several signalling pathways were reported to be
involved in the anti-apoptotic effect of glutamine
[82,148] but data remain sparse. For example, the
amino acid was shown to facilitate the inhibition of
apoptosis signal-regulating kinase (ASK1) in HeLa
cells, thereby limiting apoptosis and providing one
possible explanation for the anti-apoptotic activity of
glutamine [149]. In addition, glutamine may activate
the ERK signalling pathway in rat intestinal epithelial
cells, preventing apoptosis, although JNK and p38
activities were not modified [150]. However, glutamine
was shown to partially prevent the increase in p38 and
JNK phosphorylation in rat neutrophils, thereby
reducing apoptosis induced by exercise [151]. This
underlines the complex regulation exerted by glutamine
on signalling pathways such as the MAP kinases, i.e.
JNK activation in enterocytes [125] and inhibition in

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

indirectly involved in the metabolism of amino acids
was shown to be stimulated by glutamine in the liver
and intestine. For example, in rat liver, glutamine stim-
ulated the expression of PEPCK, glutamine synthetase
[157,158] and ASS genes [159], and these effects were
shown to be mediated, at least in part, by glutamine-
induced cell swelling [160]. Glutamine might regulate
its own synthesis by interacting at the transcriptional
and post-transcriptional levels with the 3¢-UTR of the
glutamine synthetase gene but the regulatory factors
involved are not yet identified [161]. Several reports
brought about some characterization of the molecular
mechanisms involved in the glutamine action on genes
related to metabolism, as summarized in Fig. 2. A first
study was performed in HepG2 hepatoma cells where
glutamine stimulated transcription of the GAPDH gene
[162]. Using deletion mutants and site-directed muta-
genesis of the GAPDH promoter, it was shown that
glutamine responsiveness is mediated by a specific
sequence (-126/-118) which could bind C/EBP proteins.
The corresponding binding cis element was not speci-
fied further but the metabolism of glutamine was
found to be required in this effect. In a second study
performed in cultured rat cardiomyocytes, glutamine
was shown to stimulate the expression of CPT1 and
ADSS1 [163], encoding enzymes involved in cardiac
fatty acid metabolism and adenine nucleotide metabo-
lism, respectively. Induction was mediated via the pro-
tein kinase A pathway and partly through that of
mammalian target of rapamycin, which is known to be

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

blockade of glutamine metabolism was observed in
Ehrlich tumour cells [167]. Finally, in a study per-
formed in HepG2 hepatoma cells, glutamine was
shown to activate the nuclear farnesoid X receptor
(FXR)/retinoid X receptor (RXR), favouring its bind-
ing to its responsive sequence in the promoter of the
ASCT2 gene encoding a glutamine transporter
[168,169].
Together, these studies illustrate that different tran-
scription factors, namely C/EBP, FXR/RXR, cAMP
response element modifier and Sp1, and their corre-
sponding responsive elements are required to regulate
various metabolic pathways in the hepatic, cardiac and
intestinal transcriptional response to glutamine. These
responsive cis elements are not specific of an AAR
pathway suggesting that the effects of glutamine and
potentially those of other amino acids might depend
not only on cell types but also on the structure of the
gene promoters.
Finally, Fig. 3 summarizes the different families of
transcription factors modulated by glutamine to regu-
late physiological processes. Moreover, considering the
effects of other amino acids (independently of the cell
types and the target genes), the figure highlights that
some amino acids may act similarly to glutamine, some
others exerting the opposite effect.
Concluding remarks
In summary, regulation of transcription by amino
acids appears to derive from a variety of mechanisms.
Indeed, since the initial reports identifying specific

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

the potential diversity of AAR pathways, but despite
its central regulatory role in numerous functions, the
involved intracellular metabolites and complete signal-
ling pathways remain to be identified. Finally,
Fig. 3. Families of transcription factors modulated by glutamine to
regulate physiological processes. Comparison with the effects of
other amino acids. The families of transcription factors modulated
by glutamine are written in coloured characters depending on its
effect: red, inhibition; green, activation; grey, inhibition or activation
depending on the cell types or the experimental conditions. The
effects of the other amino acids (circle connected by a line) are
presented by using the same colours.
Glutamine and transcription factors C. Brasse-Lagnel et al.
1836 FEBS Journal 276 (2009) 1826–1844 Journal compilation ª 2009 FEBS. No claim to original French government works.
although some studies were performed in vivo, it must
be noted that most of the data on the molecular mech-
anisms by which glutamine is acting were obtained
with cultured transformed cells. It would be therefore
worthwhile to demonstrate that the identified mecha-
nisms are also involved in normal cells.
Acknowledgements
We thank Dr Carole Beaumont (INSERM U773,
Paris, France) for critical reading of this manuscript.
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