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

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; signal

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: Alain.lavoinne@chu-rouen.fr

(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.

acid availability have been extensively studied in lower

eukaryotes such as yeasts [4], the control of

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 eleele-ments

(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 suzip-perfamily 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 genea

Transcription factor(s) involved

Localization of the responsive sequence(s)

Responsive

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]

and -76/-68)

AARE 5¢-TGATGCAAA-3¢

and 5¢-TTTGCATCA-3¢

[30]

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.

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 additranscrip-tional 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

xc ), [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

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]

deprivation

Albumin inhibition HNF1a inhibition 5¢-Flanking region

(-60/-46)

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)

a Accessory sites and additional factors are not cited.

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 funcaddi-tionality 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

Cultured cells

Cytoplasm

Nucleus

ATF2, Nrf2

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.

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.

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

Leu + Ile + Cys + Trp

deprivation

Increased CHOP mRNA and protein levels Mouse fibroblasts [48]

Methionine

Decreased PPAR a, c mRNA and protein levels Human monocytes [57]

Histidine

Arginine

Leu or Ile or Val

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]

Homocysteine addition Increased CHOP, Gadd45, ATF4, Id-1, SREBP

and YY1 mRNA levels

Rat kidney mesangial cells [74] Activated IjB kinases and increased

NF-jB binding

Human endothelial cells [75]

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

Deprivation Human intestinal (Caco-2) cells STAT-4 Increased DNA binding and

nuclear protein level

[91]

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 Rat colon (and pancreas) in vivo

(experimental colitis)

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]

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 HSF-1 and Sp1 Increased O-glycosylation and phosphorylation [111]

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

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)

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 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

Addition Mouse embryonic fibroblasts HSF-1 Increased phosphorylated nuclear HSF-1

and DNA binding

[144]

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-jB in 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

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, leadstimulat-ing 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

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