Dysregulation of glutathione synthesis in liver disease☆

Shelly C.Lu

Division of Digestive and Liver Diseases,Cedars-Sinai Medical Center,Los Angeles,CA,USA

Keywords:

Glutathione (GSH)

Glutamate-cysteine ligase (GCL)

Glutathione synthetase (GS)

Nuclear factor-erythroid 2 related factor 2

(NRF2)

MAF BZIP transcription factor G (MAFG)

c-MAF

Antioxidant-response element

Liver disease

ABSTRACT

Glutathione(GSH),a tripeptide that is present in all mammalian tissues,is especially highly concentrated in the liver.GSH synthesis occurs via two adenosine triphosphate (ATP)-requiring enzymatic steps:the first is rate-limiting,catalyzed by glutamate-cysteine ligase,generates γ-glutamylcysteine from glutamate and cysteine;the second is catalyzed by GSH synthetase,generates GSH from γ-glutamylcysteine and glycine.GSH defends against oxidative stress,participates in detoxification of xenobiotics,determines the redox status of the cell,and regulates vital processes such as growth and apoptosis.Hepatic GSH plays a central role in the interorgan GSH homeostasis because sinusoidal efflux of hepatic GSH determines plasma GSH level.In liver diseases GSH homeostasis is perturbed by multiple mechanisms.Hepatic GSH biosynthesis is impaired in cholestatic liver injury,endotoxemia,and fibrotic injury largely because the expression of the GSH synthetic enzymes falls.Lower hepatic GSH level further exacerbates and perpetuates ongoing liver injury.However,in hepatocellular carcinoma GSH synthetic enzymes are upregulated and this may play a role in chemoresistance.This review focuses on the current understanding of hepatic GSH synthesis in health and disease.

1.Introduction

Glutathione (GSH) is a tripeptide,γ-L-glutamyl-L-cysteinylglycine,made in the cytosol of all mammalian cells as the most abundant non-protein thiol.GSH defends against oxidative stress,participates in detoxification of xenobiotics,determines the redox status of the cell,and regulates vital processes such as growth and apoptosis.Liver has the highest GSH concentration of all tissues and hepatic GSH plays a key role in interorgan GSH homeostasis by being the main source of plasma GSH.1Hepatic GSH level falls in multiple liver diseases.The fall in hepatic GSH is due to a combination of factors,such as depletion during oxidative stress,increased utilization and export,and decreased synthesis.2Reduced hepatic GSH level can exacerbate and perpetuate liver injury.This review discusses GSH functions and homeostasis with the focus on mechanisms of GSH synthesis dysregulation in liver diseases.

2.Functions and importance of GSH

GSH is synthesized in all mammalian cells and exists in the thiol-reduced (GSH) and disulfide-oxidized (GSSG) forms.3GSH is the predominant form,existing in millimolar concentrations in most cells (liver 5-10 mM).Almost 90% of cellular GSH is in the cytosol,10% is in the mitochondria and a small amount is in the endoplasmic reticulum (ER).4,5Cytosolic GSH in the rat liver turns over rapidly with a half-life of 2-3 h.GSH has a unique structure in that the peptide bond linking glutamate and cysteine is through the γ-carboxyl group of glutamate rather than the conventional αcarboxyl group.Importantly,only one enzyme,γ-glutamyl-transpeptidase(GGT),is able to hydrolyze this unique peptide bond and it is present on the external surfaces of many cell types.6Thus,GSH is resistant to intracellular degradation and is metabolized extracellularly by organs expressing GGT(such as the liver).GGT breaks down the released GSH to glutamate and cysteinylglycine,which can be broken down to cysteine and glycine by dipeptidases.The constituent amino acids are then taken up and reincorporated into GSH.This is the so-called γ-glutamyl cycle,described by Alton Meister,7in the early 1970’s,that allows GSH to serve as a continuous source of cysteine.This is important as extracellular cysteine is unstable and rapidly auto-oxidizes to cystine,which can generate potentially toxic oxygen free radicals.

GSH serves several vital functions including:(i) detoxifying electrophiles;(ii) scavenging free radicals;(iii) maintaining the essential thiol status of proteins;(iv) providing a reservoir for cysteine;and (v) modulating critical cellular processes such as growth and death,microtubular-related processes,immune function,and fibrogenesis.These functions were reviewed in detail previously and are briefly mentioned below.8

2.1.Antioxidant function

GSH plays an important defensive role against oxidative stress,particularly in the mitochondria.9GSH peroxidase catalyzes reduction of hydrogen peroxide and lipid peroxide as GSH gets oxidized to GSSG in the process.GSSG is then reduced back to GSH by GSSG reductase at the expense of nicotinamide adenine dinucleotide phosphate(NADPH).The GSH to GSSG ratio determines the intracellular redox potential (proportional to the log of [GSH]2/[GSSG]),10which needs to be maintained in a narrow range.To prevent a major shift in the redox equilibrium,GSSG can be actively exported out of the cell or react with a protein sulfhydryl group forming a mixed disulfide.By this mechanism GSH can become depleted in severe oxidative stress.

2.2.Regulation of cell growth

GSH level regulates cell growth.Increased GSH level is associated with a proliferative response in many normal and malignant cell types.11-14For hepatocytes in culture,GSH level increases when hepatocytes are plated under low density,a condition that stimulates cells to enter the cell cycle,15and similarly,GSH level increases after 2/3 partial hepatectomy prior to the onset of deoxyribonucleic acid (DNA) synthesis.16Blocking the increase in hepatic GSH level reduced DNA synthesis by 33% during liver regeneration.16Increased GSH levels also occur in many cancers including liver cancer,which many believe contribute to chemo- and radiation resistance.17,18One often proposed mechanism for GSH to enhance DNA synthesis has to do with maintaining reduced glutaredoxin and thioredoxin,which are important for the activity of ribonucleotide reductase,the rate-limiting enzyme in DNA synthesis.19The GSH redox status can also affect the expression and activity of many factors important for cell cycle progression.Finally,GSH co-localizes to the nucleus at the onset of proliferation,which is thought to regulate the activity of many nuclear proteins including histones and a reducing condition was thought to be necessary for cell cycle to progress.20,21

2.3.Regulation of cell death

GSH also modulates cell death.Apoptosis,the best characterized programmed cell death that is initiated and executed by the caspase family,is often preceded by GSH depletion.18,22The fall in GSH level occurs because of increased reactive oxygen species (ROS),enhanced GSH export and decreased GSH synthesis.23,24Interestingly,enhanced GSH export appears necessary in order for apoptosis to occur in many cell types.25However,very high levels of ROS can cause profound GSH depletion and convert apoptotic to necrotic cell death.23GSH depletion has also been linked to other forms of programmed cell death,such as necroptosis,ferroptosis,and autophagy.18,26These different forms of cell death may occur simultaneously or in succession.How the GSH level influences the interrelationships and crosstalks between these different forms of cell death remains incompletely understood.

3.Hepatic GSH export

The liver releases GSH into both sinusoidal blood and the bile.Sinusoidal GSH export determines systemic availability of GSH and is thought to be mediated by organic anion-transporting polypeptide 1.27In contrast,biliary GSH efflux is believed to be mediated by multidrug resistance proteins.18Once released into bile GSH is rapidly broken down by GGT and is an importance source of precursors for intestinal epithelial cell GSH synthesis.28Hormones such as vasopressin,adrenalin,and phenylephrine,induced sinusoidal GSH efflux and lowered biliary GSH efflux in perfused rat livers.29We also reported glucagon increased sinusoidal GSH efflux in the same model.30These hormones are known to be elevated during stress and could contribute to hepatic GSH depletion during stress.31

4.Hepatic GSH synthesis

The liver has tremendous capacity to synthesize GSH and has the highest GSH level of all organs.32GSH synthesis occurs via two adenosine triphosphate (ATP)-requiring enzymatic steps,the first step is the formation of γ-glutamylcysteine from glutamate and cysteine catalyzed by glutamate-cysteine ligase (GCL,EC 6.3.2.2),and the second step is the formation of GSH from γ-glutamylcysteine and glycine catalyzed by GSH synthetase (GS,EC 6.3.2.3).GCL is the rate-limiting enzyme of GSH synthesis that is composed of a catalytic(GCLC,Mr ～73 kDa)and a modifier(GCLM,Mr ～31 kDa) subunit,which are encoded by different genes in rodents and humans.33-36GCLC is catalytically active by itself and is subject to feedback inhibition by GSH.37However,GCLM lowers the Kmof GCL for glutamate and raises the Kifor GSH so that the holoenzyme is catalytically much more active.34,38Consistently,Gclm knockout mice have 85-90%lower tissue GSH levels.39In contrast,GS is composed of two identical subunits(Mr ～118 kDa)and is not subject to feedback inhibition by GSH.40Although GCL is the ratelimiting enzyme,GS is important in determining overall GSH synthetic capacity in certain tissues and/or under stressful conditions.For instance,surgical trauma lowered GSH levels and GS activity in skeletal muscle while GCL activity was unchanged.41We also reported in rat hepatocytes that increased expression of GS further enhanced GSH synthesis compared to increased expression of GCLC expression alone.42Nevertheless,dysregulation of GSH synthesis in liver diseases typically involve only GCL (see below).

In the liver GSH synthesis is largely determined by the expression and activity of GCL,which is mainly determined by the availability of L-cysteine and feedback competitive inhibition by GSH(Ki= 2.3 mM).6,43The intracellular cysteine concentration is close to GCL’s Kmvalue for cysteine (0.1-0.3 mM) whereas the intracellular glutamate concentration is 10-fold higher than GCL’s Kmvalue for glutamate (1.8 mM).43

4.1.Hepatic uptake of cysteine,cystine,and methionine

The liver derives cysteine from the diet,protein breakdown,and from methionine via the trans-sulfuration pathway.8Cysteine is taken up by the alanine-serine-cysteine (ASC) system (the ubiquitous Na+-dependent neutral amino acid transporter with preferred substrates alanine,serine and cysteine) but its plasma concentration is low because cysteine is highly unstable and readily oxidizes to cystine,which is taken up via the Xc-system (the cystine/glutamate antiporter that exchanges extracellular cystine for intracellular glutamate) by some cells including hepatocytes,particularly under oxidative stress.44,45Methionine uptake is via the L system (the transporter responsible for transport of several large neutral amino acids) and once taken up it is converted to cysteine efficiently in the liver via the trans-sulfuration pathway.8,46

4.2.Trans-sulfuration pathway

The liver has one of the highest activities of the trans-sulfuration pathway,which converts methionine to cysteine through a series of enzymatic steps.47,48Liver receives up to half of the daily intake of methionine and catabolizes it to S-adenosylmethionine(SAMe),the principal biological methyl donor,in a reaction catalyzed by methionine adenosyltransferase(MAT).47As the methyl group from SAMe is transferred to a long list of acceptor molecules,S-adenosylhomocysteine (SAH) is generated.SAH is toxic,as it is a potent inhibitor of all transmethylation reactions,so it is promptly hydrolyzed to homocysteine (Hcy) and adenosine in the only reversible reaction of the trans-sulfuration pathway that is catalyzed by SAH hydrolase.In the liver Hcy can undergo three metabolic pathways,two of which regenerate methionine (via methionine synthase,which requires folate and vitamin B12,and betaine homocysteine methyltransferase,which requires betaine),and the third pathway generates cysteine.In this last pathway Hcy condenses with serine to form cystathionine in a reaction catalyzed by cystathionine β synthase (CBS).This is followed by cleavage of cystathionine by γ-cystathionase to generate cysteine.Both CBS and γ-cystathionase require vitamin B6.47,48Patients with chronic liver disease have marked abnormalities in methionine metabolism and the trans-sulfuration pathway.One key mechanism is decreased hepatic MATactivity and lower SAMe level.47The other is deficiency of co-factors such as B vitamins and lower activity of CBS,which is activated by SAMe.49Thus,patients with chronic liver disease have impaired cysteine availability largely due to decreased conversion of methionine to cysteine.Abnormalities in the methionine metabolic pathway are reviewed in detail elsewhere.47,50

Fig.1 summarizes GSH homeostasis in the liver.

5.Regulation of GCL

In liver diseases GCL activity is often altered due to a variety of mechanisms affecting both GCLC and GCLM,or only GCLC.These mechanisms occur at transcriptional,post-transcriptional and posttranslational levels.

5.1.GCLC and GCLM transcriptional regulation

Rodents and human GCLC are similarly regulated transcriptionally.8Consensus nuclear factor-kappa B(NF-kB),Sp-1,activator protein (AP)-1,AP-2,metal response,and antioxidant response element (ARE) elements have been identified in the human GCLC promoter,while AP-1 and ARE elements are present in the human GCLM promoter.8While many of the regulators influence the expression of both GCLC and GCLM in a coordinated manner,others affect only GCLC.For instance,oxidative stress is famously known to activate both GCLC and GCLM transcription,whereas hormones such as insulin and hydrocortisone only increase GCLC transcription.51-53Consistently,erythrocytes from diabetic patients have decreased GSH levels and increased susceptibility to oxidative stress.54Interestingly,in a group of 401 patients with type I diabetic mellitus,the single nucleotide polymorphism(SNP)-129C →T in GCLC(rs17883901),where the T allele determines a lower promoter activity,is significantly associated with renal disease.55Thus,lower GCLC transcription in diabetic patients may occur due to a combination of mechanisms.

GCLC transcription is also increased(GCLM is unchanged)during hepatocyte proliferation,such as following partial hepatectomy and in hepatocellular carcinoma (HCC).15-17An increase in GCLC expression alone is capable of normalizing liver GSH level in mice lacking GCLM.56This further demonstrates the importance of GCLC in the overall GSH synthesis capacity.

Fig.1.Hepatic GSH homeostasis.

Lastly,transforming growth factor-β1 (TGF-β1),a cytokine implicated in pulmonary and liver fibrosis,was also shown to lower GSH synthesis at the level of GCLC.57,58In type II alveolar epithelial cells TGF-β1 lowered GCLC transcriptional activity.57Similarly,TGFβ1 was thought to mediate its profibrogenic effects by suppressing the expression of GCLC(no effect on GCLM)and lowered GSH levels in rat hepatic stellate cells(HSCs).58The suppressive mechanism of TGF-β1 on GCLC transcription was thought to be displacement of nuclear factor-erythroid 2 related factor 2 (NRF2) bound to the ARE.59However,a more recent work showed TGF-β1 suppressed both GCLC and GCLM expression in mice and human umbilical endothelial cells by a mechanism that involved microRNA (miR)-433.59

Oxidants as well as antioxidants such as butylated hydroxyanisole,tert-butyl hydroquinone,and inducers of Phase II detoxifying enzymes all increase the transcription of GCLC and GCLM.8Induction of GCLC and GCLM transcription is a defense mechanism against stress.However,if the stress persists,GCLC and GCLM can become dysregulated as seen in multiple liver injuries (see below under dysregulation of GSH synthesis in liver injuries).

NRF2 is the best-known transcription factor that positively regulates GCLC and GCLM transcription via binding to the ARE present in human and rodent GCLC and GCLM promoters.8NRF2 belongs to the cap ‘n’ collar-basic leucine zipper proteins and is normally kept in the cytosol by Kelch-like ECH-associated protein 1(KEAP1) homodimer,the E3 ubiquitin ligase adaptor that targets NRF2 for proteasomal degradation.60Oxidative stress activates NRF2 by modifying either KEAP1 or NRF2 posttranslationally to cause their dissociation.61Another mechanism in single live cells shows NRF2 binding to KEAP1 dimers keeps KEAP1 in a closed conformation and unable to bind newly synthesized NRF2,allowing NRF2 to translocate to the nucleus.62Once inside the nucleus,NRF2 forms heterodimers with small MAF(MAFG,MAFK and MAFF)and JUN (c-JUN,JUN-D,and JUN-B) proteins to bind and activate the ARE.63NRF2/MAFG heterodimerization has also been shown to enhance NRF2 nuclear retention.64KEAP1 also shuttles between the nucleus and the cytoplasm and the nuclear import is mediated by importin α7.65KEAP1 has a strong nuclear export signal that facilitates export of KEAP1-NRF2 complex to keep ARE-dependent gene induction under control.

NRF1 is also induced by stress signals and heterodimerize with small MAFs to activate the ARE.66However,NRF1 is not regulated by KEAP1;instead NRF1 is located in the ER as an integral membrane protein.66Once in the ER,NRF1 undergoes N-linked glycosylation and remains in the ER until it is degraded or released during ER stress and translocates to the nucleus to activate target genes.66,67Deletion of NRF1 or NRF2 lower GCLC and GCLM expression,67,68whereas overexpression of NRF1 or NRF2 induced the human GCLC promoter activity.69,70

Besides NRF1 and NRF2,NF-kB and AP-1 family members also positively regulate GCLC and GCLM transcriptionally.71,72There is a complex interaction between NRF1/NRF2 and AP-1/NF-kB family members.Expression and nuclear binding activities of c-JUN,c-FOS,p50 and p65 are lower in fibroblasts deficient in NRF1 or NRF2.72Overexpression of NRF1 and NRF2 restored the rat 1.8 kb-Gclc promoter activity and response to tert-butyl hydroquinone but this was blocked if the AP-1 and NF-kB binding sites were mutated.72In addition,NF-kB activation can increase AP-1 expression and nuclear binding activity.71This explains how NF-kB activation increased GCLM promoter activity even though no NF-kB element is present.71Finally,c-MYC has also been shown to induce human GCLC and GCLM transcription via noncanonical c-MYC binding sites(CACATG,E box) present in the GCLC and GCLM promoters under oxidative stress.73However,in liver disease c-MYC upregulation can cause the opposite effect via an indirect mechanism that involves miR-27a/b(see below).

5.2.GCLC and GCLM post-transcriptional regulation

GCLC and GCLM are regulated post-transcriptionally by miRNAs,which are small noncoding RNAs of 21-25 nucleotides in length that in most instances regulate gene expression by targeting the 3＇untranslated region(3＇UTR)of messenger RNAs(mRNAs)leading to reduced protein translation and/or increased mRNA degradation.74These miRNAs can target GCLC and GCLM directly or indirectly by targeting the NRF2-KEAP1 pathway.75For instance,miR-433 was shown to directly target both GCLM and GCLC via their 3＇UTR.59In contrast,several miRNAs regulate GCLC and GCLM indirectly via NRF2.76miR-28,miR-93,miR-153,miR-27a,miR-27b,and miR-142-5p have all been shown to directly target NRF2 3＇-UTR in different cell lines and animal models.75,77We showed miR-27a and miR-27b participate in a complex circuit that leads to downregulation of both GCLC and GCLM in cholestatic liver injury (see below under cholestatic liver injury).77

In addition to miRNAs,treatment with diethyl maleate or 4-hydroxynonenal (4-HNE) increased GCLC transcription and stabilized GCLC mRNA but the mechanism is unclear.78,794-HNE also stabilized GCLM mRNA by a mechanism that required de novo protein synthesis.79

5.3.GCLC and GCLM post-translational regulation

GCLC is regulated at the protein level by phosphorylation and caspase-mediated cleavage.We and others showed GCL activity can be inhibited by several stress hormones.80,81Some of these hormones also increase sinusoidal GSH efflux.29,30,81,82Increased sinusoidal GSH efflux will increase systemic GSH availability and inhibition of GSH synthesis may conserve cysteine for synthesis of stress proteins.83One underlying mechanism of the inhibition in GCL activity is phosphorylation of GCLC by activation of protein kinase A,protein kinase C or Ca2+-calmodulin kinase II,kinases that are induced by stress hormones.84GCLC is phosphorylated in cultured hepatocytes under basal condition and phosphorylation increased upon treatment with dibutyryl cAMP (DBcAMP) or phenylephrine,suggesting GCLC may be under a basal inhibitory tone.84

During apoptosis induced by TGF-β1,tumor necrosis factor α(TNFα),and Fas,GCLC can be cleaved from the full-length 73 kDa to a 60 kDa form.85,86GCLC cleavage requires caspase 3 and occurs at Asp499 within the sequence AVVD499G,which is thought to be important for disulfide bond formation with GCLM.24,85However,GCL activity was not lower during apoptotic cell death.85The significance of GCLC cleavage remains unclear.

4-HNE was shown to directly adduct GCLC Cys553 and GCLM Cys35 in vitro.87Interestingly 4-HNE GCLC adduct had higher activity of monomeric GCLC but was unable to form GCL holoenzyme.87Since the holoenzyme is much more active this is likely to lower overall GCL activity.However,whether this occurs in vivo or not is unclear.

6.Regulation of GSH synthetase (GS)

GS regulation is much less studied but GS deficiency in humans can result in severe metabolic acidosis,hemolytic anemia and central nervous system damage because the accumulated γ-glutamylcysteine is converted to 5-oxoproline,which is toxic.88There are reports that a fall in GS alone resulted in a lower GSH level,which would contradict the notion that GCL is rate-limiting.41,89In normal liver the specific activity of GS is normally 2 to 4 times that of GCL activity,51,90but this may not be the case in other tissues.For instance,in normal human skeletal muscle,the specific activity of GS is only 36% higher than that of GCL and this was selectively lowered by surgical trauma leading to reduced GSH synthesis.41

Many inducers of both GCL subunits also induce the expression of GS.8In contrast,treatments that increase the expression of GCLC alone had no influence on GS expression.8There are exceptions,such as during rapid liver growth after partial hepatectomy and in HCC,where GS expression increased in parallel to GCLC while GCLM remained unchanged.16,17One plausible explanation is that when GCL is markedly induced the second step in GSH synthesis might become limiting.A coordinated induction of both GCLC and GS would further enhance GSH synthesis.This notion is supported by the finding that GCLC induction alone increased the GSH synthesis capacity by 50-100%,51,91whereas induction of both GCLC and GS increased the GSH synthesis capacity by 161-200%.16,90

GS transcriptional regulation is similar to the GCL subunits.8AP-1,NRF1,NRF2 are all positive regulators of the GS promoter.92,93NFkB can also activate the rat GS promoter indirectly via AP-1.71Posttranscriptional and post-translational regulation of GS have not been reported.

Table1 summarizes regulation of the GSH synthetic enzymes.

7.Dysregulation of GSH synthesis in liver injury

Reduced GSH levels occur in many human diseases and they contribute to worsening the diseases.94Oxidative injury is the leading cause of GSH depletion in these disorders but GSH deficiency due to polymorphism of GCLC and/or GCLM that result in lower GCL expression has been reported in disorders including schizophrenia,cardiovascular diseases,stroke,and asthma.85,95In liver disease,there are conflicting data regarding -129C/T polymorphism of the GCLC gene in non-alcoholic fatty liver disease.96,97Patients with chronic liver diseases have low hepatic GSH levels that up until 10 years ago have mostly been attributed to nutritional deficiency and oxidative stress.Our accumulating evidence suggests that a key reason for the low GSH level is lower expression of the genes involved in GSH synthesis.This can further contribute to liver injury by lowering the antioxidant defense,allowing inflammatory cytokines to be released and fibrosis to progress.The opposite scenario,higher GSH level,occurs in many cancers including HCC and it may provide not only a proliferative advantage,but also resistance to drug and radiation therapy.17,94Consistently,a recent report found higher GCLC expression in HCC correlated with worse outcome.98Below summarizes mechanisms leading to reduced GSH synthesis in liver injuries from chronic cholestasis,alcohol,fibrosis,and endotoxemia,and higher GSH levels in HCC.

7.1.Chronic cholestatic liver injury

Cholestasis,due to impairment in bile formation,secretion or flow,is present in many human liver diseases.99This can occur due to genetic causes such as mutations involving bile salt and phospholipid transporters,immune-mediated disorders such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis,and mechanical causes such as bile duct obstruction or scarring as seen in biliary atresia.99,100Retention of hydrophobic bile acids is thought to be the underlying mechanism for liver injury,which can lead to cell death,fibrosis,and cirrhosis.99A commonly used rodent model of chronic cholestasis is bile duct ligation (BDL).Using this model and feeding mice lithocholic acid(one of the toxic bile acids),we described changes in GSH synthetic enzymes and the underlying mechanisms as detailed below.

During BDL in mice we described an early induction in the hepatic expression of GSH synthetic enzymes,likely due to an adaptive response triggered by oxidative stress,101but this was followed by a fall in their expression and GSH levels at the later stages of BDL.102In parallel to these changes was an early(day 3)increase in NRF2 binding to the ARE followed by a fall at day 14 that persisted to day 28.102One underlying mechanism was related to an increase in the expression of several MAF proteins (c-MAF,MAFG,and MAFK) as well as increased c-MAF and MAFG nuclear binding to ARE.MAFG and MAFK are small MAFs that heterodimerize with NRF2 to activate ARE-dependent genes.61-63However,an increase in the expression of small MAFs can repress ARE-dependent gene expression because homodimers of small MAFs bind to the ARE but lack transcriptional activation domain so they act as competitive repressors of the ARE.103Large MAF proteins like c-MAF also bind to the ARE as homodimers and heterodimers with small MAFs to repress ARE-dependent gene expression.104Consistent with the notion that higher MAFG and c-MAF expression during BDL displaced NRF2 from binding and activating ARE,blocking their increase during BDL protected against the fall in the expression of GSH synthetic enzymes and GSH levels.105

Table1 Regulation of GSH synthetic enzymes.

We also described another mechanism that contributes to the fall in hepatic GSH level during cholestasis that involves c-MYC and prohibitin 1(PHB1).77Using biotinylated ARE in pull-down assays,followed by proteomic analysis,we identified c-MYC and PHB1 as interacting proteins at the ARE.77We had reported c-MYC expression is induced early during BDL,106and its sustained induction may play a role in cholestasis-associated development of cholangiocarcinoma (CCA).107PHB1,a well-known mitochondrial chaperone protein,had been reported to be induced by c-MYC.108However,we found that c-MYC actually suppressed PHB1 expression by two different mechanisms.One is indirect via miR-27a and miR-27b,which are downstream c-MYC targets that are induced during BDL and they directly inhibit PHB1 via its 3＇UTR.77Another is a direct effect of c-MYC acting as a repressor on an E-box present in the PHB1 promoter.109Importantly both c-MYC and PHB1 interact with NRF2 at the ARE,with c-MYC acting as a repressor that lowers NRF2 binding whereas PHB1 is an activator that enhances NRF2 binding.77In addition,miR-27a and miR-27b also negatively regulate NRF2 via its 3＇UTR.77Lower PHB1 expression was confirmed in cholestatic liver injury in humans,such as PBC and biliary atresia.110Lower NRF2 and GCLC expression was also found in patients with PBC.111Taken together,activation of this c-MYCmiR-27a/b-NRF2/PHB1 circuit in cholestatic liver injury culminates in lower expression of ARE-dependent genes,such as GSH synthetic enzymes (Fig.2).Note that these results contradict earlier work showing c-MYC induces GCL subunits transcriptionally in melanoma cells and fibroblasts.73However,Levy et al.112also found c-MYC negatively regulates GCLC and GCLM transcription in human bronchial alveolar epithelial cells,albeit via a different mechanism.

Another potential mechanism in the murine BDL model is miR-433,which increased in expression during BDL.59However,whether it contributes to the fall in GCLC and GCLM expression during BDL has not been critically examined.

Ursodeoxycholic acid (UDCA) and SAMe are two agents that have been used to treat cholestatic liver injury.47,113UDCA was the only food and drug administration (FDA) approved medication for the treatment of PBC until 2016 when obeticholic acid was approved for patients that had insufficient response to UDCA.114We found UDCA and SAMe prevented the increase in c-MYC,MAFG and c-MAF,and protected against the fall in expression of PHB1,GSH synthetic enzymes and GSH levels in murine models of cholestatic liver injury.77,102,105Combining UDCA and SAMe exerted additional benefit,suggesting they have different mechanisms.Obeticholic acid is a potent agonist of farnesoid X receptor(FXR)and MAFG is a downstream target of FXR that exerts transcriptional repression on bile acid synthesis as part of the enterohepatic negative feedback regulation.115This prompted us to compare these three agents in cholestatic liver injury and cancer.116We found induction of MAFG by toxic bile acid required trans-activation of AP-1,E-box and NF-kB elements present in its promoter region.UDCA and SAMe target these elements in overlapping manner,thus explaining why combining them is more effective than using them alone.In contrast,obeticholic acid induced MAFG via the FXR element.Of concern is that higher MAFG expression was found in both human HCC and CCA and higher MAFG expression correlated with worse outcome in HCC.116Using a xenograft model we showed that treatment with SAMe reduced tumor growth from HCC and CCA but treatment with obeticholic acid markedly increased tumor growth.This raises the concern of long-term use of obeticholic acid in patients who may already harbor premalignant changes in the liver.

7.2.Alcoholic liver disease (ALD)

Fig.2.Chronic cholestasis turns on c-MYC-miR27a/b circuit to inhibit ARE-dependent gene expression.

Fig.3.SUMO-1 sumoylation of NRF2 and MAFG is needed for heterodimerization and trans-activation of ARE in mouse liver,HSCs,macrophages and hepatocytes.

Patients with ALD frequently have reduced hepatic and plasma GSH levels.117,118This is one of the reasons that ALD patients are predisposed to acetaminophen toxicity.118GSH deficiency in these patients occur as a result of multiple mechanisms including nutritional deficiency,oxidative stress,and reduced cysteine availability from the methionine metabolic pathway.117,119We also reported 50%lower GCLC and GS mRNA levels in a group of patients with alcoholic hepatitis but the mechanism is unclear.120Importantly mitochondrial GSH depletion occurs early in experimental models of ALD.121These abnormalities predispose them to progressive liver injury.

7.3.Liver fibrogenesis

Liver fibrosis is characterized by activation of HSCs.122HSCs reside in the space of Disse and are the major storage sites of vitamin A in normal liver.Following chronic liver injury,HSCs lose their vitamin A,proliferate,produce a variety of collagenous and non-collagenous extracellular matrix proteins while undergoing a major phenotypical transformation to α-smooth muscle actin (α-SMA)positive activated HSCs.122Activated HSCs synthesize fibrotic matrix proteins that inhibit fibrosis degradation.One of the bestknown pro-fibrogenic factors is TGF-β,which has been shown to inhibit GCL expression in pulmonary and liver fibrosis.58,123-125On the other hand,epigallocatechin-3-gallate from green tea and curcumin have been shown to exert anti-fibrotic effects in hepatic HSCs by inducing GCL expression and GSH synthesis.58,125Consistently,preventing the fall in hepatic GSH level protected against,105whereas lowering hepatic GSH level exacerbated hepatic fibrosis triggered by BDL.126

The expression of GSH synthetic enzymes fall during HSC activation.126Keeping GCLC expression low kept HSCs in activated state.126Intriguingly activated HSCs have lower NRF2/MAFG heterodimer binding to the ARE even though nuclear MAFG level is higher.The opposite was true for quiescent HSCs,namely higher NRF2/MAFG heterodimer binding to the ARE despite lower total nuclear MAFG level.The underlying mechanism is due to a fall in sumoylation of NRF2 and MAFG during HSC activation.Specifically,we found both NRF2 and MAFG are sumoylated by small ubiquitinrelated modifier 1(SUMO-1),which promoted NRF2 binding to the ARE and heterodimerization with MAFG.During HSC activation SUMO-1 sumoylated NRF2 and MAFG levels fall and they are not able to heterodimerize or bind to the ARE.126Taken together,sumoylation of NRF2 and MAFG enhances heterodimerization,trans-activation of ARE and increases GCLC expression,which keeps HSCs in quiescent state.This mechanism is also important in endotoxemia (see below).

7.4.Effect of endotoxin on liver GSH

Lipopolysaccharide (LPS,also known as endotoxin) is a major constituent of the outer membrane of gram-negative bacteria.127The liver plays a key role in clearing gut-derived LPS,which explains why the degree of endotoxemia correlates with the severity of liver failure.128Gut-derived endotoxin worsens ALD and nonalcoholic steatohepatitis by activating Kupffer cells and triggering the synthesis and release of pro-inflammatory cytokines and inducible nitric oxide synthase.128-130In addition,endotoxemia lowers hepatic GSH levels by multiple mechanisms,including oxidative stress,increased sinusoidal GSH efflux and decreased GSH synthesis.131-133This sets up a vicious cycle because the endogenous GSH level determines susceptibility to LPS-induced injury in multiple tissues.132,134,135Studies have shown that GSH regulates the innate immune response.For instance,LPS-induced mortality and TNFα secretion were higher when GSH level was reduced.136We and others have shown endotoxemia leads to a fall in GCL expression,GCL activity and hepatic GSH levels.132,133In a follow-up work we elucidated how this occurs.We found that LPS treatment lowered Gclc and Gclm transcription because of decreased sumoylation of NRF2 and MAFG,their heterodimerization and subsequent binding and trans-activation of the ARE.137LPS treatment reduced the expression of ubiquitinconjugating enzyme 9 (UBC9),the sole E2 enzyme required for sumoylation,in RAW cells (murine macrophages),in mouse liver after in vivo treatment,and in mouse hepatocytes.Overexpressing UBC9 protected against LPS-mediated inhibition of GCLC and GCLM expression.137Thus,this is a feed forward loop where LPS lowers GSH synthetic capacity via UBC9 and the lower GSH level further allows LPS signaling to occur.

Fig.3 summarizes changes in NRF2 and MAFG sumoylation during HSC activation and in response to LPS.

7.5.GSH in HCC

While GSH levels are lower in a variety of chronic liver diseases,the situation is reversed in HCC.We and others have reported GCLC and GSH levels are higher in HCC.17,98,138We also found GS expression was higher in HCC but GCLM expression was unchanged.17The mechanism of induction lies at the transcriptional level and likely from activation of the ARE,AP-1,and NF-kB elements,as the nuclear binding activities were higher in HCC as compared to normal liver.17In the liver higher GSH levels is important for hepatocyte proliferation.Consistently,cultured hepatocytes exhibit increased growth when supplemented with GSH ester and stopped growing if GSH synthesis was blocked by buthionine sulfoximine (BSO,an irreversible inhibitor of GCL).17Similarly,blocking the early increase in GSH level by BSO during liver regeneration blunted liver regeneration.17Higher GSH level has been shown in many cancers and likely also provide a proliferative advantage,in addition to resistance to chemotherapy and radiation.139Thus,while enhancing hepatic GSH content is important to halt the progression of many chronic liver diseases,this strategy will accelerate HCC growth.

8.Concluding remarks

Over the past several decades much has been learned about factors that determine hepatic GSH synthesis.Increasing evidence shows GSH synthesis is impaired in multiple liver injuries.Despite an abundance of evidence showing lower hepatic GSH level perpetuates injury,targeting this by oral GSH supplementation in humans has not worked because of its low bioavailability(degraded by intestinal GGT).140Using N-acetylcysteine would not circumvent the block in GCL so other strategies will be needed.One group reported using sublingual GSH to bypass intestinal degradation and showed it was able to raise plasma total and reduced GSH levels in addition to increasing GSH/GSSG ratio after three weeks of treatment in human volunteers.140It will be of interest to examine whether this approach will be effective in ameliorating liver injuries where reduced hepatic GSH synthesis occur.In addition,strategies that target the underlying mechanism(s) responsible for the fall in GSH synthesis may also be more effective.Lastly,prior to any treatment intended to enhance GSH synthesis,presence of HCC should be ruled out.

Declaration of competing interest

The author declares that she has no conflict of interest.

Acknowledgements

This work was supported by the Women’s Guild Cedars-Sinai.