
 the endoplasmic reticulum ( er ) is an essential organelle of eukaryotic cells functioning in secretory protein synthesis and processing , lipid synthesis , calcium storage / release , and detoxification of drugs .
unfolded proteins are retained in the er and targeted for retrotranslocation to the cytoplasm for rapid degradation . under normal physiological conditions , there is a balance between the unfolded proteins and the er folding machinery .
disruption of the balance results in accumulation of unfolded proteins , a condition termed er stress [ 15 ] .
the er stress triggers the unfolded protein response ( upr ) , which attenuates protein translation , increases protein folding capacity , and promotes degradation of unfolded proteins , thus restoring er homeostasis . however , prolonged upr leads to an attempt to delete the cell causing injuries .
molecular chaperones such as the glucose - regulated protein 78 ( grp78/bip ) interact with three er membrane resident stress sensors : inositol - requiring enzyme-1 ( ire1 ) , transcription factor-6 ( atf6 ) , and pkr - like eukaryotic initiation factor 2 kinase ( perk ) , and play a vital role in maintaining the protein homeostasis inside the er [ 15 ] .
many human diseases such as metabolic syndrome , neurodegenerative diseases , alcohol - induced organ disorders , and inflammatory diseases involve er stress and impaired upr signaling [ 17 ] .
increasing evidence supports er stress as a key mechanism in alcohol - induced liver disease ( ald ) , a disease that affects over 140 million people worldwide .
potential molecular mechanisms underlying alcohol - induced er stress in major organs including liver , brain , pancreas , lung , and heart have been discussed previously [ 810 ] . in this review , i will focus on updates and new insights into the pathogenesis of alcohol - induced er stress and discuss an emerging role of alcohol - induced er stress in liver tumorigenesis and hepatocellular carcinogenesis .
alcohol is mainly metabolized in the liver and liver cells are rich in er , which assume synthesis of a large amount of secretory and membrane proteins .
the upr plays a pivotal role in maintaining er homeostasis in the liver under both physiological and pathological conditions [ 4 , 5 , 9 ] . in the early 80s , stress - induced er damages in the liver
were observed in ultrastructural , morphological , and histological studies [ 43 , 44 ] .
however , little was known then about occurrence and mechanisms of alcohol - induced er stress .
a role of er in alcohol metabolism began to be recognized as nadh from the hepatic alcohol oxidation by alcohol dehydrogenase ( adh ) was also found to support microsomal alcohol oxidations [ 4346 ] .
the inducible microsomal ethanol oxidizing system ( meos ) is associated with er proliferation and concomitant induction of cytochrome p4502e1 ( cyp2e1 ) in rats and in humans [ 45 , 46 ] .
free radical release , as a consequence of cyp2e1 activities in the er and subsequent oxidative stress , and lipid peroxidation generally contribute to ald .
however , alcohol - induced er stress response ( aerr ) that involves the upr was not recognized until recently .
molecular evidence for an impaired upr was first found in the mice with chronic intragastric alcohol infusion ( ciai ) ( figure 1 ; table 1 ) .
alterations of some er stress markers : grp78 , grp94 , chop ( c / ebp homologous protein ) , and bad ( the bcl-2-associated death promoter ) , in dna microarrays were associated with severe steatosis , scattered apoptosis , and necroinflammation .
srebp-1c ( sterol regulatory element - binding protein-1c ) was found to be a strong candidate linking er stress to alcoholic fatty liver , because srebp-1c knockout mice were protected against triglyceride accumulation .
chop was found to be a key factor in aerr - caused cell death , as knocking out chop resulted in minimal alcohol - induced apoptosis in the liver .
upstream of er stress , altered methionine metabolism , and elevated homocysteine were initially proposed to be responsible for aerr because alcohol - induced hyperhomocysteinemia ( hhcy ) is often seen in rodents and humans [ 4750 ] and homocysteine is known to modify proteins biochemically [ 8 , 9 , 11 ] .
first , betaine is a methyl donor for remethylation of homocysteine to methionine catalyzed partially by betaine - homocysteine methyltransferase ( bhmt ) .
simultaneous betaine feeding or transgenic expression of bhmt in ciai mice decreased the elevated homocysteine and abrogated aerr in parallel with decreased alt and amelioration of ald [ 11 , 14 , 15 ] .
second , an intragastric infusion with both high fat and alcohol induced moderate obesity and much severe ald , which resulted from synergistic effects of an accentuated er stress by the alcohol - induced hhcy in combination with mitochondrial stress , nitrosative stress , and adiponectin resistance .
consequently , the rat animals have a minimal er stress response and are more resistant to ald , which correlates with a significant induction of bhmt .
fourth , in a study with 14 inbred mouse strains with ciai , profound differences in ald were observed among the strains in spite of consistently high levels of urine alcohol .
er stress related genes were induced only in strains with the most liver injury , which were closely associated with expression patterns of methionine metabolism - related genes and plasma homocysteine levels .
thus , abnormal protein modifications by excessive homocysteine as a result of aberrant one - carbon metabolism   and methionine deficiency   are likely responsible for the alcoholic er stress and upr in ciai mice that lack a sufficient upregulation of bhmt
. however , other causes for the alcoholic er stress are present in the ciai model .
for instance , in a study with ciai rats to examine effects of selective inhibition of cyp2e1 on the development of alcoholic fatty liver , liver triglycerides were lower .
er stress indicated by the er stress marker trb3 ( a mammalian homolog of drosophila tribbles functions as a negative modulator of protein kinase b ) was increased after ethanol and was further increased upon inhibition of cyp2e1 or overall ethanol metabolism .
this suggests a contributing role of alcohol metabolites , for example , acetaldehyde , or oxidants to the alcoholic er stress response . in another study with cystathionine  synthase ( cbs ) heterozygous mice treated with ciai
, steatohepatitis was accompanied with upregulations of hepatic er stress components including grp78 , atf4 ( activating transcription factor 4 ) , chop , and srebp-1c and negatively correlated with s - adenosylmethionine ( sam ) to s - adenosylhomocysteine ( sah ) ratio .
aerr was associated with a decrease in levels of suppressor chromatin marker trimethylated histone h3 lysine-9 ( 3meh3k9 ) in the promoter regions of the er stress markers .
similarly , epigenetic mechanism for aerr might also occur in human alcoholics , as dna hypermethylation of the promoter of herp ( homocysteine - induced er protein ) gene downregulates its mrna expression in patients with alcohol dependence .
aerr occurs not only in the aforementioned ciai models but also in other chronic or acute models / systems ( table 1 ) , which have been providing additional insights into aerr and ald . in micropigs fed alcohol orally , liver steatosis and apoptosis were shown to be accompanied by increased mrna levels of cyp2e1 and selective er stress markers .
folate deficiency appeared to be responsible for the er stress and injury . in mice , however , oral alcohol feeding ad libitum does not usually result in hhcy as remarkable as seen in the ciai mice .
correspondingly , the degree of aerr and subsequent liver injury may depend on additional genetic and/or dietary factors .
for instance , in the mice with liver specific deletion of grp78/bip , a robust er stress response was observed at moderate oral alcohol doses ( e.g. , 4  g / kg ) , which was accompanied by much aggravated hepatosteatosis and hepatic fibrosis .
thus , compared to the homocysteine - er stress mechanism , the liver bip deletion represents a genetic predisposition that unmasks a distinct mechanism by which alcohol induces er stress , one that is largely obscured by compensatory changes in normal animals or presumably in the majority of human population who have low - to - moderate drinking [ 8 , 22 ] . the effect of genetic predisposition on aerr and hepatic injury is also observed in a recent study using mice with low alcohol - induced plasma homocysteine and deficient in the acid sphingomyelinase ( asmase ) .
strong aerr and enhanced susceptibility to lipopolysaccharides ( lps ) or concanavalin - a were present in asmase / mice fed alcohol orally , indicative of a mitochondrial effect on aerr .
in addition , in iron overloaded mice deficient in the hemochromatosis gene ( hfe/ ) , cofeeding ad libitum with alcohol and a high - fat diet ( hfd ) led to profound steatohepatitis and fibrosis [ 24 , 25 ] .
xbp1 splicing , activation of ire-1 and perk , and increased chop protein expression were associated with impaired autophagy response , suggesting that preconditioning with iron overloading may modulate aerr and promote liver injury through interacting with other adaptive or compensatory mechanisms .
alternatively , the contributing role of er stress to ald in oral feeding models could be secondary .
this is indicated by a time - course study with a mouse model of early - stage ald .
mice with oral alcohol feeding exhibited significant hepatic steatosis and elevated plasma alt values . at 1 to 2 weeks after alcohol feeding , oxidative stress indicated by 4-hydroxynonenal- ( 4-hne- ) modified proteins was increased , whereas hepatic glutathione ( gsh ) levels were significantly decreased as a consequence of decreased cbs activity , increased gsh utilization , and increased protein glutathionylation . except for 4-hne adduction to the er disulfide isomerase ( pdi )
, significant upregulations of other er markers and srebp pathways were not detected in vivo during the same early period of alcohol feeding [ 26 , 27 ] .
although the actual blood alcohol levels were not measured in this study , which might not reach a critical point and vary widely among individual mice at a liberal access to alcohol , the results may suggest a secondary role of aerr in this early ald model .
thus , interplay or cross - talk between aerr and other stresses might be critical in ald .
this notion is supported by a few most recent reports , which appears more evident in cell or animal models with acute alcohol exposure .
however , in a perfused rat liver system , downregulation of grp78 and activation of c - jun n - terminal kinase ( jnk ) and protein kinase b ( pkb / akt ) were enhanced by a cotreatment of acute ethanol with a classical inhibitor of adh , and an antioxidant addition reduced the activation of jnk and cell death .
high concentrations of the pharmacological er stress - inducing agents such as tunicamycin or brefeldin a activate jnk and inhibit mitochondrial respiration and cell death in hepatocytes .
second , the mice with liver specific grp78 deletion are sensitized to a variety of acute hepatic disorders by alcohol , a high - fat diet , anti - hiv drugs , or toxins .
hiv protease inhibitors inhibit the er caatpase ( serca ) and modulate calcium homeostasis in mice and primary human hepatocytes , which aggravates aerr and ald .
third , the interferon regulatory factor 3 ( irf3 ) is activated early by er stress in mice fed alcohol either orally or intragastrically , which involves an er adaptor , the stimulator of interferon genes ( sting ) .
independent of inflammatory cytokines and type - i interferons ( ifns ) , irf3 exerts its pathogenic role in ald through causing apoptosis of hepatocytes , which strongly suggests that aerr pathways and the lps - tlr4 ( toll - like receptor 4 ) pathways   are parallel or equally important in initiating ald .
in addition to rodents , aerr has also been found in other species including human alcoholics ( table 1 ) .
zebrafish larvae represent an alternative vertebrate model for studying aerr and ald because their liver possesses the pathways to metabolize alcohol that can be simply added to the water , that is , acute alcohol .
aerr is present in alcohol - treated zebrafish , which may also interact with other pathological factors . upon alcohol challenge
further , the er stress response appeared much robust in zebrafish deficient in the cdp - diacylglycerol - inositol 3-phosphatidyltransferase ( cdipt ) that primarily locates on the cytosolic aspect of the er . thus , integrity of the er or alcohol metabolism might be necessary for aerr . in supporting this , in the species caenorhabditis elegans without a liver for alcohol digestion / metabolism , little aerr has been detected .
the most important and clinically relevant studies regarding aerr are from human cells and patients .
aerr is reported in human monocyte - derived dendritic cells ( mddc ) , hepg2 cells expressing human cyp2e1 , and primary human hepatocytes .
oxidative stress resulted from the function of cyp2e1 and/or interactions with other drugs contributing to aerr in the human cells .
however , cultured human cell models may not reflect the complexity of the response in vivo .
for instance , it was reported that , upon alcohol exposure , vl-17a cells metabolized alcohol which caused er fragmentation inside the cells , but little activation of upr target genes was detected . nevertheless , striking upregulation of multiple er stress signaling molecules
was detected in human patients with ald ( table 1 ) [ 3942 ] , which is correlated with deregulated lipid metabolism , ceramide accumulation , and impaired insulin signaling , indicating that aerr is an integrated part of pathogenesis of ald in human alcoholics .
it has been well accepted that the upr is a double - edged response because both adaptive survival and eliminative apoptosis can be induced by upr components [ 16 ] .
it is beneficial or prosurvival if it happens transiently or lasts for a short period of time , whereas it is detrimental or deadly if it is prolonged .
recent studies indicate that the upr is associated with solid tumor development in many types of tissues or organs including the liver [ 55 , 56 ] . since the microenvironments of solid tumors are generally hypoxic , acidic , and nutrient deficient [ 57 , 58 ] , which individually or collectively favor activation of er stress response , it is conceivable that the upr is persistently present during tumorigenesis .
emerging evidence suggests that cancerous cells could modify and perturb the er stress - associated cell death signaling , which permits survival and growth .
for instance , the master regulator upr , grp78 , plays a dual role in tumor cells [ 22 , 59 ] .
it controls early tumor development through suppressive mechanisms such as the induction of cell cycle arrest or tumor dormancy upon perk activation . on the other hand , at more advanced stages of tumor progression , during which cells are exposed to more severe stressors
, grp78 suppresses caspase 7 activation and interacts with er stress - induced protein chaperones such as clusterin to promote cell survival and further tumor development .
the perk - eif2-atf4 pathway is often activated by the hypoxic condition in solid tumors [ 6264 ] , which activates angiogenic genes , vascular endothelial growth factor ( vegf ) , type 1 collagen inducible protein , and autophagosome components such as lc3 , ensuring cell survival over hypoxia - induced er stress [ 6567 ] . prolonged expression and activation of atf6
in addition , the ire1-xbp1 pathway interacts with antiapoptotic bcl-2 family members and the sigma-1 receptor , which is often increased in many human cell lines [ 6972 ] .
therefore , impaired and/or prolonged upr has a high potential to modulate cell fates and differentiations towards tumorigenesis .
however , the mechanisms by which alcohol exerts its carcinogenic effect are largely unknown and currently there is no effective treatment . considering that
several lines of evidence indicate that polymorphic responses of major er chaperones to alcohol and other stressors are associated with hepatocellular carcinogenesis in human populations [ 7782 ] , it is not unusual to find a role of aerr in hcc .
in fact , we recently found spontaneous hepatocellular adenomas- ( hca- ) like tumors in aged female mice with a liver specific bip deletion and under constitutive er stress [ 22 , 59 , 83 ] .
active atf6 , chop , gsk3 , and creld2 ( cysteine rich with egf - like domains 2 ) were increased in the knockout , indicative of continuous er stress response .
none of p53 , hnf1 , or gp130 was significantly changed compared between wild type and knockout .
interestingly , cyclin d was specifically reduced in the tumor portion of the knockout mice . since most liver tumors were found in female knockouts , expression of receptors for sex hormones such as estrogen receptors , er and  , and androgen receptor , ar ,
three variants of er were detected in the liver , and their molecular sizes are 66  kd , 46  kd , and 36  kd , respectively . the er variant 36  kd was remarkably increased in the tumor portion of the knockout liver . in contrast , there were no significant changes in the expression of er , ar , cyclin e , or cyclin g. these findings revealed thatinhibition of cyclin d and overexpression of er variant 36  kd are associated with the tumor development in the female knockouts under constitutive er stress .
furthermore , the tumors are highly malignant in mice with additional stresses such as high - fat diet or alcohol intake [ 83 , 84 ]
. the pathways of erk1/2 , stat3 , and p38 were activated , which are known to promote hcc progression [ 85 , 86 ] .
the constitutive er stress - induced spontaneous liver tumors that are dominant in female animals are similar to human hca [ 8790 ] , which are of clinical relevance since about 80% of hca cases are from women taking oral contraceptives for years [ 90 , 91 ] .
potential causesfor human hca are mutations in hnf1 , -catenin , gp130 , or chronic inflammation [ 8793 ] .
hepatocellular protein homeostasis has rarely been noticed to be a potential mechanism for hca development .
thus , the above findings on cyclin d and er variants may reveal a novel er stress mechanism for hca for several reasons ( figure 2 ) .
first , the in vivo inhibition of cyclin d expression upon er stress in knockouts is consistent with an earlier study , which demonstrated that activation of the upr in mouse nih 3t3 fibroblasts with tunicamycin led to a decline in cyclin d and subsequent g(1 ) phase arrest [ 9496 ] .
second , increased expression of cyclin d is usually associated with proliferation in other systems .
however , a number of studies have shown many new roles of cyclin d   and a surprising lack of the correlation of increased cyclin d with proliferation in tumors [ 99 , 100 ] .
for instance , in one subtype of human breast carcinoma , cyclin d1 protein expression was absent in the noninvasive cells [ 101 , 102 ] .
similarly , a loss of cyclin d did not inhibit the proliferative response of mouse liver to mitogenic stimuli   and mrna levels of cyclin d1 were downregulated in patients with hcc .
most recent molecular evidence further supports this er stress - cyclin d - tumorigenesis mechanism .
nrf2 ( the nuclear factor erythroid 2-related factor 2 ) activities are associated with aging .
er stress activates nrf2 and atf6 , both of which regulate the orphan nuclear receptor , shp ( small heterodimer partner ) which acts as a transcriptional corepressor modulating cyclin d1 and subsequent hepatic tumorigenesis [ 106 , 107 ] .
the er stress sensor perk has been shown to phosphorylate the forkhead transcription factor 3 ( foxo3 )   and suppressed foxo3 exacerbates alcoholic hepatitis and insulin resistance impairing cyclin d function promoting hcc [ 109111 ] . thus
, abnormal functions of cyclin d under er stress conditions most likely disturb liver cell proliferation ( figure 2 ) .
third , since the authentic er66 interacts with cyclin d physically [ 100 , 101 ] , the hepatic er variants may result from an unbalanced long - term molecular interaction between er and the suppressed cyclin d under er stress ( figure 2 ) .
alternatively , the er variants may be produced from an incomplete protein processing / maturation of er by an impaired er - associated degradation ( erad ) .
components of erad are indeed altered in the bip knockout mouse models under constitutive upr [ 22 , 59 , 83 , 84 ] , and there is a report indicating that an activation of the xbp1-hrd1 ( an e3 ubiquitin ligase also called synoviolin ) branch by the upr facilitates nrf2 ubiquitylation and degradation during liver cirrhosis .
fourth , considering that er gene polymorphism is associated with risk of hbv - related acute liver failure   and a switch from the authentic er to a predominant expression of er36 is associated with development and progression of hcc [ 114116 ] , the hepatic er variants could also play an important role in alcohol and er stress - associated hcc .
the tumorigenic signaling downstream of cyclin d and er variants can be activations of the erk1/2 , ip3k - pkc , or jak - stat pathways .
overexpressed er36 has been associated with activation of these pathways and carcinogenesis in other systems such as breast cancer and gastric cancer [ 118122 ] . in the liver , activations of erk1/2 and jak - stat pathways
were observed in the bip knockout mice fed alcohol and high - fat diet [ 83 , 84 ] .
finally , studies on hepatoma cell lines , hcc tissues , and animal models of hcc suggest a possible role of sex hormones and their receptors in hcc pathogenesis
. a male prevalence of hcc is often observed in young and middle aged patient populations in certain regions exposed to additional hcc risk factors .
however , the male prevalence of hcc tends to diminish in aged human populations   as well as in aged animals fed alcohol [ 83 , 84 ] .
therefore , alcohol - induced er stress and cell cycle impairment may exert specific effects on aging , hepatic expression of estrogen receptors , and subsequent tumorigenesis in females .
alcohol - induced hepatic er stress occurs in the liver of many species including human alcoholics , which has recently been established as an important mechanism for both acute and chronic alcohol - induced liver pathogenesis and disease development .
multiple factors commonly associated with alcohol consumption such as acetaldehyde , oxidative stress , excessive homocysteine , toxic lipid species , increased sah , aberrant epigenetic modifications , disruption of calcium homeostasis , and insulin resistance induce er stress response individually or collectively .
however , the precise contribution of each of the factors to the er stress induction is not clear and their importance to ald may depend on doses , duration and patterns of alcohol exposure , presence or absence of genetic and environmental factors , cross - talks with other pathogenic pathways , and liver disease stages .
the upr , as an integrated part of liver physiology and pathology like the immune response , may occur more or less all the time during alcohol consumption , which attempts to restore er homeostasis and protect against ald . however , this adaptive protection is not without detrimental consequences .
prolonged upr leads to excessive deletion of the damaged hepatocytes or cell cycle arrest , which triggers inflammatory response or interrupts normal cellular processes causing profound injuries . emerging evidence by us and others indicates a direct involvement of long - term alcohol and constitutive er stress in liver tumorigenesis and hepatocellular carcinogenesis .
the er stress and malfunctioning of cyclin d - caused cell cycle arrest are a well - established molecular mechanism , and the surprise overexpression of estrogen receptor  variants under constitutive upr may result from a mal - targeting of protein processing and turnover by aberrant erad , which reflect complexity and depth of prolonged upr - mediated pathogenesis .
thus , liver tumorigenesis by alcohol and er stress may involve not only cyclin d , er variants , erk1/2 , pkc , and stat pathways , but also other cell cycle targets such as il-6 , p21 , p27 , and cdk , other pathways such as src / egfr , pten - tgf , and insulin / igf , and epigenetic regulations such as mirnas targeting the upr components .
in addition , liver progenitor cell activation by alcohol may contribute to the malignant transformation of nonmalignant tumors developed under long - term er stress [ 22 , 59 ] .
future work should be directed to define the er stress mechanisms leading to hcc and to develop multiple therapeutic approaches to target er stress in human alcoholics with hcc .
 alcohol - induced liver disease increasingly contributes to human mortality worldwide . 
 alcohol - induced endoplasmic reticulum ( er ) stress and disruption of cellular protein homeostasis have recently been established as a significant mechanism contributing to liver diseases . 
 the alcohol - induced er stress occurs not only in cultured hepatocytes but also   in vivo  in the livers of several species including mouse , rat , minipigs , zebrafish , and humans . 
 identified causes for the er stress include acetaldehyde , oxidative stress , impaired one carbon metabolism , toxic lipid species , insulin resistance , disrupted calcium homeostasis , and aberrant epigenetic modifications . 
 importance of each of the causes in alcohol - induced liver injury depends on doses , duration and patterns of alcohol exposure , genetic disposition , environmental factors , cross - talks with other pathogenic pathways , and stages of liver disease . 
 the er stress may occur more or less all the time during alcohol consumption , which interferes with hepatic protein homeostasis , proliferation , and cell cycle progression promoting development of advanced liver diseases . 
 emerging evidence indicates that long - term alcohol consumption and er stress may directly be involved in hepatocellular carcinogenesis ( hcc ) . dissecting er stress signaling pathways leading to tumorigenesis will uncover potential therapeutic targets for intervention and treatment of human alcoholics with liver cancer . 