Alcohol, Signaling, and ECM Turnover

Devanshi Seth, Nympha B. D’Souza El-Guindy, Minoti Apte, Montserrat Mari,Steven Dooley, Manuela Neuman, Paul S. Haber, Gopal C. Kundu, Agus Darwanto,

Willem J. de Villiers, A. Vonlaufen, Z. Xu, P. Phillips, S. Yang, D. Goldstein, R. M. Pirola,J. S. Wilson, Anna Moles, Anna Fernandez, Anna Colell, Carmen Garcıa-Ruiz,

Jose C. Fernandez-Checa, Christoph Meyer, and Nadja M. Meindl-Beinker

Alcohol is recognized as a direct hepatotoxin, but the precise molecular pathways that are impor-tant for the initiation and progression of alcohol-induced tissue injury are not completely under-stood. The current understanding of alcohol toxicity to organs suggests that alcohol initiatesinjury by generation of oxidative and nonoxidative ethanol metabolites and via translocation ofgut-derived endotoxin. These processes lead to cellular injury and stimulation of the inflammatoryresponses mediated through a variety of molecules. With continuing alcohol abuse, the injury pro-gresses through impairment of tissue regeneration and extracellular matrix (ECM) turnover, lead-ing to fibrogenesis and cirrhosis. Several cell types are involved in this process, the predominantbeing stellate cells, macrophages, and parenchymal cells. In response to alcohol, growth factorsand cytokines activate many signaling cascades that regulate fibrogenesis. This mini-review bringstogether research focusing on the underlying mechanisms of alcohol-mediated injury in a numberof organs. It highlights the various processes and molecules that are likely involved in inflamma-tion, immune modulation, susceptibility to infection, ECM turnover and fibrogenesis in the liver,pancreas, and lung triggered by alcohol abuse.

Key Words: Fibrosis, Liver Disease, Pancreas, Lung, Bacterial and Viral Infection.

E XCESSIVE ALCOHOL CONSUMPTION has beenreported to have causal relationship with more than 60

types of diseases (WHO, 2006). Chronic alcohol abuse dam-ages nearly every organ in the body, especially the liver andpancreas. Investigations have shown that chronic alcoholinvokes complex but common pathophysiological changes

through altered metabolic, immunologic, and inflammatoryprocesses in many organs. Chronic alcohol consumption isalso responsible for approximately 3.6% of cancers world-wide (Seitz and Stickel, 2007). Alcohol as a direct hepato-toxin can induce cell damage through increased generationof reactive oxygen species (ROS) (Breitkopf et al., 2005;Parola and Robino, 2001) and by altering gut permeabilityto release bacterial endotoxin lipopolysaccharide (LPS)(Fukui et al., 1991; Nanji et al., 1994). Other mechanismsthat have emerged from studies in several organs andexperimental models of alcohol include changes in pro-inflammatory and pro-fibrogenic cytokines such as interleu-kins (IL-1a, IL-6, and IL-8) (Neuman et al., 1998), tumornecrosis factor alpha (TNF-a), and transforming growth fac-tor beta (TGF-b) (Tilg and Diehl, 2000). ROS produced dur-ing alcohol metabolism leads to inflammation mainlythrough TNF-a and TGF-b and cause cell death in the liver,lung, pancreas, heart and the brain. In addition, alcoholmodulates local innate and adaptive immune responses(Neuman et al., 1993, 1998) that can alter resistance to anumber of bacterial (Gamble et al., 2006; Szabo and Man-drekar, 2009) and viral (Graham, 2006; Neuman et al., 2008;Samet et al., 2003) infectious agents. Factors such as bacte-rial and viral infections enhance the inflammation and thedeleterious effects of alcohol. Findings also suggest thatacute and chronic alcohol exposure incur different mecha-nisms which respectively, either suppress or activate immuneresponse on bacterial infection (D’Souza El-Guindy et al.,2007). Several cell–cell signaling pathways have been

From the Drug Health Services & Centenary Institute (DS, PSH),Royal Prince Alfred Hospital, Camperdown, NSW, Australia; Depart-ment of Internal Medicine (NBDE-G, AD, WJV), University ofKentucky and Veterans Affairs Medical Center, Lexington, Kentucky;Pancreatic Research Group (MA, AV, ZX, PP, SY, DG, RMP,JSW), South Western Sydney Clinical School, Wallace Wurth Build-ing, The University of New South Wales, Sydney, Australia; Liver Unitand CIBEK (MM, AM, AF, AC, CG-R, JCF-C), IMDiM, HospitalClınic i Provincial, CIBEREHD, IDIBAPS, Instituto InvestigacionesBiomedicas de Barcelona, Consejo Superior de Investigaciones Cientıfi-cas, Barcelona, Spain; Molecular Alcohol Research in Gastroenterology(SD, CM, NMM-B), II. Medical Clinic, Universitatsmedizin Mann-heim, Medizinische Fakultat Mannheim der Ruprecht-Karls-UniversitatHeidelberg, Mannheim, Germany; In Vitro Drug Safety and Biotech-nology and Department of Pharmacology (MN), Faculty of Medicine,University of Toronto, Ontario, Canada; and National Centre for CellScience (GCK), Pune, Maharashtra, India.

Received for publication June 3, 2009; accepted August 20, 2009.Reprint requests: Devanshi Seth, Drug Health Services &

Centenary Institute, Royal Prince Alfred Hospital, Page 5 MissendenRoad, Camperdown, NSW 2050, Australia; Fax: +61 2 95157980;E-mail: devanshi.seth@email.cs.nsw.gov.au

Copyright � 2009 by the Research Society on Alcoholism.No claim to original U.S. government works

DOI: 10.1111/j.1530-0277.2009.01060.x

Alcoholism: Clinical and Experimental Research Vol. 34, No. 1January 2010

4 Alcohol Clin Exp Res, Vol 34, No 1, 2010: pp 4–18

implicated that regulate the immune response(s), triggerfibrogenesis and wound healing through activation oftranscription factors, such as, nuclear factor (NF)jB andactivation protein-1 (AP-1). With persistent alcohol abuse,the injury progresses through impairment of tissue regenera-tion, cytokine production, leukocyte infiltration and extracel-lular matrix (ECM) turnover, and fibrogenesis leading tocirrhosis. As fibrosis develops there is a shift in the subendo-thelial ECM from low-density matrix to interstitial fibrillarcollagen (Gressner and Bachem, 1990) and a significantincrease in total collagen content (Rojkind et al., 1979).Several cell types are involved in this process, predominantlystellate cells and macrophages. Recent studies demonstratethat parenchymal cells, such as hepatocytes also play amajor role in alcohol-induced pathogenesis (Dooley et al.,2008; Zeisberg et al., 2007). Advances in delineating themolecular mechanisms in alcohol-mediated tissue injury havebeen made mostly with alcoholic liver disease (ALD) whichhas also served as a model for alcohol damage in otherorgans.

NOVEL MECHANISMS OF ALCOHOL INJURY INDIVERSE ORGANS AND TISSUES

Recent progress in alcohol research has revealed novelmechanisms, such as the discovery of sphingomyelinasesplaying a role in the TNF-a induced heptaocyte cell deathand fibrogenesis (Marı and Fernandez-Checa, 2007; Marıet al., 2004); TFG-b-mediated regulation of Cyp21A1 andADH1 contributing toward liver fat accumulation and oxi-dative stress, and the antagonistic action of Smad 7 at differ-ent stages and locations of TGF-b-mediated signaling inALD (Dooley et al., 2001, 2008). Regulation of plasminhomeostasis through PAI-1 (Arteel, 2008; Seth et al., 2008)and other fibrinolysis regulating molecules, such as osteo-pontin (Opn) (Apte et al., 2005b; Seth et al., 2006a,b) arealso some of the recent discoveries that provide insights inthe common mechanisms involved in alcohol-induced organinjury. More recently, Opn is also suspected to have a role inalcohol-mediated lipid accumulation in the hepatocytes bymodulating peroxisome-proliferator activated receptor alpha(PPAR-a) (Nath and Szabo, 2009). PPAR-a, a transcriptionfactor regulating genes involved in fatty acid oxidation, isknown to suppress lipid accumulation and is downregulatedwith chronic alcohol consumption. However, chronic alcoholfed Opn knockout mice display increased PPAR-a, lipidaccumulation and liver injury (Lee et al., 2008). In otherforms of alcohol-mediated injury, alcohol as a co-factormodulates immune responses and can alter resistance to bac-terial and viral infections. Mechanisms include increased celltoxicity and immune modulation via TNF-a led signalingevents during viral infection. Various strains of Friend viruscan induce erythroleukemias through activation of erythro-poietin (Epo) (Shaked et al., 2005), and Epo is also associ-ated with enhanced tumorigenicity of erythroleukemias(Cocco et al., 1996). A link between the severity of leukemia

and alcohol consumption has also been reported (Ido et al.,1996), and mice treated with alcohol had enhanced Epo-R-mediated signaling and acceleration of preleukemic stage,suggesting that Epo pathway is important in enhancing pre-leukemic stage, and that alcohol plays a role in this process.These systems are highlighted in Fig. 1 and described belowin more detail.

Toxicity of Alcohol Metabolite Acetaldehyde

Alcohol, directly or via its metabolites, acetaldehyde andacetate, can evoke mechanisms that promote cellular and tis-sue injury. The metabolism of alcohol to acetaldehyde andacetate by way of alcohol and acetaldehyde dehydrogenases(ADH and ALDH, respectively), and microsomal cyto-chrome P450 2E1 (Cyp2E1), leads to persistent redox stressassociated with lipid peroxidation, increased collagen produc-

Fig. 1. Overview of novel pathways in alcohol induced tissue injury in theliver. Alcohol mediates cellular injury in liver cells through endotoxin, oxida-tive stress, and its metabolites, such as acetaldehyde, mainly via TNF-a(gray box) and TGF-b (pink box). This invokes downstream signaling in aseries of molecules (red) leading to activation of transcription factors NFjBand AP-1. The activated transcription factors regulate expression of variousmolecules (blue box) involved in immune response, inflammation, apopto-sis, susceptibility to pathogens mainly via TNF-a, and lipid accumulation,fibrinolysis, fibrogenesis, and ECM remodeling via TGF-b. These processesoccur in many cell types including Kupffer cells, stellate cells, lymphocytes,and hepatocytes (brown boxes). AP-1, activation protein 1; ALK, serine ⁄threonine kinase receptor; ASMases, acidic sphingomyelinases; CTGF,connective tissue growth factor; Cyp2e1, cytochrome P450 2e1; EMT, epi-thelial mesenchymal transition; EpoR, erythropoietin receptor; Erk, extracel-lular signal-regulated kinases; IRAK1, Interleukin 1 receptor associatedkinase; IjB: Inhibitor of jB; Jak ⁄ STAT: Janus kinases ⁄ Signal Transducersand Activators of Transcription; MAPK, mitogen associated protein kinases;MMPs, matrix metalloproteinases; MyD88, myeloid differentiated factor 88;NFjB, nuclear factor jB; OPN, osteopontin; PAI-1, plasminogen activatorinhibitor-1; PI3K, phosphatidylinositol kinase; PKC, protein kinase C;PPARa, peroxisome proliferator activated receptor alpha; PPARc, peroxi-some proliferator activated receptor gamma; Smad, homolog of drosophilamothers against decapentaplegic; TNFR, TNF-a receptor; TRAF, TNFRassociated factor; TBRII, TGF-b receptor II; uPA, urokinase plasminogenactivator; ZO-1, zonula occludens.

ALCOHOL, SIGNALING, AND ECM TURNOVER 5

tion (Tsukamoto, 1993) and fibrogenesis. Acetaldehyde formsneo-antigenic protein- and DNA-adducts and also generatesIgAs that are deposited in the tissues and are responsible forinflammatory and immune responses in the cells (Seitz andHomann, 2007; Tuma and Casey, 2003). DNA adducts maysubsequently lead to replication errors and point mutations.Acetaldehyde may directly bind to proteins related withDNA repair and methylation and thus interfere with pro-cesses controlling gene activity and integrity of DNA. In addi-tion, acetaldehyde-induced chromosome aberrations arelikely to eventually lead to cancer development. There is nowconvincing evidence that the carcinogenic effect of alcohol isdue to the DNAmutagenic properties of acetaldehyde operat-ing at multiple levels. These diverse mutagenic mechanismsare summarized in detail in a recent review by Seitz andStickel (2007). However, as acetaldehyde is rapidly metabo-lized in the liver, it only plays a minor role in hepatocellularcarcinoma development, whereas these mechanisms arepivotal in other alcohol-related cancers, like that of the upperaerodigestive tracts, breast and colorectal cancer (Seitz andBecker, 2007). Acetaldehyde also disrupts the barrier functionprovided by tight junctions in epithelial cells. In the hepato-cytes, tight junctions are disrupted by acetaldehyde therebyincreasing paracellular permeability. In vitro studies usingintestinal epithelial Caco-2 cells have shown that the effect ofacetaldehyde on tight junctions is mediated via tyrosinekinase dependent inhibition of protein tyrosine phosphataseactivity (Atkinson and Rao, 2001). Acetaldehyde-induced dis-ruption of tight junctions is also associated with increasedtyrosine phosphorylation of several related proteins, such aszonula occludens (ZO)-1 and b-catenin, indicating thatacetaldehyde can modify intracellular signaling pathways todestabilize the tight junction protein complex leading toincreased permeability to endotoxin (Atkinson and Rao,2001) and fibrogenesis. Acetaldehyde also induces collagen 1synthesis in the hepatic stellate cells (HSCs) (Greenwel, 1999)and promotes ECM remodeling by altering matrix metallo-proteinases (MMPs) (Casini et al., 1994). Acetaldehyde-induced downstream signaling occurs through severaltranscription factors and complex pathways (Tommaso et al.,2008). For example, collagen a1 and a2 promoters haveacetaldehyde-responsive elements within binding sites fortranscription factors, as well as overlapping with TGF-b andTNF-a response elements (Greenwel, 1999) evident ofcomplexities and multilayered controls. Acetaldehyde triggersphosphorylation of protein kinase C (PKC) involving extra-cellular receptor kinase (Erk)1 ⁄2 and phosphatidylinositolkinase (PI3K) signaling and activation of transcription factorAP-1, leading to collagen induction (Svegliati-Baroni et al.,2001, 2005).

LPS-Mediated Alcohol-Induced Tissue Injury

One of the main features of alcohol-induced tissue injury isvia LPS. The onset of ALD is steatohepatitis characterized byprogressive tissue damage initiated by lipid accumulation in

the liver. This is accompanied by infiltration of inflammationpromoting cells that migrate toward the liver in response toactivation of Kupffer cells (KC), the liver resident macro-phages. Alcohol augments the translocation of gut-derivedLPS which is central to the activation of KCs in ALD(Wheeler et al., 2000).When activated, KCs produce signalingmolecules, such as cytokines, that promote inflammation andincrease ROS. Inactivating KCs prevents alcohol-inducedliver injury underscoring the significance of these cells inALD pathogenesis (Adachi et al., 1994). The LPS and LPSbinding protein (LBP) complexes with CD14 and toll likereceptor-4 (TLR-4) on activated KC surface (Su, 2002), trig-gering the pro-inflammatory signaling cascades that areimportant for the development of ALD. TLR-4 also mediatesinflammatory signaling by LPS in HSCs (Paik et al., 2003). Inactivated stellate cells, TLR-4 is upregulated, further sensiti-zing this cell type leading to fibrogenic responses in ALD(Paik et al., 2003). The importance of this mechanism inALD has been impressively documented by Uesugi and col-leagues (2001) and is further supported by the finding ofTLR-4 gene polymorphisms connected to liver fibrosis (Guoet al., 2009; Huang et al., 2007). Similarly, development ofchronic pancreatitis was enhanced in the alcohol-fed rats withrepeated injections of LPS as evidenced by acinar atrophyand pancreatic fibrosis (Vonlaufen et al., 2007) (Fig. 2A).Bacterial toxins are known to be injurious to the lung by

impairing microcirculation and, similar to the liver and pan-creas, by promoting chemical mediators release and inducingoxidative stress. However, unlike the liver, the lung is con-stantly exposed to particulate matter from the external envi-ronment in performing the function of gas exchange. Livemicroorganisms and LPS are common biological constituentsof the particulate matter. These are usually cleared by themechanical (e.g., mucociliary apparatus) and chemical pro-cesses in the upper respiratory tract. When these processes areimpaired or fail the particulate matter enters into the alveoli,evoking a self resolving inflammatory response by the alveo-lar macrophage (AM). Alcohol abuse perturbs the mucocili-ary clearance of the pathogens by cilia, the AM recognitionand phagocytosis of the stimuli, generation of chemical medi-ators, and the recruitment of auxiliary defenses, as well as thesecretion of surfactant by lung epithelial cells (D’Souza El-Guindy et al., 2007; D’Souza et al., 1996; Gamble et al.,2006; Joshi and Guidot, 2007; Szabo and Mandrekar, 2009;Wyatt et al., 2004). These adverse effects of acute and chronicalcohol intoxication on the lung have been reviewed exten-sively elsewhere (Boe et al., 2009; Happel and Nelson, 2005).Like the liver KC, the AM is the primary defender of thelung. It recognizes the stimulus, that escapes expulsion by theupper respiratory tract, through pattern recognition receptorsincluding the TLRs, and then releases chemical mediators(e.g., cytokines, chemokines, and ROS). These are bacterici-dal in nature and ⁄or facilitate the recruitment of other leuko-cytes for effective removal of the stimulus and restoration oflung homeostasis. In addition to the AM, other innateimmune cells including neutrophils, monocytes, dendritic, and

6 SETH ET AL.

natural killer cells are also equipped with the pattern recogni-tion receptors and can aid in defense against a variety of lunginfections.

Role of Stellate Cells in Alcohol-Induced Fibrogenesis andCancer

Pancreatic injury in response to alcohol can occur viapancreatic stellate cells (PSCs) that are shown to be the keyplayers in alcoholic pancreatitis (Apte et al., 1998; Bachemet al., 1998). In normal tissue, PSCs play an important role inmaintaining a delicate balance between ECM synthesis anddegradation. During alcoholic pancreatic injury, PSCs areactivated in a paracrine fashion, by growth factors, pro-inflammatory cytokines, and oxidant stress (Apte et al.,2005a) and are the major source of collagen deposition infibrotic pancreas (Haber et al., 1999). The likely activatingfactors include proliferative and pro-fibrogenic growth factorssuch as platelet derived growth factor (PDGF) and TGF-b,as well as oxidant stress generated within the tissues by oxida-tive metabolism of alcohol to acetaldehyde and subsequentlyto acetate (Apte et al., 1999; Masamune et al., 2002). Nota-bly, PSCs can also synthesize endogenous cytokines resultingin an autocrine loop of cell activation (Shek et al., 2002;Sparmann et al., 2005). Due to the generally accepted factthat chronic alcohol administration is insufficient to causeovert pancreatic (and other tissue) injury in experimentalanimals, additional triggers such as LPS, may be needed toproduce demonstrable tissue injury. In vivo models of pancre-atic injury using alcohol and LPS exhibited increased collagenstaining around ducts and near acinar cells, associated withincreased hydroxyproline levels and increased expression ofpro-collagen and alpha smooth muscle actin (a-SMA), anindication of PSC activation (Perides et al., 2005). Given thatPSCs are now established as key players in the fibrosis ofchronic pancreatitis, it is not surprising that the putative roleof these cells in pancreatic cancer has attracted the attentionof researchers. It is well known that one of the important riskfactors for pancreatic cancer is chronic pancreatitis, and asnoted earlier, the 2 diseases have a striking histopathologicfeature in common i.e., extensive fibrosis (desmoplasia).Indeed, microarray analyses have demonstrated that thestromal compartments of chronic pancreatitis and pancreaticcancer have at least 107 genes in common (Binkley et al.,2004). Using in vitro and in vivo models, it has also beendemonstrated that PSCs are responsible for producing thedesmoplastic reaction in pancreatic cancer (Apte et al., 2004).A bidirectional interaction exists between PSCs and pancre-atic cancer cells, indicating that cancer cells recruit PSCswhich, in turn, facilitate local tumor progression and distantmetastasis (Apte et al., 1998; Bachem et al., 1998). In vitrostudies using PSCs either co-cultured with or exposed to con-ditioned media from cancer cells have established that (i) pan-creatic cancer cells induce PSC activation (as indicated byincreased proliferation, extracellular matrix synthesis andmigration) and that (ii) PSCs induce cancer cell proliferationbut inhibit apoptosis, thereby effectively enhancing the sur-vival of cancer cells (Fig. 2B) (Apte et al., 2004; Bachemet al., 2005; Vonlaufen et al., 2008). Thus pancreatic cancercells are able to recruit host PSCs to their immediate vicinity,

A

B

Fig. 2. (A) Effect of alcohol±LPS on histological and biochemical indicesof pancreatic fibrosis. Pancreatic sections and tissue homogenates from 4rat groups were analyzed: non-alcohol-fed no LPS (Control, C), alcohol-fedno LPS (A), nonalcohol fed, LPS (CLr) and alcohol-fed LPS (ALr). Panels(a) and (b) represent data for morphometric analysis of sections stained withMasson’s trichrome and Sirius Red (for collagen) respectively. Panel (c)depicts data for hydroxyproline content of the pancreas. Alcohol-fed ratssubjected to repeated LPS injections showed significant increases in Mas-son’s trichrome and Sirius Red staining as well as in pancreatic hydroxypro-line levels, compared to the C, A, or CLr animals. (B) Interactions betweenpancreatic cancer cells and pancreatic stellate cells. Pancreatic cancer cellsincrease PSC proliferation, extracellular matrix synthesis, and migration. Inturn, PSCs increase cancer cell proliferation and migration but decreasecancer cell apoptosis.

ALCOHOL, SIGNALING, AND ECM TURNOVER 7

and in turn, PSCs can establish a growth permissive environ-ment for tumor cells. Activation of PSCs by cancer cells ismediated by fibroblast growth factor (FGF) and PDGFsecreted by cancer cells (Bachem et al., 2005) and Apte’sgroup recently demonstrated that the proliferative effect ofPSC secretions on pancreatic cancer cells is mediated, at leastin part, by PDGF (Vonlaufen et al., 2008). An in vivo ortho-topic tumor model shows accelerated pancreatic tumorgrowth in the presence of PSCs, fibrosis within tumor tissue,increased cancer cell proliferation, decreased apoptosis andsignificantly higher regional and distant metastasis (Vonlaufenet al., 2008). These novel observations show that PSC acti-vation is a central feature of alcoholic pancreatic fibrosis aswell as pancreatic cancer. The orthotopic tumor model ofalcoholic pancreatitis presents an important tool for (i) theassessment of the chronological sequence of events in thepancreas during injury, (ii) the evaluation of efficacy oftherapeutic strategies, and (iii) determination of the potentialreversibility of pancreatic acinar injury and fibrosis uponwithdrawal of alcohol.Alcohol-induced factors such as ROS, LPS, cytokines, che-

mokines, and growth factors drive the liver response by acti-vating hepatic stellate cells (HSCs), similar to PSCs in thepancreas. Role of HSCs in liver injury of any etiology is wellestablished as these cells undergo activation through stages ofinitiation and perpetuation similar to PSCs (Friedman, 1993).Initiation results from paracrine stimulation from neighboringcells and the ECM disruption by metalloproteinases (Li andFriedman, 1999). Perpetuation involves the maintenance ofthe activated state through enhanced cytokine expressionresulting from both autocrine and paracrine stimulation andcontinued ECM remodeling (Friedman, 2000).Similar mechanisms, to those in the pancreas, may also be

operating in the liver during alcohol-mediated carcinogenesis,where HSCs are known to mediate fibrosis and interact withepithelial cells on alcohol activation. Strikingly again like pan-creatitis, cirrhotic livers have a higher incidence of developingcancer (Kojiro and Roskams, 2005). The inflammatory andfibrotic milieu, driven through several cytokines includingTGF-b, TNF-a, PDGF, and connective tissue growth factor(CTGF) triggering several signaling pathways, seem to besimilar in both organs. The profibrotic TGF-b plays animportant role during pathogenesis of liver disease (premalig-nant stages) and malignant transformation by a switch to atumor progression (Dooley et al., 2009). In addition, TNF-a-mediated NFjB ⁄ IjB kinase 2 (IKK2) activation elicits cellsurvival and anti-apoptotic signals favoring tumorigenesis.Maeda et al. showed that IKK2 knockout mice were pro-tected from chemical-induced carcinogenesis (Maeda et al.,2005) and by extension, suggests a role for this pathway evenfor alcohol-mediated carcinogenesis. In the liver, otheralcohol metabolic products also seem to be involved in carci-nogenesis. ROS produced by a number of enzymatic reactionsduring alcohol metabolism by ADH1 and Cyp2E1, is thedriving force in malignant transformation, together withongoing cirrhosis. Elevated ROS levels increase lipid

peroxidation and generate reactive molecules such asmalondialdehyde, which then react with DNA bases. Thisresults in highly mutagenic exocyclic DNA adducts (Hu et al.,2002), which increasingly occur during ALD progression(Frank et al., 2004). Cyp2E1 activity is also elevated up to 20-fold in patients chronically consuming alcohol (Oneta et al.,2002). This amplifies ROS formation upon alcohol challengeand accelerates DNA damage. Elevated Cyp2E1 activityfurther contributes to transformation by activation of pro-carciongens like nitrosamines (Seitz and Stickel, 2007). Addi-tionally, reactive nitrogen species is also known to precede thedevelopment of hepatocellular carcinoma. Cyp2E1 is alsoimplicated in retinoic acid receptor-mediated c-jun kinase(JNK) signaling pathway and AP-1 transcription factorexpression in experimental models of alcohol, favoring hepa-tic cell proliferation and survival in malignant transformation(Wang et al., 1998b). Moreover, genetic predispositions, suchas polymorphisms in genes coding for ADH, ALDH,Cyp2E1, CD14, TNF-a, and others can considerablycontribute to the risk of alcohol abuse and addiction, andthus indirectly to development of alcohol-induced cancer(Edenberg, 2007; Stickel and Osterreicher, 2006).Both HSCs and PSCs, when activated have a myofibroblast

phenotype, increased cytokine production and secrete exces-sive amounts of ECM proteins encouraging development offibrosis (Friedman, 1999b, 2000). Histological, immunohisto-chemical, and in situ hybridisation techniques have estab-lished that PSCs and HSCs are the predominant source of thecollagen deposited in fibrotic pancreas (Haber et al., 1999)and liver, respectively. Other mesenchymal cell types havealso been identified that can differentiate into active pro-fibrogenic fibroblast and contribute to tissue injury via TGF-bsignaling (Beaussier et al., 2007) likely toward carcinogenesis.

Role of Hepatocytes: Epithelial to Mesenchymal Transition

Emerging evidence implicates hepatocytes as a source ofpro-fibrogenic fibroblastoid cells by virtue of undergoing epi-thelial to mesenchymal transition (EMT) in chronic liverinjury (Dooley et al., 2008; Kaimori et al., 2007; Zeisberget al., 2007). This change is driven by active TGF-b signaling.TGF-b synergizes with alcohol in inducing oxidative stress(Zhuge and Cederbaum, 2006) and increases the inflamma-tory response of endotoxins (Seki et al., 2007). Noteworthy isthe TGF-b-mediated anti-proliferative and pro-apoptoticchange in hepatocyte characteristics under certain conditions.For example, in cancer, TGF-b growth inhibitory capacity isoften lost (Inagaki et al., 1993) leading to promotion of prolif-eration and migration, making the cells more aggressive ininfiltrating surrounding tissue and enhancing metastasis. Arecent report provides evidence for laminin-5 and TGF-b act-ing together for transition of noninvasive hepatocellular carci-noma cells to an invasive hepatocellular carcinoma type. Thischange in phenotype is driven by TGF-b and is sustained byincrease in expression of PDGF (Giannelli et al., 2005;Gotzmann et al., 2006). The TGF-b-mediated switch of

8 SETH ET AL.

phenotype is not only valid in hepatocellular carcinoma cells,but also in adult, nontransformed hepatocytes. A recent studydemonstrated that TGF-b-induced apoptosis occurred onlyin a minor fraction of cultured hepatocytes (Dooley et al.,2008). The majority of hepatocytes lost epithelial characteris-tics and acquired a mesenchymal phenotype, called EMT,regulated by a switch in the cell’s expression pattern. Thisillustrates the potentiality of diverse responses of hepatocytesto TGF-b, as well the importance and influence of surround-ing cellular matrix in a 3-dimensional context.In intact liver, hepatocytes show a strong apical-basal polar

phenotype enabling them to fulfill their multiple and spatiallydirected physiological functions. These functions, amongothers, include uptake from blood, carbohydrate homeostasis,catabolism, and detoxification, as well as bile secretion. Thisis achieved by an impermeable layer of hepatocytes connectedby cell–cell junctions, mainly tight junctions. Upon TGF-b-induced cell transition this epithelial phenotype is lost, tightjunctions are dissolved and several factors like zonulaoccludens-1 and E-cadherin are downregulated. In parallel,mesenchymal marker proteins are induced, such as, vimentinand type I collagen. Another property of the cells undergoingEMT is a gain in migratory capacity accompanied with achange in morphology toward a fibroblastoid shape (Fig. 3).Similar characteristics of EMT are also shown by Zeisbergand colleagues in hepatocytes treated with TGF-b (Zeisberget al., 2007). In their very elegant study using double trans-genic mice in a carbon tetrachloride (CCL4)-induced liverfibrosis model they demonstrated that a considerable fractionof fibroblast specific protein 1 (FSP-1) positive fibroblastswas derived from hepatocytes. Gene expression profiling in

hepatocyte cells treated with TGF-b revealed a precise anddetailed picture of TGF-b-induced pro-fibrotic and EMT tar-get genes (Breitkopf et al., 2005). Using this approach, knowntargets involved in growth control, like p21 and apoptosis,were identified. Further they found differentially regulatedgenes related to EMT (vimentin, Snail, E-cadherin, ZO-1, b-catenin, and others), fibrosis (CTGF, collagen type I, TIMP-1, and others), and also to alcohol metabolism (ADH1 andCYPs). Studies from Dooley’s group and others reveal thatupon induction with TGF-b, Snail-a TGF-b target gene anda critical regulator of E-cadherin expression, facilitates thedownregulation of cell junction protein E-cadherin. Theseinvestigations also demonstrate TGF-b dependent upregula-tion of Snail in hepatocytes around inflamed and fibrotic tis-sue, further supporting the idea of hepatocyte transition andprofibrogenic action in liver diseases.High CTGF levels have been associated with severity of

fibrotic diseases and might reflect a therapeutic node for treat-ing chronic liver fibrosis (Grotendorst, 1997; Lasky et al.,1998). CTGF is a strong pro-fibrotic mitogen and an earlytarget gene of TGF-b signaling. Until recently CTGF wasaccepted to be expressed by activated stellate cells in liver dis-eases (Paradis et al., 2002). The involvement of CTGF inhepatocytes of hepatitis B virus infected livers as well as inCCL4 treated mice (Weng et al., 2007) is a novel finding. It isnoteworthy that expression of CTGF was mainly mediatedby TGF-b ⁄ serine ⁄ threonine kinase receptor (ALK)5 ⁄Smad3signaling, although additional pathways have been describedto contribute to TGF-b-mediated CTGF expression in othercells types (Arnott et al., 2008; Gressner and Gressner, 2008;Gressner et al., 2008; Woods et al., 2008).

SIGNALING PATHWAYS IMPORTANT INALCOHOL-MEDIATED TISSUE INJURY

Alcohol toxicity invokes several signaling pathways ⁄molecules. These include mitogen associated protein kinases(MAPK), PI3K, TGF-b signaling cascade, PKC, PPARc andthe JAK-STAT pathway regulating expression of a numberof inflammatory and ECM molecules through transcriptionfactors NFjB and AP-1 in both liver (Dooley et al., 2000;Furukawa et al., 2003; Mandrekar and Szabo, 2009) and pan-creas (Apte et al., 2006). Recent research has revealed novelmolecules and their roles in mediating these signaling path-ways leading to progression of tissue damage, including acti-vation of KCs and transformation of quiescent to activatedstellate cells. Some of these novel mechanisms are explainedwith special reference to hepatic cells.

LPS-TLR-4-Mediated Signaling

Endotoxin-mediated activation of KCs via LPS-LPB-CD14-TLR-4 complex initiates a variety of signaling cascadesin the cell. One such signaling pathway involves IL-1 receptorassociated kinase (IRAK), myeloid differentiated factor 88(MyD88), and TNFR associated factor (TRAF) to modulate

Fig. 3. Effects of ethanol uptake in liver cells. Ethanol metabolites inducelipid peroxidation and ROS with subsequent damage of hepatocytes andHSC activation. Alcohol challenge is accompanied with an impaired gut bar-rier function that leads to elevated endotoxin levels in the liver. LPS then trig-gers HSC and Kupffer cell activation by TLR-4 and CD14 signaling. TGF-bstimulates hepatocytes to undergo apoptosis or epithelial mesenchymaltransition (EMT). Apoptotic hepatocytes can be phagocytosed by hepaticstellate cells and trigger their activation (in part by TLR-9 signaling), therebycontributing to inflammation and fibrosis. The recently described mechanismof hepatocyte EMT upon TGF-b signaling actively drives inflammatory andfibrotic processes. A considerable part of the active, profibrogenic fibroblastsderive from hepatocytes (see text for details). FN, fibronectin.

ALCOHOL, SIGNALING, AND ECM TURNOVER 9

NFjB transcription factor activity. NFjB is maintained in aninactive state by inhibitory molecule IjB. Signals generatedon endotoxin binding to CD14 ⁄TLR-4 receptor complexreleases IjB, resulting in activated form of NFjB. The conse-quence is generation of superoxide anions and production ofcytokines which further damage the tissue. IRAK1 is alsorapidly inactivated in response to macrophage activationdecreasing signaling, suggesting a negative feedback mecha-nism (Li et al., 2000). Consistent with this, alcohol adminis-tration in mice rapidly suppressed the production and activityof IRAK1 in the liver and IRAK1 expression correlated withthe degree of liver cell response (decreased TNF-a) to endo-toxin levels after acute alcohol exposure inducing tolerance toLPS (Yamashina et al., 2000). In contrast, long term exposureto alcohol increased IRAK and TNF-a, thereby inducing sen-sitization to LPS (Yamashina et al., 2000). It is hypothesizedthat the role of IRAK1 may be central to the dual effects ofalcohol observed with acute alcohol typically inducing toler-ance and chronic alcohol sensitizing the liver to the effects ofendotoxin (Mathurin et al., 2000). Evidence strongly indicatesthat these effects are brought upon by changes in levels andactivity of IRAK1 and CD14 (Wheeler and Thurman, 2003;Yamashina et al., 2000). The critical role of TLR-4 in thispathway is also demonstrated in mice strain C3H ⁄HeJ thathas a mutated form of TLR-4 gene and cannot initiate thesesignals. These mice are resistant to endotoxin effects and showno signs of chronic alcohol induced liver injury compared towild type mice that develop severe alcoholic hepatitis (Uesugiet al., 2001). Other experimental models that lacked CD14 orLBP also demonstrate that LPS-mediated signaling pathwaysare critical for alcoholic liver injury (Yin et al., 2001). On LPSinduction, early growth response-1 (Egr-1), another transcrip-tion factor, signals through MAPK (Erk1 ⁄2) via TNF-a inhepatic macrophages on chronic alcohol exposure (Kishoreet al., 2002). Egr-1 is known to contribute toward LPS-induced increased sensitivity of macrophages in chronic alco-hol models (Pritchard and Nagy, 2005). The lack of steatosisor increased ALT and TNF-a in Egr-1 knockout mice afterchronic alcohol (McMullen et al., 2005) exposure also sup-ports this proposition.

TGF-b-Mediated Signaling

The TGF-b signaling pathway has recently been studied ingreat detail by Dooley’s group and a comprehensive pictureof the TGF-b pathway interactome is provided by Taylor andWrana (Taylor andWrana, 2008). TGF-b can induce apopto-sis via TNF-a related pathway through activation of tran-scription factor AP-1 and via Smad signaling. Smads(homolog of drosophila mothers against decapentaplegic) area class of proteins that modulate the activity of TGF-bligands and act as nuclear transcription factors on complexingwith other Smads. TGF-b forms a complex with TGF-breceptor II (TBRII) and ALK5, phosphorylating Smad 2 andSmad 3. This complex in association with Smad 4 translocatesto the nucleus, regulating gene transcription (Shi and

Massague, 2003; Taylor and Wrana, 2008). Among others,Smad target genes include ECM-related molecules such as(plasminogen activator inhibitor-1) PAI-1, urokinase plasmi-nogen activator (uPA), and collagen. TGF-b signaling acti-vates several positive and negative feedback mechanisms,particularly through Smad7, a key negative feedback partici-pant that modulates TGF-b signaling in a site- and stage-specific manner (Dooley et al., 2008; Nakao et al., 1997; Shiand Massague, 2003; Shi et al., 2004; Zhang et al., 2007). Inhepatocytes, these feedback mechanisms tightly regulateTGF-b signaling in order to check excessive apoptosis andinhibition of proliferation. This mechanism presents an extre-mely sophisticated way to prevent hepatic failure that wouldotherwise result due to substantial cell loss (Schrum et al.,2001). Importantly, only a small proportion of hepatocytesgo through apoptosis but the majority have been shown toundergo EMT induced by TGF-b signaling (Dooley et al.,2008). The involvement of hepatocytes in modulating fibrosisin this manner is novel and intriguing and has important con-sequences in ALD pathogenesis. ALK5 dependent phosphor-ylation of Smad 2 translocates it to the nucleus where it bindsto p38-MAPK-dependent phosphorylated Smad 3 and Smad4. This hetero-complex can then bind with high affinity andspecificity to the Smad binding element (SBE) in the promoterregion of PAI-1 and stimulates PAI-1 transcription (Furukawaet al., 2003). Recent reports increasingly suggest a role forPAI-1 in liver injury (also see plasminogen system below).Besides the canonical Smad signaling, TGF-b can transducesignaling via other pathways (Moustakas and Heldin, 2008).As TGF-b plays a prominent role in ⁄during development andtissue homeostasis, its signaling is tightly controlled. The com-plexity of TGF-b signaling manifests in cofactors’ binding toand associating with TGF-b signaling cascade components aswell as in cross talks with other signaling molecules, such asthrough Akt and Erk pathways.

TNF-a-Mediated Signaling

TNF-a is a pro-inflammatory cytokine induced primarilyin macrophages and is considered to be a major player inALD as a therapeutic target. It has been shown in experimen-tal models of alcohol, that anti-TNF-a antibodies attenuatehepatic necrosis and inflammation. Also, in the intragastricinfusion model, TNF receptor 1 (TNFR1) knockout micewere resistant to ALD. In human, anti-TNF-a antibodyshowed initial promise as a specific molecular treatment forALD, however, a randomized controlled trial closed aftercases of serious sepsis emerged in treated patients.In addition to the signaling pathways described above, novel

mechanisms involving several sphingolipids have been impli-cated in TNF-a-mediated hepatocellular death. For example,Sphingosine-1-phosphate is shown to have an anti-apoptoticrole (Marı and Fernandez-Checa, 2007). Glycosphingolipidswhich act as second messengers mediate stress and deathligand-induced cell death, differentiation, proliferation andgene regulation (Marı and Fernandez-Checa, 2007; Morales

10 SETH ET AL.

et al., 2007). Sphingomyelinases (SMases), specifically acidicSMase (ASMase), is required for hydrolysis of sphingomyelinand is involved in the activation of the mitochondrial pathwayof apoptosis (Hannun and Luberto, 2000; Kolesnick andKronke, 1998). Ceramide is generated on activation of SMasesand plays a crucial role in determining the fate of cells inresponse to stress such as induced by alcohol and death recep-tor-mediated signaling. In hepatocytes, TNF-a binding toTNFR activates ASMase dependent signaling and induces celldeath (Garcıa-Ruiz et al., 2003; Marı et al., 2004). Culturedhepatocytes depleted of mitochondrial glutathione (mGSH)are sensitive to TNF-a, and undergo apoptotic cell death butare rescued by Cyclosporin A treatment. In contrast, hepato-cytes from ASMase) ⁄ ) mice are insensitive to TNF-a, butresponsive to exogenous ASMase-induced down-regulation ofmethionine adenosyltransferases (MAT)1A. Moreover, inan in vivo model of lethal hepatitis by TNF-a, depletion ofS-adenosyl-l-methionine (SAM) preceded activation ofcaspase-8 and -3 with massive liver damage and death of themice. In contrast, minimal hepatic SAM depletion, caspaseactivation, and liver damage are seen in ASMase) ⁄ ) mice.More importantly, therapeutic treatment with SAM abrogatescaspase activation and liver injury, thus rescuing ASMase+ ⁄+

mice from TNF-a-induced lethality. This demonstrates a dualrole for ASMase in TNF-a-induced liver failure by targetingglycosphingolipids to the mitochondria (Garcıa-Ruiz et al.,2003) and through downregulation of MAT1A and subse-quent SAM depletion (Marı et al., 2004) (Fig. 4). In ASMasenull mice there was minimal TNF-a as well as LPS-inducedliver injury (Garcıa-Ruiz et al., 2003) suggesting that thismechanism may also be important in alcohol-mediated injury.TNF–TNFR1 interactions are responsible for generating sur-vival and death signals through NFjB and caspase-8, respec-

tively (Micheau and Tschopp, 2003; Muppidi et al., 2004).NFjB prevents apoptosis by inhibiting caspase-8 (Thorne andTschopp, 2001), upregulating inhibitors of apoptosis (Wanget al., 1998a) and downregulating ROS-mediated JNK activa-tion (Kamata et al., 2005; Sakon et al., 2003). Studies haveshown that stress induced by selective depletion of mito-chondrial glutathione (mGSH) sensitizes hepatocytes to TNFin an unconventional pathway that is independent of NFjBinactivation. These observations highlight the overall relevanceof sphingolipids ⁄ASMase pathway and imply that such mech-anisms may also be important in alcohol-induced liver andother organ injury.

Alcohol-Induced Fibrogenesis Modulated viaPlasmin-Plasminogen System

The mechanisms by which hepatocytes alter fibrosis havebeen shown in in vitro studies (Seki, 1996; Seki et al., 2007).One such mechanism is by dysregulating plasmin-plasmino-gen homeostasis and altering fibrinolysis. The expression andactivities of pro- and anti-fibrinolytic molecules are meant tobalance the formation and resolution of fibrotic scar therebymodulating fibrogenesis. The major players involved in thispathway are the pro-fibrinolytic uPA, tissue-type plasmino-gen activators (tPA) and anti-fibrinolytic PAI-1. The first hintof involvement of hepatocytes and TGF-b in fibrosis andmatrix remodeling came from a study in AML12 cells, animmortalized nontransformed hepatocyte cell line, showingthat TGF-b strongly induces PAI-1 in a rapid and transientmanner (Seki, 1996). PAI-1 is an acute-phase protein and amajor inhibitor of uPA and tPA that regulate liver matrixremodeling through the activation of plasminogen to plasmin.The activation of plasminogen to plasmin breaks down fibrinthrough fibrinolysis and both are increased in alcoholic cir-rhosis (Leiper et al., 1994; Shanmukhappa et al., 2006).Recently, Seth et al. have shown increased PAI-1, uPA, tPA,plasminogen, plasmin and fibrinolysis in in vivo mouse and invitro hepatic cell culture models of alcohol (Seth et al., 2008).The data from their study suggest a pro-fibrinolytic profilewith exposure to low dose alcohol and a propensity for anti-fibrinolytic response with high-dose alcohol presumably, dueto several log-fold increase in PAI-1 expression (Seth et al.,2008). Similarly, PAI-1 is strongly induced and is knownto regulate ECM remodeling in experimental models ofalcoholic as well as other forms of liver injury (Arteel, 2008;Bergheim et al., 2006; Seth et al., 2008). PAI-1 affects activityof MMPs further contributing to ECM remodeling (Bergheimet al., 2006; Ramos-DeSimone et al., 1999). In other experi-mental models of chronic liver injury, increased ECM accu-mulation was shown in plasminogen knockout mice (Pohlet al., 2001) suggesting the significance of this pathway in liverfibrogenesis. Plasmin activation is also known to be regulatedthrough Osteopontin (Opn), a Th1 cytokine. As shown bycomprehensive signaling studies in cancer cells, Opn interactswith several integrins (avb3, a4b1, a9b1) and CD44 receptorsinitiating a variety of signaling cascades that regulate gene

Fig. 4. Mechanisms used by ASMase in mediating the cytotoxic effectsof TNF-a. Upon binding to its receptor, TNF activates ASMase resulting onone hand in the downregulation of MAT1A, and consequently in SAM andGSH depletion; and on the other hand ASMase fuels the synthesis of com-plex glycosphingolipids, such as gangliosides, able to target mitochondriaand inducing mitochondrial oxidative stress. Both processes promote thedamaging effects induced by TNF. SAM treatment would replenish GSHstores, including those in mitochondria, producing favorable conditions inwhich hepatocytes withstand the harmful effects of TNF.

ALCOHOL, SIGNALING, AND ECM TURNOVER 11

transcription via NFjB and AP-1 (Das et al., 2004, 2005;Philip and Kundu, 2003; Rangaswami et al., 2005). Bindingof Opn to its receptors induces PI3K ⁄Akt-dependent NFjBactivation and uPA secretion in cancer cells (Das et al., 2005).uPA ⁄ tPA, MMP9, and plasmin are also activated via MAPKand Erk pathways activating transcription factors AP-1 inbreast and prostate cancer cells (Angelucci et al., 2002; Daset al., 2004; Philip and Kundu, 2003). Intriguingly, findingsin human ALD show for the first time a significant increasein expression of Opn, Opn receptors (integrins, CD44), Opnmodifiers (thrombin, MMPs, tissue inhibitors of MMP[TIMPs]) and downstream effectors (uPA, tPA, plasminogen)(Seth et al., 2003, 2006a,b). Increased Opn plasma levels are amarker for fibrosis (Szalay et al., 2009), matrix remodeling(Simoes et al., 2009), and metastatic progression in severalcancers (Bellahcene et al., 2008; Patani et al., 2008), includinghepatocellular carcinoma (Kim et al., 2006; Zhang et al.,2006). Hepatic Opn expression is also increased in CCL4

model of liver injury and the methionine choline deficient dietmodel of nonalcohol steatohepatitis (NASH) (Lee et al.,2004; Sahai et al., 2004). Opn upregulation was shown todirectly mediate stellate cell activation in vitro in the CCL4

rat model (Lee et al., 2004). Alcohol also induced Opn in thelung and liver, along with a panel of other inflammatorymediators, such as TNF-a, IL-10, macrophage inflammatoryprotein (MIP)-2, and MMP-9 (Xu et al., 2007). There is com-pelling evidence that suggests these mechanisms may also beoccurring in liver and other organs in response to alcohol-induced injury.

Alcohol Modulates Immune Response During Bacterial andViral Infection

The recognition that alcoholics are at a greater risk foracquiring and dying from tuberculosis and bacterial pneumo-nias dates back to the clinical observations made by BenjaminRush in 1784 (Rush, 1943). These observations have sincethen been confirmed by epidemiological and laboratory stud-ies (MacGregor et al., 1978; Mason et al., 2004; Saitz et al.,1997; Schmidt and De Lint, 1972; Zaridze et al., 2009). Morerecently, alcohol abuse has also been associated withincreased incidence and severity of acute respiratory distresssyndrome (ARDS) (Esper et al., 2006; Moss et al., 1996),increased risk for viral and fungal infections and, subsequentsusceptibility to secondary bacterial infections (Fong et al.,1994; Jerrells et al., 2007). The increased susceptibility of alco-hol abusers to a variety of lung infections is attributed largelyto alcohol-mediated alterations in innate and adaptive immu-nity (Happel and Nelson, 2005; Joshi and Guidot, 2007;Szabo and Mandrekar, 2009). As is the case with otherorgans, the effects of alcohol on the lung may be direct ormediated via its metabolites (Sisson 2007). Findings using invivo animal models of acute alcohol intoxication and ⁄orin vitro exposure of isolated AM to alcohol indicate that asingle exposure to intoxicating levels (acute) of alcohol cansuppress lung innate immune responses to acute immune

stimulus. Studies performed using either lungs or immunecells isolated from human and rodents suggest that acutealcohol can suppress innate immune responses by altering thebalance between pro- and anti-inflammatory mediators(D’Souza El-Guindy et al., 2007; Gamble et al., 2006; Szaboand Mandrekar, 2009). It is suspected that alcohol inhibitsinflammatory cytokine secretion by affecting the NF-jB sig-naling pathway (Happel and Nelson, 2005). More recently,using a murine model of acute alcohol intoxication, alcohol isshown to impair LPS-induced pulmonary chemokine LIXproduction and alveolar neutrophil influx (Walker et al.,2009). On the other hand, excessive and prolonged alcoholconsumption is suspected to induce a state of low gradeinflammation, making the lungs hyporesponsive to bacterialinsult and increasing susceptibility to the infection. In thiscontext, chronic alcohol feeding in drinking water to mice isreported to increase basal levels of secreted TNF-a, osteopon-tin, MMP-9, Il-12p40, MIP-2, and blunt the inflammatoryresponse to subsequent infection with live Streptococcus pneu-moniae. This is accompanied by a high pathogen burden bothin the lung and systemic circulation (Xu et al., 2007).Impaired phagocytosis of inactivated bacteria by AM,decreased cellular glutathione, decreased ROS generation,impaired lipid peroxidation, TNF-a secretion and increasedapoptosis are also reported in AM isolated from rat andmouse models of chronic alcohol feeding (Brown et al.,2007a; D’Souza et al., 1996). Interestingly, supplementingalcohol diet with acetylcysteine, a precursor of glutathione, isreported to prevent the alcohol-induced defects in phagocyto-sis and loss of cell viability (Brown et al., 2007a), offering apotential mechanism to restore alcohol-induced defects inlung cell functions. Studies performed on bronchoalveolarlavage (BAL) and immune cells isolated from lungs of sub-jects (human and rodents) ingesting alcohol or with naıvelung immune cells exposed in vitro to alcohol suggest thatalcohol modulates not only innate but also adaptive immunecell functions (Szabo and Mandrekar, 2009). Throughoutbacterial invasion, the antigen presenting cells (dendritic cells,monocytes, and macrophages) assist the transition of innateto adaptive immune responses by T-lymphocyte activation(Bartlett et al., 2008; Tsai and Grayson, 2008). IL-23, a cyto-kine produced by activated AM and involved in the activa-tion of memory T-cell proliferation and T-cell derived IL-17production, is suppressed by acute alcohol intoxication inmice infected with Klebsiella pneumoniae (Happel and Nelson,2005). Using murine model of chronic alcohol intake and Kpneumonia infection, alcohol abuse is also reported to sup-press IL-12, a critical cytokine driving T-cell IFN-c response,and IFN-c expression in the lung (Shellito et al., 2001). In thissame model of infection, the expression of IL-17, a cytokinewhich serves as a link between innate and adaptive immu-nity, is shown to be inhibited by chronic alcohol intake(Ye et al., 2001). In mouse models of tuberculosis, excessivealcohol feeding impaired pathogen clearance, blunted CD4+and CD+ T lymphocyte responses, decreased lymphocyteproliferation, and IFN-c levels in CD4+ T (Mason et al.,

12 SETH ET AL.

2004). Similarly, decreased ability to clear respiratory syncy-tial virus (RSV) infection despite increased IFN-a and IFN-binduction, progressive loss of CD+ T cells, increased injuryand lethality along with increased inflammation reported inanimal models of chronic alcohol feeding (Jerrells et al.,2007).Both acute and chronic alcohol exposure can also direct

cytotoxicity via increased production of pro-inflammatorycytokines and chemokines and inhibiting T-cell activationand IL-12 production (Szabo and Mandrekar, 2009). In par-ticular, alcohol-induced cell toxicity is mediated via TNF-aled signaling events that activate transcription factors that canalter resistance to viral infections (Neuman et al., 1999, 2008).Alcohol is a known co-factor promoting hepatitis C virus(HCV) infection. It accelerates disease progression ofHCV-related cirrhosis and increases risk of hepatocellularcarcinoma. Alcohol modifications of cytokine profiles canmanipulate the transformation of cells from a preleukemic toleukemic state. In the Friend murine leukemia virus(F-MuLV), the role of alcohol was examined by Neuman’sgroup. They showed that alcohol could induce erythroleuke-mia in a BALBc mouse model. The alcohol fed mice showedan accelerated progression of the preleukemic stage withenhanced proliferation of erythroblasts and splenic infiltration(Shaked et al., 2005). In this model, the TNF-a and IL-8 weregreatly increased in the liver and spleen of mice exposed toalcohol. In addition, erythropoietin-receptor (Epo-R) trans-duction pathway was also upregulated altering gp55 ⁄Epo-R-mediated signaling, early initiation of tumorigenesis andenhancing preleukemic stage. Activation of Epo is known tobe associated with increased tumorigenicity of erythroleuke-mias (Cocco et al., 1996). Studies in ALD and other viralinfections also reveal that exposure to alcohol alters theimmune responses to infection and accelerate disease pro-gression. Early studies provide clinical evidence in patientswith chronic ALD showing reduced IL-2 indicating a defi-cient Thl activity (Saxena et al., 1986), and elevated serumlevels of IgA and IgE, suggestive of Th2 activation. Similarobservations are recorded in a murine retroviral model ofacquired immune deficiency syndrome (MAIDS) (Fitzpatricket al., 1995), and this model has been successfully usedto evaluate the effects of alcohol on retrovirus-inducedimmunodeficiency. In this model, chronic alcohol altered thecourse of the disease and in the noninfected mice, chronicalcohol feeding led to immune enhancement rather than sup-pression consistent with the hypothesis that chronic alcoholabuse depresses Th1 function and activates Th2 responses.The study of Friend erythroleukemia has made a contri-bution to the understanding of tumor progression eventsassociated with human cancer (Ben-David and Bernstein,1991; Shaked et al., 2005). Neuman et al. have demonstratedthat alcohol changes the splenic microenvironment promot-ing a hematologic malignancy via Epo ⁄Epo-R pathway(Neuman et al., 2008), and paves the way to test possiblepharmacological treatments for alcohol-induced tumor pro-gression in human leukemia.

Alcohol, ECM Remodeling, and Fibrogenesis: Role forASMase, MMPs, TIMPs

During liver fibrosis and cirrhosis, ECM remodeling isaccompanied not only by scar formation and increaseddeposition of fibril forming collagens (like type I collagen andothers), but also by degradation of normal liver ECM. Theseorchestrated processes are regulated by expression and activa-tion of MMPs as well as by TIMPs (Arthur, 1995, 2000; Sieg-mund et al., 2005). Remarkably, among others, acetaldehydeand TGF-b directly contribute to this process, in part bydownregulating MMPs and upregulating type I collagen(Siegmund et al., 2005).As described above, ASMase-mediated ceramide genera-

tion modulates the fibrogenic potential of HSC during fibro-genesis, via regulation of cathepsins (Cts) (Moles et al.,2009). Cathepsins are known to be involved in tumorigene-sis, cell death and are necessary for HSC transdifferentiationinto myofibroblasts. During in vitro mouse HSC activation,CtsB and CtsD are increased in parallel to a-SMA andTGF-b levels (Moles et al., 2009). Inhibition of CtsB ortransfection with CtsB siRNAs in vitro blunted Akt phos-phorylation and HSC proliferation and decreased expressionof a-SMA and TGF-b mRNA in activated HSC. Further-more, in in vivo murine model of CCl4-induced fibrogenesis,CtsB expression increased in HSCs and its inactivation abro-gated HSC activation, inflammation, and collagen deposition(Moles et al., 2009), suggesting that the antagonism ofcathepsins in HSC may be of relevance for the treatmentof liver fibrosis.Little is known on the effects of alcohol abuse on lung

ECM regulation. Limited published studies performed inrodent models of alcohol suggest that alcohol abuse canincrease the expression of fibronectin by alveolar type II cells,and activate lung MMP-9 and MMP-2 without affecting theirproduction. The studies also suggest these effects of alcoholmay be mediated by alcohol-induced oxidative stress (Brownet al., 2007b; Lois et al., 1999). A recent clinical study reportsincreases in fibronectin gene expression by alveolar macro-phages and epithelial lining fluid recovered from alcoholabusing individuals (Burnham et al., 2007). ECM turnover isregulated by endogenous neutral endopeptidases in the lung,amongst which are the Zinc (Zn) dependent MMPs andTIMPs: the balance between MMPs and TIMPs determineseither tissue homeostasis or disease state (Chirco et al., 2006;Greenlee et al., 2007; Murphy and Nagase, 2008). Recentstudies suggest that pathogens can bind to ECM moleculesvia adhesins and use the ECM for survival and dissemination.The pathogens also release endopeptidases that can disinte-grate the ECM and release ECM protein fragments withpro-inflammatory properties (Morwood and Nicholson,2006). In many pulmonary diseases, including ARDS andbacterial pneumonias, abnormal remodeling or destruction ofECM has been reported (Elkington and Friedland, 2006;O’Reilly et al., 2008; Okamoto et al., 2004). Not only do thepro-inflammatory and apoptotic mediators such as TNF-a,

ALCOHOL, SIGNALING, AND ECM TURNOVER 13

IL-1-b and FAS regulate the expression and activation ofMMPs and TIMPs, but the MMPs and TIMPs can also regu-late the expression of pro-inflammatory and apoptotic media-tors as well as the recruitment of inflammatory cells to theinflammation site. The ability of the TIMPs to balance theMMPs activity may allow these molecules to regulate com-munication between immune cell signaling networks and theECM (Elkington and Friedland, 2006; Greenlee et al., 2007).Elevated levels of MMPs (MMP-2, MMP-8, and MMP-9,specifically) are reported in BAL recovered from patients withacute lung injury and hospital acquired pneumonia (Fligielet al., 2006; Schaaf et al., 2008). Also, increased activitationof lung MMP-9 and MMP-2 in response to acute endotox-emia have been reported in rodent model of chronic alcoholingestion (Lois et al., 1999). Taken together, these studies sug-gest that activation of tissue remodeling may contribute tothe increased susceptibility for ARDS in individuals consum-ing excessive alcohol.Described above are several lines of investigation that

reveal common molecular pathways that are important forthe initiation and progression of alcohol-induced tissue injury.However, it is prudent to note that some pathways may onlybe important under certain conditions, in specific organs andarbitrated through a diverse range of molecules. These mole-cules and pathways are complex and context dependent, andcollectively contribute to alcohol-induced abnormalities,impaired immune and wound repair responses. Comprehen-sive and diverse approaches are required for exploration andunderstanding of these mechanisms of alcohol-mediatedpathogenesis.

ACKNOWLEDGMENTS

The work described in this review was supported byNational Health and Medical Research Council (NH&MRC)of Australia ⁄Department of Veteran’s Affairs (DVA) (Seth);NIH, NIAAA grant R01AA013168 (D’Souza El-Guindy);NH&MRC and the Cancer Council of New South Wales,Australia (Apte); CIBEREHD and grant PI070193 (Institutode Salud Carlos III, Spain), and by grant P50-AA-11999(Research Center for Liver and Pancreatic Diseases, U.S.National Institute on Alcohol Abuse and Alcoholism) (Mari);BMBF (HepatoSys) and the European Alcohol ResearchFoundation (ERAB) (Dooley).

REFERENCES

Adachi Y, Bradford BU, Gao W, Bojes HK, Thurman RG (1994) Inactiva-

tion of Kupffer cells prevents early alcohol-induced liver injury. Hepatology

20:453–460.

Angelucci A, Festuccia C, D’Andrea G, Teti A, Bologna M (2002)

Osteopontin modulates prostate carcinoma invasive capacity through

RGD-dependent upregulation of plasminogen activators. Biological

Chemistry 383:229–234.

Apte UM, Banerjee A, McRee R, Wellberg E, Ramaiah SK (2005b) Role of

osteopontin in hepatic neutrophil infiltration during alcoholic steatohepati-

tis. Toxicol Appl Pharmacol 207:25–38.

Apte MV, Haber PS, Applegate TL, Norton ID, McCaughan GW, Korsten

MA, Pirola RC, Wilson JS (1998) Periacinar stellate shaped cells in rat

pancreas – identification, isolation, and culture. Gut 43:128–133.

Apte MV, Haber PS, Darby SJ, Rodgers SC, McCaughan GW, Korsten MA,

Pirola RC, Wilson JS (1999) Pancreatic stellate cells are activated by proin-

flammatory cytokines: implications for pancreatic fibrogenesis. Gut 44:534–

541.

Apte MV, Park S, Phillips PA, Santucci N, Goldstein D, Kumar RK, Kumar

RK, Ramm GA, Buchler M, Friess H, McCarroll JA, Keogh G, Merrett

N, Pirola R, Wilson JS (2004) Desmoplastic reaction in pancreatic cancer:

role of pancreatic stellate cells. Pancreas 29:179–187.

Apte MV, Pirola RC, Wilson JS (2005a) Molecular mechanisms of alcoholic

pancreatitis. Dig Dis Sci 23:232–240.

Apte M, Pirola RC, Wilson JS (2006) Battle-scarred pancreas: role of alcohol

and pancreatic stellate cells in pancreatic fibrosis. J Gastroenterol Hepatol

21(Suppl. 3):S97–S101.

Arnott JA, Zhang X, Sanjay A, Owen TA, Smock SL, Rehman S, DeLong

WG, Safadi FF, Popoff SN (2008) Molecular requirements for induction of

CTGF expression by TGFbeta1 in primary osteoblasts. Bone 42:871–885.

Arteel GE (2008) New role of plasminogen activator inhibitor-1 in alcohol-

induced liver injury. J Gastroenterol Hepatol 23(Suppl. 1):S54–S59.

Arthur MJP (1995) Collagenases and liver fibrosis. J Hepatol 22(Suppl. 2):43–

48.

Arthur MJ (2000) Fibrogenesis II. Metalloproteinases and their inhibitors in

liver fibrosis. Am J Physiol Gastrointest Liver Physiol 279:G245–G249.

Atkinson KJ, Rao RK (2001) Role of protein tyrosine phosphorylation in

acetaldehyde-induced disruption of epithelial tight junctions. Am J Physiol

Gastrointest Liver Physiol 280:G1280–G1288.

Bachem MG, Schunemann M, Ramadani M, Siech M, Beger H, Buck A,

Zhou S, Schmid-Kotsas A, Adler G (2005) Pancreatic carcinoma cells

induce fibrosis by stimulating proliferation and matrix synthesis of stellate

cells. Gastroenterology 128:907–921.

Bachem MG, Schneider E, Gross H, Weidenbach H, Schmid RM, Menke A,

Siech M, Beger H, Grunert A, Adler G (1998) Identification, culture, and

characterization of pancreatic stellate cells in rats and humans. Gastroenter-

ology 115:421–432.

Bartlett JA, Fisher AJ, McCray PBJ (2008) Innate immune functions of the

airway epithelium. Contrib Microbiol 15:147–163.

Beaussier M, Wendum D, Schiffer E, Dumont S, Rey C, Lienhart A,

Housset C (2007) Prominent contribution of portal mesenchymal cells to

liver fibrosis in ischemic and obstructive cholestatic injuries. Lab Invest

87:292–303.

Bellahcene A, Castronovo V, Ogbureke KUE, Fisher LW, Fedarko NS (2008)

Small integrin-binding ligand N-linked glycoproteins (SIBLINGs): multi-

functional proteins in cancer. Nature Review Cancer Research 8:212–226.

Ben-David Y, Bernstein A (1991) Friend virus-induced erythroleukemia and

the multistage nature of cancer. Cell 66:831–834.

Bergheim I, Guo L, Davis MA, Duveau I, Arteel GE (2006) Critical role of

plasminogen activator inhibitor-1 in cholestatic liver injury and fibrosis.

J Pharmacol Exp Ther 316:592–600.

Binkley CE, Zhang L, Greenson JK, Giordano TJ, Kuick R, Misek D,

Hanash S, Logsdon CD, Simeone DM (2004) The molecular basis of pan-

creatic fibrosis: common stromal gene expression in chronic pancreatitis and

pancreatic adenocarcinoma. Pancreas 29:254–263.

Boe DM, Vandivier RW, Burnham EL, Moss M (2009) Alcohol abuse and

pulmonary disease. J Leukoc Biol [Epub ahead of print]. DOI: 10.1189/

jlb.0209087.

Breitkopf K, Haas S, Wiercinska E, Singer MV, Dooley S (2005) Anti-TGF-

beta strategies for the treatment of chronic liver disease. Alcohol Clin Exp

Res 29(11 Suppl.):121S–131S.

Brown LA, Ping XD, Harris FL, Gauthier TW (2007a) Glutathione availabil-

ity modulates alveolar macrophage function in the chronic ethanol-fed rat.

Am J Physiol Lung Cell Mol Physiol 292:L824–L832.

Brown LA, Ritzenthaler JD, Guidot DM, Roman J (2007b) Alveolar type II

cells from ethanol-fed rats produce a fibronectin-enriched extracellular

matrix that promotes monocyte activation. Alcohol 41:317–324.

14 SETH ET AL.

Burnham EL, Moss M, Ritzenthaler JD, Roman J (2007) Increased fribronec-

tin expression in lung in the setting of chronic alcohol abuse. Alcohol Clin

Exp Res 31:675–683.

Casini A, Ceni E, Salzano R, Milani S, Schuppan D, Surrenti C (1994) Acetal-

dehyde regulates the gene expression of matrixmetalloproteinase-1 and -2 in

human fat-storing cells. Life Sci 55:1311–1316.

Chirco R, Liu X, Jung K (2006) Novel functions of TIMPs in cell signaling.

Cancer Metastasis Rev 25:99–113.

Cocco P, Rapallo M, Targhetta R, Biddau PF, Fadda D (1996) Analysis of

risk factors in a cluster of childhood acute lymphoblastic leukemia. Arch

Environ Health 51:242–244.

D’Souza El-Guindy NB, de Villiers WJ, Doherty DE (2007) Acute alcohol

intake impairs lung inflammation by changing pro- and anti-inflammatory

mediator balance. Alcohol Alcohol 41:335–345.

D’Souza NB, Nelson S, Summer WR, Deaciuc IV (1996) Alcohol modulates

alveolar macrophage tumor necrosis factor-alpha, superoxide anion, and

nitric oxide secretion in the rat. Alcohol Clin Exp Res 20:156–163.

Das R, Mahabeleshwar GH, Kundu GC (2004) Osteopontin induces AP-1-

mediated secretion of urokinase-type plasminogen activator through c-Src-

dependent epidermal growth factor receptor transactivation in breast cancer

cells. J Biol Chem 279:11051–11064.

Das R, Philip S, Mahabeleshwar GH, Bulbule A, Kundu GC (2005) Osteo-

pontin: it’s role in regulation of cell motility and nuclear factor kappa B-

mediated urokinase type plasminogen activator expression. IUBMB Life

57:441–447.

Dooley S, Delvoux B, Lahme B, Mangasser-Stephan K, Gressner AM (2000)

Modulation of transforming growth factor beta response and signaling dur-

ing transdifferentiation of rat hepatic stellate cells to myofibroblasts. Hepa-

tology 31:1094–1106.

Dooley S, Delvoux B, Streckert M, Bonzel L, Stopa M, ten Dijke P, Gressner

AM (2001) Transforming growth factor beta signal transduction in hepatic

stellate cells via Smad2 ⁄ 3 phosphorylation, a pathway that is abrogated dur-

ing in vitro progression to myofibroblasts. TGFbeta signal transduction

during transdifferentiation of hepatic stellate cells. FEBS Lett 502:4–10.

Dooley S, Hamzavi J, Ciuclan L, Godoy P, Ilkavets I, Ehnert S, Ueberham E,

Gebhardt R, Kanzler S, Geier A, Breitkopf K, Weng H, Mertens PR (2008)

Hepatocyte-specific Smad7 expression attenuates TGF-beta-mediated fibro-

genesis and protects against liver damage. Gastroenterology 135:642–659.

Dooley S, Weng H, Mertens PR (2009) Hypotheses on the role of transform-

ing growth factor-beta in the onset and progression of hepatocellular carci-

noma. Dig Dis Sci 27:93–101.

Edenberg HJ (2007) The genetics of alcohol metabolism: role of alcohol dehy-

drogenase and aldehyde dehydrogenase variants. Alcohol Res Health 30:5–

13.

Elkington PTG, Friedland JS (2006) Matrix metalloproteinases in destructive

pulmonary pathology. Thorax 61:259–266.

Esper A, Burnham EL, Moss M (2006) The effect of alcohol abuse on ARDS

and multiple organ dysfunction. Minerva Anestesiol 72:375–381.

Fitzpatrick EA, Rhoads CA, Espandiari P, Kaplan AM, Cohen DA (1995)

Ethanol as a possible cofactor in the development of murine AIDS. Alcohol

Clin Exp Res 19:915–922.

Fligiel SE, Standiford T, Fligiel HM, Tashkin D, Strieter RM, Warner RL,

Johnson KJ, Varani J (2006) Matrix metalloproteinases and matrix metallo-

proteinase inhibitors in acute lung injury. Hum Pathol 37:422–430.

Fong IW, Read S, Wainberg MA, Chia WK, Major C (1994) Alcoholism and

rapid progression to AIDS after seroconversion. Clin Infect Dis 19:337–338.

Frank A, Seitz HK, Bartsch H, Frank N, Nair J (2004) Immunohistochemical

detection of 1,N6-ethenodeoxyadenosine in nuclei of human liver affected

by diseases predisposing to hepato-carcinogenesis. Carcinogenesis 25:1027–

1031.

Friedman SL (1993) Seminars in medicine of the Beth Israel Hospital, Boston.

The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies.

N Engl J Med 328:1828–1835.

Friedman SL (1999b) The virtuosity of hepatic stellate cells. Gastroenterology

117:1244–1246.

Friedman SL (2000) Molecular regulation of hepatic fibrosis, an integrated cel-

lular response to tissue injury. J Biol Chem 275:2247–2250.

Fukui H, Brauner B, Bode JC, Bode C (1991) Plasma endotoxin concentra-

tions in patients with alcoholic and non-alcoholic liver disease: reevaluation

with an improved chromogenic assay. J Hepatol 12:162–169.

Furukawa F, Matsuzaki K, Mori S, Tahashi Y, Yoshida K, Sugano Y,

Yamagata H, Matsushita M, Seki T, Inagaki Y, Nishizawa M, Fujisawa J,

Inoue K (2003) MAPK mediates fibrogenic signal through Smad3 phos-

phorylation in rat myofibroblasts. Hepatology 38:879–889.

Gamble L, Mason CM, Nelson S (2006) The effects of alcohol on immunity

and bacterial infection in the lung. Medecine et maladies infectieuses 36:72–

77.

Garcıa-Ruiz C, Colell A, Marı M, Morales A, CalvoM, Enrich C, Fernandez-

Checa JC (2003) Defective TNF-alpha-mediated hepatocellular apoptosis

and liver damage in acidic sphingomyelinase knockout mice. J Clin Invest

111:197–208.

Giannelli GBC, Fransvea E, Sgarra C, Antonaci S (2005) Laminin-5 with

transforming growth factor-beta1 induces epithelial to mesenchymal transi-

tion in hepatocellular carcinoma. Gastroenterology 129:1375–1383.

Gotzmann J, Fischer AN, Zojer M, Mikula M, Proell V, Huber H, Jechlinger

M, Waerner T, Weith A, Beug H, Mikulits W (2006) A crucial function of

PDGF in TGF-betamediated cancer progression of hepatocytes. Oncogene

25:3170–3185.

Graham CS (2006) The Data Collection on Adverse Events of Anti-HIV

Drugs Study Group. Liver-related deaths in persons infected with the

human immunodeficiency virus: the D:A:D Study. Arch Intern Med

166:1632–1641.

Greenlee KJ, Werb Z, Kheradmand F (2007) Matrix metalloproteinases in

lung: multiple, multifarious, and multifaceted. Physiol Rev 87:69–98.

Greenwel P (1999) Acetaldehyde-mediated collagen regulation in hepatic

stellate cells. Alcohol Clin Exp Res 23:930–933.

Gressner AM, Bachem MG (1990) Cellular sources of non-colagenous matrix

proteins: role of fat storing cells in fibrogenesis. Semin Liver Dis 10:30–46.

Gressner OA, Gressner AM (2008) Connective tissue growth factor: a fibro-

genic master switch in fibrotic liver diseases. Liver Int 28:1065–1079.

Gressner OA, Lahme B, Siluschek M, Rehbein K, Weiskirchen R, Gressner

AM (2008) Intracrine signalling of activin A in hepatocytes upregulates con-

nective tissue growth factor (CTGF ⁄CCN2) expression. Liver Int 28:1207–

1216.

Grotendorst GR (1997) Connective tissue growth factor: a mediator of TGF-

beta action on fibroblasts. Cytokine Growth Factor Rev 8:171–179.

Guo J, Loke J, Zheng F, Hong F, Yea S, Fugita M, Tarocchi M, Abar OT,

Huang H, Sninsky JJ, Friedman SL (2009) Functional linkage of cirrhosis-

predictive single nucleotide polymorphisms of toll-like receptor 4 to hepatic

stellate cell responses. Hepatology 49:960–968.

Haber PS, Keogh GW, Apte M, Moran CS, Stewart NL, Crawford D, Pirola

RC, McCaughan GW, Ramm GA, Wilson JS (1999) Activation of pancre-

atic stellate cells in human and experimental pancreatic fibrosis. Am J

Pathol 155:1087–1095.

Hannun YA, Luberto C (2000) Ceramide in the eukaryotic stress response.

Trends Cell Biol 10:73–80.

Happel KI, Nelson S (2005) Alcohol, immunosuppression, and the lung. Proc

Am Thorac Soc 2:428–432.

Hu W, Feng Z, Eveleigh J, Iyer G, Pan J, Amin S, Chung FL, Tang MS

(2002) The major lipid peroxidation product, trans-4-hydroxy-2-nonenal,

preferentially forms DNA adducts at codon 249 of human p53 gene, a

unique mutational hotspot in hepatocellular carcinoma. Carcinogenesis

23:1781–1789.

Huang H, Shiffman ML, Friedman S, Venkatesh R, Bzowej N, Abar OT,

Rowland CM, Catanese JJ, Leong DU, Sninsky JJ, Layden TJ, Wright TL,

White T, Cheung RC (2007) A 7 gene signature identifies the risk of devel-

oping cirrhosis in patients with chronic hepatitis C. Hepatology 46:297–306.

Ido M, Nagata C, Kawakami N, Shimizu H, Yoshida Y, Nomura T,

Mizoguchi H (1996) A case-control study of myelodysplastic syndromes

among Japanese men and women. Leuk Res 20:727–731.

Inagaki M, Moustakas A, Lin HY, Lodish HF, Carr BI (1993) Growth inhibi-

tion by transforming growth factor-beta (TGF-beta) type I is restored in

TGF-beta-resistant hepatoma cells after expression of TGF-beta receptor

type II cDNA. Proc Natl Acad Sci USA 90:5359–5363.

ALCOHOL, SIGNALING, AND ECM TURNOVER 15

Jerrells TR, Pavlik JA, DeVasure J, Vidlak D, Costello A, Strachota JM,

Wyatt TA (2007) Association of chronic alcohol consumption and increased

susceptibility to and pathogenic effects of pulmonary infection with respira-

tory syncytial virus in mice. Alcohol 41:357–369.

Joshi PC, Guidot DM (2007) The alcoholic lung: epidemiology, pathophysiol-

ogy, and potential therapies. Am J Physiol Lung Cell Mol Physiol

292:L813–L823.

Kaimori A, Potter J, Kaimori JY, Wang C, Mezey E, Koteish A (2007)

Transforming growth factor-beta1 induces an epithelial-to-mesenchymal

transition state in mouse hepatocytes in vitro. J Biol Chem 282:22089–

22101.

Kamata H, Honda S, Maeda S, Chang L, Hirata H, Karin M (2005) Reactive

oxygen species promote TNFalpha-induced death and sustained JNK acti-

vation by inhibiting MAP kinase phosphatases. Cell 120:649–661.

Kim J, Ki SS, Lee SD, Han CJ, Kim YC, Park SH, Cho SY, Hong Y-J, Park

HY, Lee M, Jung HH, Lee KH, Jeong S-H (2006) Elevated plasma Osteo-

pontin levels in patients with hepatocellular carcinoma. Am J Gastroenterol

101:1–9.

Kishore R, Hill JR, McMullen MR, Frenkel J, Nagy LE (2002) ERK1 ⁄ 2 and

Egr-1 contribute to increased TNF-alpha production in rat Kupffer cells

after chronic ethanol feeding. Am J Physiol Gastrointest Liver Physiol

282:G6–G15.

Kojiro M, Roskams T (2005) Early hepatocellular carcinoma and dysplastic

nodules. Semin Liver Dis 25:133–142.

Kolesnick RN, Kronke M (1998) Regulation of ceramide production and

apoptosis. Annu Rev Physiol 60:643–665.

Lasky JA, Ortiz LA, Tonthat B, Hoyle GW, Corti M, Athas G, Lungarella

G, Brody A, Friedman M (1998) Connective tissue growth factor mRNA

expression is upregulated in bleomycin-induced lung fibrosis. Am J Physiol

Gastrointest Liver Physiol 275(2 Pt 1):L365–L371.

Lee JH, Banerjee A, Ueno Y, Ramaiah SK (2008) Potential relationship

between hepatobiliary osteopontin and peroxisome proliferator-activated

receptor {alpha} expression following ethanol-associated hepatic injury in

vivo and in vitro. Toxicol Sci 106:290–299.

Lee SH, Seo GS, Park YN, Yoo TM, Sohn DH (2004) Effects and regulation

of osteopontin in rat hepatic stellate cells. Biochem Pharmacol 68:2367–

2378.

Leiper K, Croll A, Booth NA, Moore NR, Sinclair T, Bennett B (1994) Tissue

plasminogen activator, plasminogen activator inhibitors, and activator-

inhibitor complex in liver disease. J Clin Pathol 47:214–217.

Li L, Coursat S, Hu J, McCall CE (2000) Characterization of IRAK in nor-

mal and endotoxin tolerant cells. J Biol Chem 275:2410–2414.

Li D, Friedman SL (1999) Liver fibrogenesis and the role of hepatic stellate

cells: new insihts and prospects for therapy. J Gastroenterol Hepatol

14:618–633.

Lois M, Brown LA, Moss IM, Roman J, Guidot DM (1999) Ethanol inges-

tion increases activation of matrix metalloproteinases in rat lungs during

acute endotoxemia. Am J Respir Crit Care Med 160:1354–1360.

MacGregor RR, Gluckman SJ, Senior JR (1978) Granulocyte function and

levels of immunoglobulins and complements in patients admitted for with-

drawal from alcohol. J Infect Dis 138:747–755.

Maeda S, Kamata H, Luo JL, Leffert H, Karin M (2005) KKb couplet hepa-

tocyte death to cytokine-driven compensatory proliferation that promotes

chemical hepatocarcinogenesis. Cell 121:977–990.

Mandrekar P, Szabo G (2009) Signalling pathways in alcohol-induced liver

inflammation. J Hepatol 50:1258–1266.

Marı M, Colell A, Morales A, Paneda C, Varela-Nieto I, Garcıa-Ruiz C,

Fernandez-Checa JC (2004) Acidic sphingomyelinase downregulates the

liver-specific methionine adenosyltransferase 1A, contributing to tumor

necrosis factor-induced lethal hepatitis. J Clin Invest 11:895–904.

Marı M, Fernandez-Checa JC (2007) Sphingolipid signalling and liver dis-

eases. Liver Int 27:440–450.

Masamune A, Kikuta K, Satoh M, Sakai Y, Satoh A, Shimosegawa T (2002)

Ligands of peroxisome proliferator-activated receptor-gamma block activa-

tion of pancreatic stellate cells. J Biol Chem 277:141–147.

Mason CM, Dobard E, Zhang P, Nelson S (2004) Alcohol exacerbates murine

pulmonary tuberculosis. Infect Immun 72:2556–2563.

Mathurin P, Deng QG, Keshavarzian A, Choudhary S, Holmes EW,

Tsukamoto H (2000) Exacerbation of alcoholic liver injury by enteral

endotoxin in rats. Hepatology 32:1008–1017.

McMullen MR, Pritchard MT, Wang Q, Millward CA, Croniger CM,

Nagy LE (2005) Early growth response-1 transcription factor is essential

for ethanol-induced fatty liver injury in mice. Gastroenterology 128:2066–

2076.

Micheau O, Tschopp J (2003) Induction of TNF receptor I-mediated apopto-

sis via two sequential signaling complexes. Cell 114:181–190.

Moles A, Tarrats N, Fernandez-Checa JC, Marı M (2009) Cathepsins B and

D drive hepatic stellate cell proliferation and promote their fibrogenic poten-

tial. Hepatology 49:1297–1307.

Morales A, Lee H, Goni FM, Kolesnick R, Fernandez-Checa JC (2007)

Sphingolipids and cell death. Apoptosis 12:923–939.

Morwood SR, Nicholson LB (2006) Modulation of the immune response by

extracellular matrix proteins. Arch Immunol Ther Exp 54:367–374.

Moss M, Bucher B, Moore FA, Moore EE, Parsons PE (1996) The role of

chronic alcohol abuse in the development of acute respiratory distress syn-

drome in adults. JAMA 275:50–54.

Moustakas A, Heldin CH (2008) Dynamic control of TGF-beta signaling and

its links to the cytoskeleton. FEBS Lett 582:2051–2065.

Muppidi JR, Tschopp J, Siegel RM (2004) Life and death decisions: secondary

complexes and lipid rafts in TNF receptor family signal transduction.

Immunity 21:461–465.

Murphy G, Nagase H (2008) Progress in matrix metalloproteinase research.

Mol Aspects Med 29:290–308.

Nakao A, Afrakhte M, Moren A, Nakayama T, Christian JL, Heuchel R,

Itoh S, Kawabata N, Heldin NE, Heldin CH, Dijke P (1997) Identification

of Smad7, a TGF beta-inducible antagonist of TGF-beta signalling. Nature

389:631–635.

Nanji AA, Khettry U, Sadrzadeh SM (1994) Lactobacillus feeding reduces

endotoxemia and severity of experimental alcoholic liver disease. Proc Soc

Exp Biol Med 205:243–247.

Nath B, Szabo G (2009) Alcohol-induced modulation of signalling pathways

in liver parenchymal and nonparenchymal cells: implications for immunity.

Semin Liver Dis 29:166–177.

Neuman MG, Koren G, Tiribelli C (1993) In vitro, assessment of the ethanol-

induced hepatotoxicity on Hep G2 cell line. Biochem Biophys Res Commun

197:932–941.

Neuman MG, Sha K, Esguerra R, Winkler RE, Cooper C, Hilzenrat N, Wise

J, Zahari S, Seth D, Gorrell M, Haber P, McCaughan G, Leo MA, Lieber

CS, Voiculescu M, Buzatu E, Ionescu C, Dudas J, Saile B, Ramadori G

(2008) Inflammation and repair in viral hepatitis C. Dig Dis Sci 53:1468–

1487.

Neuman MG, Shear NH, Bellentani S, Tiribelli C. (1998) Role of cytokines in

ethanol-induced hepatocytotoxicity in Hep G2 cells. Gastroenterology

114:157–169.

Neuman MG, Shear NH, Cameron RG, Katz GG, Tiribelli C (1999)

Ethanol-induced apoptosis in vitro. Clin Biochem 32:547–555.

O’Reilly PJ, Gaggar A, Blalock JE (2008) Interfering with extracellular matrix

degradation to blunt inflammation. Curr Opin Pharmacol 8:242–248.

Okamoto T, Akuta T, Tamura F, van der Vliet A, Akaike T (2004) Molecu-

lar mechanisms for activation and regulation of matrix metalloproteinases

duringbacterial infections and respiratory inflammation. BiolChem385:997–

1006.

Oneta CM, Lieber CS, Li J, Ruttimann S, Schmid B, Lattmann J, Rosman

AS, Seitz HK (2002) Dynamics of cytochrome P4502E1 activity in man:

induction by ethanol and disappearance during withdrawal phase. J Hepatol

36:47–52.

Paik YH, Schwabe RF, Bataller R, Russo MP, Jobin C, Brenner DA (2003)

Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopoly-

saccharide in human hepatic stellate cells. Hepatology 37:1043–1055.

Paradis V, Dargere D, Bonvoust F, Vidaud M, Segarini P, Bedossa P (2002)

Effects and regulation of connective tissue growth factor on hepatic stellate

cells. Lab Invest 82:767–774.

Parola M, Robino G (2001) Oxidative stress-related molecules and liver fibro-

sis. J Hepatol 35:297–306.

16 SETH ET AL.

Patani N, Jouhra F, Jiang W, Mokbel K (2008) Osteopontin expression pro-

files predict pathological and clinical outcome in breast cancer. Anticancer

Res 28:4105–4110.

Perides G, Tao X, West N, Sharma A, Steer ML (2005) A mouse model of

ethanol dependent pancreatic fibrosis. Gut 54:1461–1467.

Philip S, Kundu GC (2003) Osteopontin induces nuclear factor kappa

B-mediated promatrix metalloproteinase-2 activation through I kappa B

alpha ⁄ IKK signaling pathways, and curcumin (diferulolylmethane) down-

regulates these pathways. J Biol Chem 278:14487–14497.

Pohl JF, Melin-Aldana H, Sabla G, Degen JL, Bezerra JA (2001) Plasminogen

deficiency leads to impaired lobular reorganization and matrix accumulation

after chronic liver injury. Am J Pathol 159:2179–2186.

Pritchard MT, Nagy LE (2005) Ethanol-induced liver injury: potential roles

for egr-1. Alcohol Clin Exp Res 29:146S–150S.

Ramos-DeSimone N, Hahn-Dantona E, Sipley J, Nagase H, French DL,

Quigley JP (1999) Activation of matrix metalloproteinase-9 (MMP-9) via a

converging plasmin ⁄ stromelysin-1 cascade enhances tumor cell invasion.

J Biol Chem 274:13066–13076.

Rangaswami H, Bulbule A, Kundu GC (2005) JNK1 differentially regulates

osteopontin-induced nuclear factor-inducing kinase ⁄MEKK1-dependent

activating protein-1-mediated promatrix metalloproteinase-9 activation.

J Biol Chem 280:19381–19392.

Rojkind M, Giambrone MA, Biempica L (1979) Collagen types in normal

and cirrhotic liver. Gastroenterology 76:710–719.

Rush B (1943) An inquiry into the effects of ardent spirits upon human body

and mind. Q J Stud Alcohol 4:321–341.

Sahai A, Malladi P, Melin-Aldana H, Green RM, Whitington PF (2004)

Upregulation of osteopontin expression is involved in the development of

nonalcoholic steatohepatitis in a dietary murine model. Am J Physiol

Gastrointest Liver Physiol 287:G264–G273.

Saitz R, Ghali WA, Moskowitz MA (1997) The impact of alcohol-related

diagnoses on pneumonia outcomes. Arch Intern Med 157:1446–1452.

Sakon S, Xue X, Takekawa M, Sasazuki T, Okazaki T, Kojima Y, Piao J-H,

Yagita H, Okumura K, Doi T, Nakano H (2003) NF-kappaB inhibits

TNF-induced accumulation of ROS that mediate prolonged MAPK activa-

tion and necrotic cell death. EMBO J 22:3898–3909.

Samet JH, Horton NJ, Traphagen ET, Lyon SM, Freedberg KA (2003) Alco-

hol consumption and HIV disease progression: are they related? Alcohol

Clin Exp Res 27:862–867.

Saxena S, Nouri-Aria KT, Anderson MG, Eddleston AL, Williams E (1986)

Interleukin 2 activity in chronic liver disease and the effect of in vitro alpha

interferon. Clin Exp Immunol 63:541–548.

Schaaf B, Liebau C, Kurowski V, Droemann D, Dalhoff K (2008) Hospital

acquired pneumonia with high-risk bacteria is associated with increased

matrix metalloproteinase activity. BMC PulmMed 12:8–12.

Schmidt W, De Lint J (1972) Causes of death of alcoholics. Q J Stud Alcohol

33:171–185.

Schrum LW, Bird MA, Salcher O, Burchardt ER, Grisham JW, Brenner DA,

Rippe RA, Behrns KE (2001) Autocrine expression of activated transform-

ing growth factor-beta(1) induces apoptosis in normal rat liver. Am J Phys-

iol Gastrointest Liver Physiol 280:G139–G148.

Seitz HK, Becker P (2007) Alcohol metabolism and cancer risk. Alcohol Res

Health 30:38–41.

Seitz HK, Homann N (2007) The role of acetaldehyde in alcohol-associated

cancer of the gastrointestinal tract. Novartis Found Symp 285:110–119; dis-

cussion 119-114, 198-119.

Seitz HK, Stickel F (2007) Molecular mechanisms of alcohol-mediated carci-

nogenesis. Nat Rev Cancer 7:599–612.

Seki T (1996) Interleukin-1 induction of type-1 plasminogen activator inhibitor

(PAI-1) gene expression in the mouse hepatocyte line, AML 12. J Cell Phys-

iol 168:648–656.

Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA,

Schwabe RF (2007) TLR4 enhances TGF-beta signaling and hepatic fibro-

sis. Nat Med 13:1324–1332.

Seth D, BeardM, Gorrell M,McCaughan GW, Haber P (2006a) Osteopontin:

an early marker for alcoholic liver injury? Alcohol Clin Exp Res 30s2(9):

89A.

Seth D, Cordoba S, Gorrell MD, McCaughan GW, Haber PS (2006b)

Intrahepatic gene expression in human alcoholic hepatitis. J Hepatol

45:306–320.

Seth D, Hogg PJ, Gorrell MD, McCaughan GW, Haber PS (2008) Direct

effects of alcohol on hepatic fibrinolytic balance: implications for alcoholic

liver disease. J Hepatol 48:614–627.

Seth D, Leo MA, McGuinness PH, Lieber CS, Brennan Y, Williams RB,

Wang XM, McCaughan GW, Gorrell MD, Haber PS (2003) Gene expres-

sion profiling of alcoholic liver disease in the baboon (Papio hamadryas)

and human liver. Am J Pathol 163:2303–2317.

Shaked Y, Cervi D, NeumanM, Chen L, Klement G, Michaud CR, Haeri M,

Pak BJ, Kerbel RS, Ben-David Y (2005) The splenic microenvironment is a

source of pro-angiogenesis ⁄ inflammatory mediators accelerating the expan-

sion of murine erythroleukemic cells. Blood 105:4500–4507.

Shanmukhappa K, Sabla GE, Degen JL, Bezerra JA (2006) Urokinase-type

plasminogen activator supports liver repair independent of its cellular recep-

tor. BMCGastroenterol 6:40.

Shek FW, Benyon RC, Walker FM, McCrudden PR, Pender SL, Williams EJ

(2002) Expression of transforming growth factor-beta 1 by pancreatic stel-

late cells and its implications for matrix secretion and turnover in chronic

pancreatitis. Am J Pathol 160:1787–1798.

Shellito JE, quanZheng M, Ye P, Ruan S, Shean MK, Kolls J (2001) Effect of

alcohol consumption on host release of interleukin-17 during pulmonary

infection with Klebsiella pneumoniae. Alcohol Clin Exp Res 25:872–881.

Shi Y, Massague J (2003) Mechanisms of TGF-beta signaling from cell mem-

brane to the nucleus. Cell 113:685–700.

Shi W, Sun C, He B, Xiong W, Shi X, Yao D, Cao X (2004) GADD34-PP1c

recruited by Smad7 dephosphorylates TGFbeta type I receptor. J Cell Biol

164:291–300.

Siegmund SV, Dooley S, Brenner DA (2005) Molecular mechanisms of alco-

hol-induced hepatic fibrosis. Dig Dis Sci 23:264–274.

Simoes DC, Xanthou G, Petrochilou K, Panoutsakopoulou V, Roussos C,

Gratziou C (2009) Osteopontin deficiency protects from airway remodeling

and hyperresponsiveness in chronic asthma. Am J Respir Crit Care Med

179:894–902.

Sisson JH (2007) Alcohol and airways function in health and disease. Alcohol

41:293–307.

Sparmann G, Glass A, Brock P, Jaster R, Koczan D, Thiesen HJ (2005)

Inhibition of lymphocyte apoptosis by pancreatic stellate cells: impact of

interleukin-15. Am J Physiol Gastrointest Liver Physiol 289:G842–G851.

Stickel F, Osterreicher CH (2006) The role of genetic polymorphisms in alco-

holic liver disease. Alcohol Alcohol 41:209–224.

Su GL (2002) Lipopolysaccharides in liver injury: molecular mechanisms of

Kupffer cell activation. Am J Physiol Gastrointest Liver Physiol 283:G256–

G265.

Svegliati-Baroni G, Inagaki Y, Rincon-Sanchez AR, Else C, Saccomanno S,

Benedetti A, Ramirez F, Rojkind M (2005) Early response of alpha2(I) col-

lagen to acetaldehyde in human hepatic stellate cells is TGF-beta indepen-

dent. Hepatology 42:343–352.

Svegliati-Baroni G, Ridolfi F, Di SA, Saccomanno S, Bendia E, Benedetti A,

Greenwel P (2001) Intracellular signaling pathways involved in acetalde-

hyde-induced collagen and fibronectin gene expression in human hepatic

stellate cells. Hepatology 33:1130–1140.

Szabo G, Mandrekar P (2009) A recent perspective on alcohol, immunity, and

host defense. Alcohol Clin Exp Res 33:220–232.

Szalay G, Sauter M, Haberland M, Zuegel U, Steinmeyer A, Kandolf R,

Klingel K (2009) Osteopontin. A fibrosis-related marker molecule in cardiac

remodeling of enterovirus myocarditis in the susceptible host. Circ Res

104(7): 851–859.

Taylor IW, Wrana JL (2008) SnapShot: the TGFbeta pathway interactome.

Cell 133:378.

Thorne M, Tschopp J (2001) Regulation of lymphocyte proliferation and

death by FLIP. Nat Rev Immunol 1:50–58.

Tilg H, Diehl AM (2000) Cytokines in alcoholic and nonalcoholic steatohepa-

titis. N Engl J Med 343:1467–1476.

Tommaso M, Elisabetta C, Calogero S, Andrea G (2008) Alcohol induced

hepatic fibrosis: role of acetaldehyde. Mol Aspects Med 29:17–21.

ALCOHOL, SIGNALING, AND ECM TURNOVER 17

Tsai KS, Grayson MH (2008) Pulmonary defense mechanisms against pneu-

monia and sepsis. Curr Opin PulmMed 14:260–265.

Tsukamoto H (1993) Oxidative stress, antioxidants and alcoholic liver fibro-

genesis. Alcohol Clin Exp Res 10:465–467.

Tuma DJ, Casey CA (2003) Dangerous byproducts of alcohol breakdown –

focus on adducts. Alcohol Res Health 27:285–290.

Uesugi T, Froh M, Arteel GE, Bradford BU, Thurman RG (2001) Toll-like

receptor 4 is involved in the mechanism of early alcohol-induced liver injury

in mice. Hepatology 34:101–108.

Vonlaufen A, Joshi S, Qu C, Phillips PA, Xu ZH, Parker N (2008) Pancreatic

stellate cells: partners in crime with pancreatic cancer cells. Cancer Res

68:2085–2093.

Vonlaufen A, Joshi S, Qu C, Phillips PA, Toi CS, Xu ZH, Pirola R, Wilson J,

Goldstein D, Apte MV (2007) Bacterial endotoxin - a trigger factor for alco-

holic pancreatitis? Findings of a novel physiologically relevant model. Gas-

troenterology 133:1293–1303.

Walker JEJ, Odden AR, Jeyaseelan S, Zhang P, Bagby GJ, Nelson S, Happel

KI (2009) Ethanol exposure impairs LPS-induced pulmonary LIX expres-

sion: alveolar epithelial cell dysfunction as a consequence of acute intoxica-

tion. Alcohol Clin Exp Res 33:357–365.

Wang D, Liu C, Chung J (1998b) Chronic alcohol intake reduces retinoic acid

concentration and enhances AP-1 (c-jun and c-fos) expression in rat liver.

Hepatology 28:744–750.

Wang CY, Mayo MW, Korneluk RG, Goeddel DV, Baldwin AS (1998a)

NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1

and c-IAP2 to suppress caspase-8 activation. Science 281:1680–1683.

Weng HL, Ciuclan L, Liu Y, Hamzavi J, Godoy P, Gaitantzi H, Kanzler S,

Heuchel R, Ueberham U, Gebhardt R, Breitkopf K, Dooley S (2007) Pro-

fibrogenic transforming growth factor-beta ⁄ activin receptor-like kinase 5

signaling via connective tissue growth factor expression in hepatocytes. Hep-

atology 46:1257–1270.

Wheeler MD, Yamashima S, Froh M, Rusyn I, Thurman RG (2000) Adeno-

virus can transduce Kupffer cells: role of oxidants in NFkB activation and

TNFa production. J Leuk Biol 69:622–630.

Wheeler MD, Thurman RG (2003) Up-regulation of CD14 in liver caused by

acute ethanol involves oxidant-dependent AP-1 pathway. J Biol Chem

278:8435–8441.

WHO (2006) World Health Organization Global Status Report on Alcohol

2004. substance_abuse ⁄publications ⁄ global_status_report_2004_overview.

Woods A, Pala D, Kennedy L, McLean S, Rockel JS, Wang G, Leask A,

Beier F (2008) Rac1 signaling regulates CTGF ⁄CCN2 gene expression via

TGFbeta ⁄Smad signaling in chondrocytes. Osteoarthritis Cartilage 17:406–

413.

Wyatt TA, Gentry-Nielsen MJ, Pavlik JA, Sisson JH (2004) Desensitization of

PKA-stimulated ciliary beat frequency in an ethanol-fed rat model of ciga-

rette smoke exposure. Alcohol Clin Exp Res 28:998–1004.

Xu Q, Schleter JB, Cox KM, de Villiers WJS, D’Souza El-Guindy NB (2007)

Alcohol-induced inflammation makes the lungs refractory to bacterial

insult. Alcohol Alcohol 42(Suppl. 1):i43.

Yamashina S, Wheeler MD, Rusyn I, Ikejimaa K, Satob N, Thurman RG

(2000) Tolerance and sensitization to endotoxin in Kupffer cells caused by

acute ethanol involve interleukin-1 receptor-associated kinase. Biochem Bio-

phys Res Commun 277:686–690.

YeP,GarveyPB,ZhangP,Nelson S, BagbyG, SummerWR, Schwarzenberger

P, Shellito JE, Kolls JK (2001) Interleukin-17 and lung host defense

against Klebsiella pneumoniae infection. Am J Respir Cell Mol Biol

25:335–340.

Yin M, Bradford BU, Wheeler MD, Takehiko U, Froh M, Goyert SM,

Thurman RG (2001) Reduced early alcohol-induced liver injury in CD14-

deficient mice. J Immunol 166:4737–4742.

Zaridze D, Brennan P, Boreham J, Boroda A, Karpov R, Lazarev A,

Konobeevskaya I, Igitov V, Terechova T, Boffetta P, Peto R (2009) Alcohol

and cause-specific mortality in Russia: a retrospective case-control study of

48,557 adult deaths. Lancet 373:2201–2214.

Zeisberg M, Yang C, Martino M, Duncan MB, Rieder F, Tanjore H, Kalluri

R (2007) Fibroblasts derive from hepatocytes in liver fibrosis via epithelial

to mesenchymal transition. J Biol Chem 282:23337–23347.

Zhang S, Fei T, Zhang L, Zhang R, Chen F, Ning Y, Han Y, Feng XH,Meng

A, Chen YG (2007) Smad7 antagonizes transforming growth factor beta

signaling in the nucleus by interfering with functional Smad-DNA complex

formation. Mol Cell Biol 27:4488–4499.

Zhang H, Ye Q, Ren N, Zhao L, Wang Y, Wu X, Sun H, Wang L, Zhang B,

Liu Y, Tang Z, Qin L (2006) The prognostic significance of preoperative

plasma levels of osteopontin in patients with hepatocellular carcinoma.

J Cancer Res Clin Oncol 132:709–717.

Zhuge J, Cederbaum AI (2006) Increased toxicity by transforming growth

factor-beta 1 in liver cells overexpressing CYP2E1. Free Radic Biol Med

41:1100–1112.

18 SETH ET AL.