LIVER FAILURE/CIRRHOSIS/PORTAL HYPERTENSION

Occludin Is Regulated by Epidermal Growth FactorReceptor Activation in Brain Endothelial Cells and

Brains of Mice with Acute Liver FailureFeng Chen,1 Tomohide Hori,1 Norifumi Ohashi,1 Ann-Marie Baine,1 Christopher B. Eckman,1

and Justin H. Nguyen2

Mechanisms of brain edema in acute liver failure (ALF) are not completely understood. Werecently demonstrated that matrix metalloproteinase 9 (MMP-9) induces significant altera-tions to occludin in brain endothelial cells in vitro and in brains of mice with experimentalALF (Hepatology 2009;50:1914). In this study we show that MMP-9-induced transactiva-tion of epidermal growth factor receptor (EGFR) and p38 MAPK/NFjB (mitogen-activatedprotein kinase/nuclear factor-kappa B) signals participate in regulating brain endothelialoccludin level. Mouse brain endothelial bEnd3 cells were exposed to MMP-9 or p38 MAPKup-regulation in the presence and absence of EGFR inhibitor, p38 MAPK inhibitor, NFjBinhibitor, and/or appropriate small interfering RNA. Reverse-transcription polymerase chainreaction (RT-PCR) and western blotting were used for messenger RNA and protein expres-sion analyses. Immunohistochemical staining and confocal microscopy were used to demon-strate cellular EGFR activation. Intraperitoneal azoxymethane was use to induce ALF inmice. Brains of comatose ALF mice were processed for histological and biochemical analyses.When bEnd3 cells were exposed to MMP-9, EGFR was significantly transactivated, followedby p38 MAPK activation, I-kappa B alpha (IjBa) degradation, NFjB activation, and sup-pression of occludin synthesis and expression. Similar EGFR activation and p38 MAPK/NFjB activation were found in the brains of ALF mice, and these changes were attenuatedwith GM6001 treatment. Conclusion: EGFR activation with p38 MAPK/NFjB signalingcontributes to the regulation of tight junction integrity in ALF. EGFR activation may thusplay an important role in vasogenic brain edema in ALF. (HEPATOLOGY 2011;53:1294-1305)

Tight junction (TJ) proteins regulate paracellularpermeability in various organs, particularlyin the brain neurovascular unit.1 In brain

capillaries the endothelial cell (EC) spreads itself over

the entire capillary basal lamina with the two plasma-lemmal surfaces aligning to form the TJ. The TJ pro-teins, including occludin, claudin-5, junctional associ-ated molecules, and their intracellular accessory factorszona occludins (ZO), seal the paracellular space. Col-lectively, the EC and its TJ, basal lamina, and associ-ated astrocyte end-feet form the blood-brain barrier(BBB) that tightly regulates what enters and exits theneurovascular unit of the brain.2

TJ proteins, particularly occludin and claudin-5, areimportant in the paracellular barrier function of theBBB and their roles have been described in variouspathological conditions.3-5 Occludin is a tetraspanmembrane protein with two extracellular loops withinthe TJ and with the amino- and carboxy-terminalchains in the cytoplasm. The C-terminal domain bindsto ZO-1 and -2, which serves as the link betweenoccludin and the cytoskeleton.6 Claudin-5 has a simi-lar distribution. Although the barrier function is typi-cally compromised by structural breakdown of theBBB, recent evidence has suggested that subtle

Abbreviations: ALF, acute liver failure; BBB, blood-brain barrier; EC,endothelial cell; EGFR, epidermal growth factor receptor; IjBa, I-kappa Balpha; MAPK, mitogen-activated protein kinase; MMP, matrixmetalloproteinase; NFjB, nuclear factor-kappa B; RT-PCR, reversetranscription-polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TJ, tight junction.From the 1Department of Neuroscience; and 2Division of Transplant Surgery,

Department of Transplantation, Mayo Clinic, Jacksonville, FL.Received July 20, 2010; accepted December 21, 2010.

Supported by the Deason Foundation, Sandra and Eugene Davenport, Mayo

Clinic CD CRT-II, AHA 0655589B, and R01NS051646-01A2 (to J.H.N.).

Address reprint requests to: Justin H. Nguyen, M.D., Division of Transplant

Surgery, Department of Transplantation, Mayo Clinic, 4500 San Pablo Road,

Jacksonville, FL 32224. E-mail: nguyen.justin@mayo.edu; fax: 904-956-3359.

CopyrightVC 2011 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.24161Potential conflict of interest: Nothing to report.

1294

alterations in either occludin or claudin-5—withoutobvious structural changes—can result in selective per-meability to small molecules.7-9 These findings supportthe concept that vasogenic edema might result from asubtle modification in TJ composition.10

In acute liver failure (ALF), brain edema is lethaland remains a major determinant of patient sur-vival.11,12 However, the exact alterations in BBB integ-rity that lead to brain edema in ALF are not known.In 2006 we reported that specific monoclonal antibod-ies against matrix metalloproteinase-9 (MMP-9)attenuate brain extravasation and edema in ALFmice.13 We recently showed that MMP-9 significantlyalters the TJ proteins, particularly occludin, in brainECs in vitro and in brains of mice that have experi-mentally induced ALF.5 However, the signal transduc-tions associated with MMP-9 that mediate the altera-tions in occludin remain unknown.The role of epidermal growth factor receptor (EGFR)

in cancer development and treatment is well known.14-16

EGFR belongs to the ErbB family of receptor tyrosinekinases. Upon ligand stimulation, they dimerize, anddimerization is then followed by receptor internalizationand autophosphorylation of the intracytoplasmic EGFRtyrosine kinase domains, which serve as binding sites forrecruiting signal transducers and activators of the intracel-lular signal transduction cascade. Recently, EGFR wasimplicated in the regulation of cellular barrier function.17

EGFR has also been shown to participate in microvascu-lar injury in diabetes,18 lung injury,19,20 and intestinalpermeability.21,22

Ligation of EGFR activates the mitogen-activatedprotein kinase (MAPK) cascades.23,24 MAPKs havebeen implicated in endothelial paracellular permeabil-ity associated with oxidative insults25-27 and with alter-ations of TJ proteins28; thus, MAPKs are important inmaintaining cellular and TJ integrity.29 Previously, weobserved that p38 MAPK is up-regulated in isolatedmicrovessels from the brains of ALF mice.30 However,the role of p38 MAPK and EGFR in BBB permeabil-ity in ALF has not been explored.In this study we investigated the role of p38MAPK/

NFjB signaling after EGFR transactivation by MMP-9 in altering the TJ element occludin in brain ECs invitro and in brains of mice with ALF.

Materials and Methods

The mouse brain endothelial cell line, bEnd3, was pur-chased from the American Type Culture Collection(CRL-2299, Manassas, VA). We purchased transfection-

ready green fluorescent protein (GFP)-tagged humanMMP-9 complementary DNA (cDNA) (RG202872)and p38 MAPK mouse cDNA (MC200120) from Ori-Gene (Rockville, MD); anti-phospho p38 MAPK(sc9211), anti-p38 MAPK (sc9212), anti-MMP-9, anti-EGFR, and anti-phospho-Tyr EGFR antibodies (sc-13520) from Santa Cruz Biotechnology (Santa Cruz,CA); rabbit anti-claudin (Zy34-1600), anti-occludin(Zy71-1500), and anti-ZO-1 (Zy40-2300) from Invitro-gen-Zymed Laboratories (Carlsbad, CA); and anti-ZO-2from BD Transduction Laboratories (San Jose, CA). Weobtained nontargeted SignalSilence control small interfer-ing RNA (siRNA) (Cat. No. 6568), and p38 MAPKsiRNA (Cat. No. 6564), EGFR siRNA (Cat. No. 6481)from Cell Signal Tech (Danvers, MA); anti-I-kappa Balpha (IjBa) (Cat. No. I0505) and p38 MAPK inhibitorSB203580 (Cat. No. S8307), MEK1/2 inhibitor PD98059 (Cat. No. p215) from Sigma-Aldrich; NFjB in-hibitor, MMP inhibitor GM6001, and EGFR inhibitorAG1478 from Calbiochem (San Diego, CA).RNA Extraction and Reverse-Transcription Poly-

merase Chain Reaction (RT-PCR) Analysis. TotalRNA was extracted from bEnd3 cells using PurelinkRNA Mini Kit (12183-018A, Invitrogen). RNA wastranscribed into single-stranded DNA by SuperScriptIII First Strand reverse transcriptase (18080-051, Invi-trogen). The yielded cDNA was used as a template.PCRs for MMP-9, occludin, ZO-1, ZO-2, and clau-din-5 were performed using Taq PCR Master Mix kit(201443 Qiagen).The primers were, for MMP-9, 50-AGACGACATA

GACGGCATCC-30 (sense) and 50-GCCCTGGATCTCAGCAATAG-30 (antisense); for occludin (220 base-pairs [bp]), 50-CACACTTGCTTGGGACAGAGG-30

(sense) and 50-TGAGCCGTACATAGATCCAGGAGC-30 9 (antisense); for ZO-1 (290 bp), 50-AGGCGCAGCTCCACGGGCTTCAGGAACTTG-30 (sense)and 50-CAGAAGCAGAAGTAGGGAGAGGTGCCGATC-30 (antisense); for claudin-5 (200 bp), 50-GCTGGCGCTGGTGGCACTCTTTGT (sense) and 50-GGCGAACCAGCAGAGCGGCAC-30 (antisense); andfor ZO-2 (240 bp), 50-TCAAACCCCTCATCCGCTGCTGGTA-30 (sense) and 50-AGTGTTCCGTTTCAATGTCTCTTTTAC-30 (antisense). PCR condi-tions were 40 cycles at 94�C for 30 seconds, 60�C for30 seconds, and 72�C for 1 minute, and a final cycleof 72�C for 1 minute. The 1.8% agarose gel and Mul-tiDoc-It digital imaging System (UVP, Upland, CA)were used.Overexpressing MMP-9 and p38 MAPK in bEnd3

Cells. The bEnd3 cells were grown to confluence on100-cm2 culture plates in Dulbecco’s modified Eagle’s

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medium with 4.5 g/L glucose, 3.7 g/L sodium bicar-bonate, 4 mM glutamine, 10% fetal bovine serum,100 U/mL penicillin, and 100 g/mL streptomycin.Cells were transfected with GFP-tagged MMP-9 andp38 MAPK cDNA using Lipofectamine 2000 (Invitro-gen). Cells transfected with expression vector served ascontrols. Transfection efficiency was monitored by flu-orescence microscopy.

Preparation of Cell Extracts and Western BlottingAnalysis. Cells were washed with phosphate-bufferedsaline (PBS) and collected into 1 mL of lysate buffer(50 mM Tris-HCl pH 7.3, 150 mM NaCl, 3 mMMgCl, 1 mM DTT, 1 mM EDTA, 1 mM EGTA,1.0% Triton X-100), and supplemented with proteaseand phosphatase inhibitors. The extraction supernatantwas collected and 30 lg of protein from each samplewas resolved on 4%-20% sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) gels,transferred, and immunoblotted onto nitrocellulosemembrane. Anti-claudin-5, anti-occludin, anti-ZO-1,anti-ZO-2, anti-phospho p38 MAPK, anti-p38MAPK, anti-IjBa, and anti-GAPDH antibodies wereused as primary antibodies.

Immunoprecipitation. The cells were lysed withice-cold immunoprecipitation buffer (50 mM HEPES,pH 7.5, 50 mM NaCl, 1% Triton X-100, 1 mMEDTA, 10 mM sodium pyrophosphate, 1 mMNa3VO4, 30 mM 2-(p-nitrophenyl) phosphate, 100mM NaF, 10% glycerol, 1.5 mM MgCl2, and proteaseinhibitor cocktail). Lysates were centrifuged at 14,000gfor 5 minutes and the supernatant was immunopreci-pitated with anti-EGFR and Dynabeads M-280 mag-netic protein bead separation system (Invitrogen) over-night at 4�C.

Brains from Mice with ALF. ALF was inducedwith an intraperitoneal (ip) injection with azoxyme-thane (AOM) (Sigma-Aldrich) as described in our pre-vious report.13 Control mice received saline. At 12hours after AOM injection, ALF mice received 2 mg/kg of GM6001 or vehicle by way of ip injection. Con-trol mice received vehicle. A heating pad was used tomaintain body temperature at 37�C. The use of ani-mals was institutionally approved in accordance withthe National Institutes of Health Guide for the Careand Use of Laboratory Animals. The progression of he-patic encephalopathy was determined clinically.13 Atthe comatose stages the study mice were killed andtheir brains were removed. Hemibrains were homoge-nized at 150 mg tissue/mL of lysate buffer and proc-essed for analysis.

ResultsOccludin Alterations in bEnd3 Cells Exposed to

MMP-9 Are Mediated by p38 MAPK. We recentlyreported that MMP-9 disrupts TJ proteins in mousebrain EC in vitro and in brains of mice with ALF.5

We also observed a significant increase in p38 MAPKactivation in ALF mice.30 Thus, we investigatedwhether p38 MAPK contributes to occludin alterationsin bEnd3 cells exposed to MMP-9. Similar to themethods used in our previous report,5 we overex-pressed MMP-9 in bEnd3 cells. We observed thatMMP-9 significantly up-regulated p38 MAPK phos-phorylation, i.e., activation, as well as decreased theamount of occludin and its associate ZO-1 (Fig. 1A).The decrease of occludin and ZO-1 was reversed bythe p38 MAPK-specific inhibitor SB203580 but notby the p42/44 MAPK inhibitor PD98059 (notshown). In contrast, alterations in other TJ proteins(claudin-5 and ZO-2) were not reversed by SB20358(not shown). To further corroborate the effect of p38MAPK, we inhibited p38 MAPK expression by usingsiRNA. We found that blocking p38 MAPK up-regu-lation effectively mitigated the reduction of occludinand ZO-1 (Fig. 1B,C). These results showed that p38MAPK is important for occludin regulation.

Up-Regulating p38 MAPK Leads to IjBa Degra-dation, NFjB Activation, and Occludin Decrease inbEnd3 Cells. To investigate the effect of p38 MAPKon the transcription and expression of occludin, wetransfected bEnd3 cells with p38 MAPK cDNA for18 hours, then treated the cells with or withoutSB203580 at 1 lM for 3 hours. Using RT-PCR,we found that overexpressing p38 MAPK resulted insignificant suppression of messenger RNA (mRNA)levels of occludin and ZO-1 but not claudin-5 andZO-2 (Fig. 2A,C). The effect was reversed withSB203580. Importantly, up-regulating p38 MAPKdid not change the MMP-9 mRNA level in thebrain EC (Fig. 2A,C).With western blotting, we found that occludin and

ZO-1 were significantly reduced by p38 MAPK up-regulation, and the reduction was partly restored withthe p38 MAPK inhibitor SB203580 (Fig. 2B,D). Incontrast, claudin-5 and ZO-2 were not affected.Because p38 MAPK is associated with IjBa degra-

dation and NFjB activation,31,32 we investigated thestatus of IjBa protein and NFjB in bEnd3 cells inwhich p38 MAPK cDNA was overexpressed. ThebEnd3 cells were transfected with p38 MAPK cDNAthen treated with 10 nM of NFjB activation inhibitor.We found that p38 MAPK up-regulation reduced

1296 CHEN ET AL. HEPATOLOGY, April 2011

IjBa, occludin, and ZO-1 levels. Importantly, wedemonstrated that p38 MAPK activation induces IjBadegradation, which was reversed by treatment with thep38 MAPK inhibitor SB203580 (Fig. 2E). Moreover,we found that after administering NFjB inhibitor thedegradation of IjBa became enhanced in p38 MAPK-up-regulated cells, and the decrease of occludin andZO-1 was reversed (Fig. 2E). Overall, our results indi-cate that p38 MAPK induces the degradation of IjBa,which leads to the release of NFjB activation that reg-ulates occludin and ZO-1 expression in mouse brainEC cells.MMP-9-Induced EGFR Transactivation Leads

to p38 MAPK Signaling and Occludin Altera-tions. MMP-9 has been shown to transactivateEGFR.33,34 Ligation of EGFR results in the activationof MAPK cascades23 and potentially in modulating TJ

proteins.35 We thus speculated that EGFR activationmight be important in MMP-9-induced alteration ofoccludin.We first confirmed whether MMP-9 could trans-

activate EGFR in bEnd3 cells. As shown in Fig.3A,B, when bEnd3 cells were exposed to MMP-9,EGFR was phosphorylated (p-TyrEGFR), i.e., acti-vated, as determined by western blotting. We pre-treated the bEnd3 cells with GM6001 (an MMP-9inhibitor) then overexpressed MMP-9; we foundthat MMP-9 inhibition attenuated the EGFR activa-tion. Using confocal microscopy, we corroboratedthe findings that activated EGFR was up-regulatedin the bEnd3 cells and that EGFR activation wasprevented with GM6001 (Fig. 3C). These findingsconfirmed that EGFR is transactivated by MMP-9in bEnd3 cells.

Fig. 1. Phospho-p38 MAPK (p-p38), p38 MAPK, occludin, and ZO-1 in bEnd3 cells in which MMP-9 was up-regulated in the presence orabsence of (A) p38 MAPK inhibitor SB203580 (1 lM) or (B) p38 MAPK siRNA. (C) Densitometric analysis of p-p38, p38, occludin, and ZO-1levels; GAPDH served as control. *P < 0.05.

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We then determined whether EGFR would directlyinfluence the p38 MAPK activation with subsequentoccludin alterations. As shown in Fig. 4, the specificEGFR inhibitor AG1478 significantly reduced the p38MAPK activation and occludin loss in a dose-depend-ent manner. Importantly, p38 MAPK activation andsuppression of occludin were similarly blocked byEGFR siRNA (Fig. 4). Overall, EGFR inhibitionwith AG1478 or EGFR deletion with siRNA blockedp38 MAPK phosphorylation and restored occludin inbrain EC.

Activated EGFR, p38 MAPK Activation, IjBaReduction, and NFjB Activation Are Associatedwith Occludin Alterations in the Brains of ALFMice. Previously, we demonstrated that in ALF mice,occludin was significantly perturbed.5 In the presentstudy, we assessed the role of EGFR activation andits associated p38 MAPK/NFjB signaling in brainsof ALF mice. We showed by western blotting thatoccludin was significantly altered in the brains of

ALF mice and the alteration was restored withGM6001 treatment. These results are consistent withour previous report.5 Importantly, we observed EGFRactivation along with p38 MAPK activation and IjBadegradation in the brains of ALF mice (Fig. 5A,B).With confocal microscopy, we substantiated that signif-icant EGFR activation occurred in brains of ALF miceand that EGFR activation was attenuated withGM6001 treatment (Fig. 5C). In contrast, brains ofnormal control mice showed no EGFR activation.We observed spontaneous hypothermia in AOM-

induced ALF mice (Fig. 6A). With heat support, thebody temperature of AOM mice was maintained atnormothermia. Treatment with GM6001 did not alterthe body temperature of the study mice (Fig. 6B). Asshown in Fig. 6C,D, we observed that the occludinalteration in AOM-induced ALF mice was independ-ent of body temperature and was reversed withGM6001. In addition, to investigate whether theoccludin alteration occurs in other model of ALF, we

Fig. 2. (A) RT-PCR analysis ofMMP-9, occludin, claudin-5, ZO-1,and ZO-2 in bEnd3 cells in whichp38 MAPK was up-regulated in thepresence or absence of SB203580.(B) Western blot analysis of occlu-din, claudin-5, ZO-1, ZO-2, andIjBa in bEnd3 cells in which p38MAPK was up-regulated in the pres-ence or absence of SB203580. (C)Densitometric measurements for(A). (D) Densitometric measure-ments for (B). (E) Western blot anal-ysis of occludin, ZO-1, and IjBa inbEnd3 cells in which p38 MAPKwas up-regulated in the presence orabsence of the inhibitor of NFjBactivation; GAPDH served as con-trol. *P < 0.01.

1298 CHEN ET AL. HEPATOLOGY, April 2011

employed a well-established model using tumor necro-sis factor-alpha (TNFa) and D-galactosamine (Gal).36

We found that occludin was decreased in brains of theGal/TNFa-induced ALF mice and that the occludinalteration was reversed with GM6001 treatment andwas independent of body temperature (Fig. 6E,F).

These results from AOM-induced ALF mice areconsistent with the findings in vitro, suggesting thatMMP-9 induced EGFR transactivation and that p38MAPK/NFjB signaling plays an important role in reg-ulating BBB TJ proteins in ALF. Collectively, our find-ings suggest that in addition to its direct proteolytic

Fig. 3. EGFR activation in bEnd3 cells that were treated with human recombinant MMP-9 (100 ng/mL for 6 hours) in the presence or absenceof the MMP-9 inhibitor GM6001 (100 nM). (A) Cell lysates were immunoprecipitated with anti-EGFR antibody and analyzed by immunoblottingwith anti-p-TyrEGFR and anti-EGFR antibodies. (B) Densitometric analysis of the p-TyrEGFR, **P < 0.01. (C) For confocal microscopy, bEnd3 cellswere fixed and immunostained for p-TyrEGFR (green color) and for nuclei with DAPI (blue).

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action,5 MMP-9 influences the TJ protein occludin inan indirect way through the following series of steps:first by transactivating EGFR on the bEnd3 cellularsurface, second by up-regulating p38 MAPK, third byIjBa degradation and NFjB activation, and finally bysuppressing occludin expression. This model is sum-marized in Fig. 7.

Discussion

In this study we have shown that MMP-9 transacti-vates EGFR in brain microvascular ECs with subse-quent p38 MAPK/NFjB signaling, resulting in sup-pressed transcription/translation and protein expressionof the TJ protein occludin. These effects were attenu-ated with specific inhibition of EGFR in brain ECs invitro. Moreover, we observed EGFR activation, p38MAPK activation, and the loss of occludin in brains ofmice with experimentally induced ALF. Together theseresults suggest that EGFR plays a role in activating thepathobiology of brain injury in ALF.Brain edema in ALF is unique. It occurs in the co-

matose stages of encephalopathy in ALF. The onset ofencephalopathy in ALF patients presages impendingbrain edema and a lethal course of the disease. Onceliver failure is resolved, either by liver transplantationor by spontaneous recovery of the injured liver, thebrain edema is resolved. However, if the brain edemais inadequately controlled, it will ultimately lead toherniation and brain death. Even with significant brainedema, both light and electron microscopic evaluationsof these brains reveal that the BBB and its TJs remainrelatively intact. However, there are certain subtle

changes, including fine perturbations at the endothelialcellular plasma membrane and thickening of the basallamina.37 In the absence of obvious structural break-down of the BBB, the prominent and consistent swel-ling of astrocytic foot processes has led to the domi-nant theory that cytotoxic mechanisms cause brainedema in ALF.38 Although vasogenic elements havebeen implicated,39,40 evidence for vasogenic edema inALF has been lacking.Increasing evidence has suggested that even with a

relatively intact BBB, subtle alterations in TJ composi-tion can result in highly selective permeability to smallmolecules, such as water and ammonia. In mice thatare selectively deficient in claudin-5, a component ofTJ proteins, the BBB and TJ appear intact under elec-tron microscopic examination. However, these micehave increased permeability to molecules that are lessthan 800 Da.8 Similarly, when occludin at the TJ istargeted with a specific peptide or is modified by pro-teolysis, TJ permeability is significantly increased with-out any obvious structural change.7,9,41 Collectively,these data indicate that altered permeability of theBBB can result from very subtle changes in the BBBand/or TJ composition.We recently observed that there are significant bio-

chemical alterations in occludin, claudin-5, and ZO-1in brains of mice with experimentally induced ALF5

and that these changes were attenuated when MMP-9was inhibited.5,13 We observed similar findings whenmurine brain EC were exposed to MMP-9 in vitro.5

However, TJs make up only a small part of the braincapillary surface area. The endothelial cellular plasma-lemma interacts with MMP-9 or other inciting factorswithin the capillary circulation to a greater extent thanthe TJs. Therefore, we speculated that endothelial cel-lular interactions with MMP-9 by way of surfacereceptors might play a major role in regulating theBBB integrity in ALF.EGFR is well known for its role in cancer invasion

and metastasis.15 EGFR is essential in epithelial cellu-lar integrity in response to injury.17 Recently, EGFRwas shown to participate in altered microvascular per-meability in intestinal disorders,21,22 lung injury,19,20

and diabetic vascular damage.18

In this study we investigated the role of EGFR acti-vation and associated signaling in occludin regulation.We found that MMP-9 transactivates EGFR in brainECs, which activates p38 MAPK, decreases IjBa, andleads to the activation of NFjB with subsequent sup-pression of the transcription and translation of occlu-din at the TJs. In a mouse model of ALF that recapit-ulates the human form of ALF, we observed similar

Fig. 4. p38 MAPK activation and occludin expression in bEnd3 cellsthat were exposed to human recombinant MMP-9 (100 ng/mL for6 hours) in the presence or absence of the EGFR inhibitor AG1478(0.1 lM and 1 lM, respectively, for 3 hours), or EGFR siRNA transfection.

1300 CHEN ET AL. HEPATOLOGY, April 2011

effects of EGFR activation and signaling on changes inoccludin expression.13,42 These results suggest thatEGFR activation and p38 MAPK/NFjB signalingplay important roles in regulating the TJ integrity inALF.

The effects of MMP-9 on BBB permeability in ALFmight thus involve more than one pathway. Directdegradation of the extracellular components of occlu-din and other TJ proteins is an important element.However, because the TJ architecture does not change

Fig. 5. EGFR activation and p38 MAPK/NFjB activation in brains of ALF mice. (A) Western blot of brain samples from control mice, andAOM-induced ALF mice treated with vehicle or with GM6001. (B) Densitometric quantification of p38 MAPK, IjBa, and p-TyrEGFR expression inthe brains of control mice and ALF mice treated with and without GM6001. Values are expressed as the mean 6 standard error of the mean(SEM) with t test. (n ¼ 3, *P < 0.05). (C) Confocal microscopic evaluation of the brains of control versus ALF mice that were treated with vehi-cle or GM6001. Activated EGFR is seen as green and the nucleus as blue.

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in ALF, the exposure of occludin is limited. It followsthat MMP-9’s direct action on occludin, being mostapical and closest to the capillary lumen, would be re-stricted.5 Because TJs make up a small portion of theBBB, quantitatively it might appear that MMP-9’seffects on the EC surface could be more important.Although we do not know which path, i.e., transcellu-lar or paracellular, is the more important regulator orcontributor to the overall BBB dysfunction in ALF, theresults from this study broaden the impact of MMP-9on BBB integrity. Furthermore, EGFR might mediate

a transcellular transport, and its cascade of intracellularsignals could serve to fine tune the overall BBB integ-rity. Fine regulation of the TJ composition by way ofthe EGFR cascade may represent a subtle modulationof the BBB in ALF.In this study we limited our focus to EGFR transac-

tivation with MMP-9. However, MMP-2 and otherMMPs, TNFa, and IL-1 may contribute to the overalldisease process.43,44 It should be noted that occludinalteration was not observed in AOM-treated mice in arecent report.45 The observed difference remains to be

Fig. 6. (A) Body temperature of AOM-induced mice showed a spontaneous hypothermia without (w/o) heating pad. Heating support provideda steady normothermia in the control and ALF mice. (B) Treatment with GM6001 did not alter the body temperature of the study mice. The con-trol, AOM, and AOMþGM6001 mice were maintained at 37�C (n ¼ 8 each group). There was no difference in body temperature among studygroups. (C) In the AOM-induced ALF mice with normothermia, occludin was observed to decrease in the AOM mice and was reversed to normallevel with GM6001. (D) Histogram of occludin alteration in the control, AOM, and AOM-induced ALF mice treated with GM6001 (n ¼ 8 in eachgroup; **P < 0.001). (E) In mice that had ALF induced with D-galactosamine and TNF at normothermia, occludin was observed to decrease inthe ALF mice and was reversed to normal level with GM6001. (F) Histogram of occludin alteration in the control, Gal/TNF, and Gal/TNF-inducedALF mice treated with GM6001 (n ¼ 9 in each group; **P < 0.001).

1302 CHEN ET AL. HEPATOLOGY, April 2011

investigated. In contrast, occludin is shown to be sig-nificantly altered in mice with ALF that is inducedwith D-galactosamine and liposaccharide.43 Similarly,we observed significant occludin perturbations in thebrains of mice that had Gal/TNFa-induced ALF, sug-gesting that occludin alteration is independent ofinduction agents. Moreover, a similar occludin altera-tion has also been shown in rats with portocaval shuntand hepatic artery ligation and the occludin reductionwas not reversed with hypothermia, consistent withour observation that occludin regulation is independ-ent of hypothermia.46 Collectively, these observationssuggest that occludin may be the common link in thebrain injury associated with ALF.Because both vasogenic and cytotoxic mechanisms

are implicated in the pathogenesis of brain edema inALF, which mechanism precedes and which is more

important in the onset of edema formation remainunresolved. Earlier evidence suggested that increasedpermeability to the small molecules precedes encephal-opathy and edema.47 However, the cytotoxic pathwaycould be the leading event. It is most likely that bothvasogenic and cytotoxic mechanisms are involved. Fur-ther study is required to elucidate the extent and orderof involvement of the vasogenic and cytotoxic mecha-nisms in ALF.In conclusion, we have shown that EGFR activation

with p38MAPK/NFjB signal transduction contributesto the regulation of BBB TJ integrity in ALF. Thesefindings not only provide evidence for vasogenic mech-anisms in the pathogenesis of brain edema, but alsoprovide a potential target for therapeutic measures toachieve effective control of the development and pro-gression of brain edema in ALF.

Fig. 7. (A) Representation of aneurovascular unit consisting of acapillary endothelial cell and itstight junction, basal lamina, andastrocytic endfoot in a normal brain.(B) Neurovascular unit in brain of asubject with ALF showing a subtlealteration in the TJ protein occludinwith EGFR being transactivated andconsequent p38 MAPK/NFjB acti-vation. Perturbations in endothelialcellular plasmalemma are sug-gested by the interrupted lines, andswollen astrocytic endfoot by sizeexpansion. EGFR, epidermal growthfactor receptor; p-p38, phospho-p38 MAPK; MMP-9, matrix metallo-proteinase-9; NFjB, nuclear factor-kappa B; IjBa, I-kappa B alpha;JAM, junction associated molecules.

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Acknowledgment: The authors thank Kathleen Nor-ton and Lisa Maroski for editorial assistance.

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