c-jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

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1 Submit Mnuscript: Help Desk: DOI: /wjg.v22.i World J Gstroenterol 2016 July 28; 22(28): ISSN (print) ISSN (online) 2016 Bishideng Publishing Group Inc. All rights reserved. Bsic Study ORIGINAL ARTICLE c-jun N-terminl kinse-medited expression enhnces heptocyte lipopoptosis nd promotes heptocyte bllooning Akiko Suzuki, Keisuke Kkisk, Yuji Suzuki, Ting Wng, Ysuhiro Tkikw Akiko Suzuki, Keisuke Kkisk, Yuji Suzuki, Ting Wng, Ysuhiro Tkikw, Division of Heptology, Deprtment of Internl Medicine, Iwte Medicl University, Moriok , Jpn Author contributions: Suzuki A performed in vivo nd in vitro studies nd wrote the pper; Kkisk K designed the experiments. Suzuki Y nd Wng T nlyzed the dt; Tkikw Y supervised the study nd revised the pper; ll uthors drfted the rticle nd mde criticl revisions relted to the intellectul content of the mnuscript, nd pproved the finl version of the rticle to be published. Supported by KAKENHI Grnt, No. 16K Institutionl niml cre nd use committee sttement: All of the niml experiments were pproved by the Animl Cre nd Use Committee of Iwte Medicl University (Moriok, Jpn; ). Conflict-of-interest sttement: There is no conflict-of-interest. Dt shring sttement: No dditionl dt re vilble. Open-Access: This rticle is n open-ccess rticle which ws selected by n in-house editor nd fully peer-reviewed by externl reviewers. It is distributed in ccordnce with the Cretive Commons Attribution Non Commercil (CC BY-NC 4.0) license, which permits others to distribute, remix, dpt, build upon this work non-commercilly, nd license their derivtive works on different terms, provided the originl work is properly cited nd the use is non-commercil. See: licenses/by-nc/4.0/ Mnuscript Source: Invited mnuscript Correspondence to: Keisuke Kkisk, MD, PhD, Assistnt Professor, Division of Heptology, Deprtment of Internl Medicine, Iwte Medicl University, 19-1 Uchimru, Iwte, Moriok , Jpn. keikki@iwte-med.c.jp Telephone: Fx: Received: April 5, 2016 Peer-review strted: April 6, 2016 First decision: My 12, 2016 Revised: My 24, 2016 Accepted: June 13, 2016 Article in press: June 13, 2016 Published online: July 28, 2016 Abstrct AIM: To clrify the reltionship between utophgy nd lipotoxicity-induced poptosis, which is termed lipopoptosis, in non-lcoholic stetoheptitis. METHODS: Mle C57BL/6J mice were fed high-ft diet (HFD) for 12 wk, fter which the liver histology nd expression of proteins such s p62 or LC3 were evluted. Alph mouse liver 12 (AML12) cells treted with plmitte () were used s n in vitro model. RESULTS: LC3-Ⅱ, p62, nd Run domin Beclin-1 intercting nd cysteine-rich contining () proteins incresed in both the HFD mice nd in AML12 cells in response to tretment. expression ws decresed upon c-jun N-terminl kinse (JNK) inhibition t both the mrna nd the protein level in AML12 cells. knockdown in AML12 cells with decresed the protein levels of both LC3-Ⅱ nd p62. expression peked t 4 h of tretment in AML12, nd then decresed. Tretment with cspse-9 inhibitor meliorted the decrese in protein expression t 10 h of nd resulted in enlrged AML12 cells under tretment. The enlrgement of AML12 cells by with cspse-9 inhibition ws cnceled by knockdown. CONCLUSION: The JNK- xis enhnced 6509 July 28, 2016 Volume 22 Issue 28

2 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis lipopoptosis, nd cspse-9 inhibition nd hd effects tht were cytologiclly similr to heptocyte bllooning. As bllooned heptocytes secrete fibrogenic signls nd thus might promote fibrosis in the liver, the inhibition of heptocyte bllooning might provide ntifibrosis in the NASH liver. Key words: Bllooned heptocyte; Cspse 9; c-jun N-terminl kinse; ; SP The Author(s) Published by Bishideng Publishing Group Inc. All rights reserved. Core tip: Autophgy is interrupted in both in vivo nd in vitro non-lcoholic stetoheptitis (NASH) models, nd impired utophgy is medited by Run domin Beclin-1 intercting nd cysteine-rich contining () protein expression vi c-jun N-terminl kinse phosphoryltion. expression ppers prior to poptosis nd enhnces plmitte toxicity in the heptocytes, nd cspse-9 decreses t the protein level during lipopoptosis. Cspse-9 inhibition with expression induces both heptocyte enlrgement nd endoplsmic reticulum stress ccumultion. The present study extends our knowledge on the precise blnce between lipopoptosis nd utophgy vi expression in NASH nd revels possible pthophysiology of bllooned heptocytes in NASH. Suzuki A, Kkisk K, Suzuki Y, Wng T, Tkikw Y. c-jun N-terminl kinse-medited expression enhnces heptocyte lipopoptosis nd promotes heptocyte bllooning. World J Gstroenterol 2016; 22(28): Avilble from: URL: DOI: INTRODUCTION The prevlence of non-lcoholic ftty liver disese (NAFLD) is drsticlly incresing in the Western countries [1]. In some NAFLD ptients, persistent inflmmtion nd progressive fibrosis develop in the liver, condition termed non-lcoholic stetoheptitis (NASH) [2]. As NASH will progress to liver cirrhosis nd end-stge liver disese, its pthogenesis needs to be clrified [3]. NASH is histologiclly chrcterized by lipid ccumultion with inflmmtion in the liver, which is considered to be result of lipotoxicity-induced heptocyte poptosis [2]. Infiltrtion of immune cells following heptocyte deth ctivtes heptic stellte cells, inducing the genertion of collgen fiber nd the development of liver fibrosis [2]. Therefore, heptocyte poptosis is considered to be n importnt step in development of NASH [4,5]. Lipid-induced poptosis, which is termed lipopoptosis, hs been estblished to cuse NASH [6,7]. Sturted free ftty cids (FFAs), such s plmitte (), induce heptocyte lipopoptosis vi endoplsmic reticulum (ER) stress, c-jun N-terminl kinse (JNK) phosphoryltion, nd mitochondril dysfunction [8,9]. Recently, it hs been reported tht sturted FFAs cn inhibit utophgy [10] which is process of cellulr selfdigestion [11]. In ddition, it hs been demonstrted tht utophgy is inhibited in the NASH liver [12]. As utophgy removes both ggregted proteins nd dmged orgnelles, it mintins orgnelle qulity nd prevents poptosis [11]. It hs been reported tht impired utophgy is ssocited with lipopoptosis nd tht chemicl gent-induced poptosis inhibits utophgy [10,13]. Accumulting evidence suggests tht inhibition of utophgy is ssocited with heptocyte poptosis in the NASH liver. However, the underlying mechnism remins uncler, nd gining clerer understnding of the inhibition of utophgy by lipopoptosis my led to the development of novel therpeutic strtegies for NASH. In line with the bove concept, we focused this study on molecule ssocited with utophgy inhibition: Run domin Beclin-1 intercting nd cysteine-rich contining () [14]. hs been found to inhibit the fusion of lysosomes to utophgosomes. However, whether is expressed in the NASH liver nd which role it would ply in NASH hs not been elucidted yet. Another histopthologicl hllmrk of NASH is the presence of bllooned heptocytes. The number of bllooned heptocytes generlly correltes with the severity of liver inflmmtion nd fibrosis in NASH [15-17]. Therefore, the prevlence of bllooned heptocytes is considered to be mrker of NASH ctivity. Bllooned heptocytes cn be histologiclly identified on the bsis of severl fetures, such s swelling, centrl nuclei, rrefied cytoplsm, nd Mllory- Denk bodies [18]. Mllory-Denk bodies contin severl proteins, such s kertin, chperones, kinses, nd protein degrdtion mchinery [19]. Although proteins in Mllory-Denk bodies should be decomposed vi the protein degrdtion pthwy, they re ccumulted in bllooned heptocytes. These findings indicte tht the protein degrdtion pthwy relted to utophgy my be impired in bllooned heptocytes. The ims of the present study were s follows: (1) to confirm the presence of n utophgic stte in n in vivo NASH model using mice fed high-ft diet (HFD); (2) to evlute the intrcellulr signling ssocited with both utophgy nd lipopoptosis; nd (3) to clrify the reltion between utophgy inhibition nd bllooning of heptocytes during lipopoptosis. MATERIALS AND METHODS Animls Mle 5-wk-old C57BL/6J mice were obtined from Chrles River Lbortories (Chrles River, Yokohm, Jpn) nd were mintined on 12-h light/12- drk cycle in humidity-controlled rooms t 22 with d libitum ccess to drinking wter. After 1 wk of 6510 July 28, 2016 Volume 22 Issue 28

3 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis hbittion, 5 mice were ssigned to ech of norml chow nd HFD (HFD-60, Orientl Yest CO., Tokyo, Jpn) groups nd were fed their respective diets for 12 wk. All of the mice were scrificed using isoflurne nesthesi fter overnight fsting t 20 wk of ge. All of the niml experiments were pproved by the Animl Cre nd Use Committee of Iwte Medicl University (Moriok, Jpn; ). Cells Alph mouse liver 12 (AML 12) cells, heptocyte cell line from mouse trnsgenic for humn trnsforming growth fctor α, were kindly supplied by Professor Itru Kojim, Gunm University. Becuse utophgic sttus ws confirmed in the NASH mouse model, we employed cell line generted from the sme species, AML 12, to investigte utophgic sttus in NASH in detil. Antibodies nd regents: The ntibodies used in this study were obtined from the following sources: nti-p62 (1:1000; #5114), nti-lc3 (1:1000; #4108), nti- (1:1000; #8465), nti-cleved cspse-3 (1:1000; #9661), nd rbbit nti-phospho- JNK (1:1000; #9251) were obtined from Cell Signling Technology, Tokyo, Jpn; mouse nti-c/ EBP homologous protein (CHOP) (1:500; sc-575), mouse nti-phospho-c-jun (1:1000; sc-822), nd got nti-β-ctin (1:1000; sc-1616) were obtined from Snt Cruz Biotechnology, Snt Cruz, CA, United Sttes). Alex Fluor 488-conjugted IgG ws from Life Technologies (Tokyo, Jpn). The JNK inhibitor SP (#420119) ws obtined from Clbiochem (Sn Diego, CA, United Sttes). Cspse-9 inhibitor Z-LEHD-FMK (b142026), nd pn-cspse inhibitor QVD-OPh (b141421) were obtined from Abcm Biochemicls (Tokyo, Jpn). Histologicl nlysis: Liver tissues were collected from the mice, fixed in 10% neutrl buffered formlin, prffin-embedded, nd sectioned. Liver specimens were stined with hemtoxylin nd eosin ccording to stndrd procedures. All smples were evluted in blinded mnner by single pthologist, who scored inflmmtion, stetosis, nd bllooning using nonlcoholic stetoheptitis ctivity scores [20]. Heptocyte tretments: Plmitte (; #P5585, Sigm Aldrich, Tokyo, Jpn), dissolved in isopropnol t stock concentrtion of 160 mmol/l, ws used to investigte signl trnsduction during lipopoptosis. AML12 cells were treted for the indicted time periods or with the indicted concentrtions of. For inhibition studies, AML12 cells were treted with (400 µmol/l or 800 µmol/l) for 4 h or 10 h in the presence or bsence of 30 µmol/l SP600125, 20 mol/l z-lehd-fmk, or 20 µmol/l QVD-OPh. All inhibitors simultneously dded with tretment. Biochemicl nlysis of the in vivo NASH model: Blood smples were obtined from the mice by crdic puncture. Serum ws obtined from the blood by centrifugtion t 3000 g for 10 min. The serum levels of lnine trnsminse (ALT) were nlyzed using n utonlyzer (JCA-BM2250; JEOL, Tokyo, Jpn). Cell prolifertion ssy For AML12 prolifertion studies, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrzolium monosodium slt] (Ncli Tesque, Kyoto, Jpn) incorportion experiments were performed using microplte reder (Multiskn FC; Thermo Fisher Scientific, Yokohm, Jpn). All prolifertion ssys were performed t lest 4 times for ech group. The results re presented s the rtio of incorportion in the cells tht received the indicted tretment to tht in vehicle-treted cells. Immunocytochemistry for p62 AML12 cells seeded in 6-well pltes were fixed with 4% prformldehyde in PBS nd permebilized with 125% (w/v) CHAPS in PBS. The primry ntibody for nti-p62 ws used t dilution of 1:500. The secondry ntibody ws Alex Fluor 488-conjugted IgG, nd ProLong Antifde with DAPI (Moleculr Probes, Eugene, OR, United Sttes) ws used s the mounting medium. Imges were cquired using n EVOS microscope (AMF 4300; Life Technologies) with excittion nd emission wvelengths of 488 nm nd 507 nm, respectively. The experiments were repeted three times. Mesurement of cell size AML12 cells seeded in 6-well pltes received the indicted tretments. Imges were cquired with the AMF 4300 microscope. Individul cells were identified nd the sizes of 25 rndomly selected cells were mesured using the Imge J softwre progrm ver (NIH, Bethesd, MD, United Sttes). The results were presented s the rtio of the size of treted cells to tht of control cells. Quntittive rel-time PCR Totl cellulr RNA of AML 12 cells ws extrcted using n RNesy Mini Kit (Qigen, Tokyo, Jpn) nd ws reverse-trnscribed into complementry (c) DNA with Moloney murine leukemi virus reverse trnscriptse (Invitrogen, Cmrillo, CA, United Sttes) nd rndom primers (Invitrogen, Cmrillo, CA, United Sttes) s described previously [21]. Quntifiction of the cdna templte ws conducted on 7500 Rel-Time PCR system (Applied Biosystems, Wlthm, MA, United Sttes) nd nlyzed using the 7500 Softwre progrm. The PCR primers were s follows: for mouse p62 (NM_011018): forwrd 5 -GAAGCTGCCCTATACCCACA-3 nd reverse 6511 July 28, 2016 Volume 22 Issue 28

4 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis 5 -TGGGAGAGGGACTCAATCAG-3 (65 bp); for mouse (NM_ ): forwrd 5 -GATGGG- GAGCGTCTGCTA-3 nd reverse 5 -TCCACAGTCGTCT- TCAAATTACC-3 (74 bp). Mouse Actin (NM_ ), mplified with the following primers: forwrd 5 -TAA- GGCCAACCGTGAAAAG-3 nd reverse 5 -ACCAGA- GGCATAGGGACA-3 (104 bp), ws used s n internl control. The trget mrna expression levels were expressed reltive to Actin for ech smple s described previously [21]. The experiment ws repeted three times. Smll interfering RNA trnsfection The smll interfering RNAs (sirnas) used for the knockdown of endogenous protein nd the negtive control sirna were purchsed from Life Technologies. sirna (5 pmol) ws trnsfected into cells using Lipofectmine RNAiMAX (Life Technologies), ccording to mnufcturer s instructions. The exmintions were performed fter 30 h of trnsfection. Immunoblotting nlysis Whole cell lystes were prepred s described previously [21]. Equl mounts of protein (10-50 µg) were resolved by sodium dodecyl sulfte-polycrylmide gel electrophoresis on 4%-12% crylmide gels, trnsferred to polyvinylidene difluoride membrnes, nd incubted with primry ntibodies. Then, the membrnes were incubted with the pproprite horserdish peroxidse-conjugted secondry ntibodies (BioSource Interntionl, Cmrillo, CA, United Sttes). Bound ntibody ws visulized using chemiluminescent substrte (ECL Prime; Amershm, Buckinghmshire, United Kingdom). Sttisticl nlysis All dt represent the results of t lest three independent experiments nd re expressed s the men ± SD. The differences between the groups were compred using Student s t-test nd one-wy nlysis of vrince with post-hoc Dunnett s test. P-vlues < 5 were considered to be sttisticlly significnt. RESULTS Autophgic process is inhibited in mice with stetoheptitis The mice were divided into 2 groups: those fed norml chow diet (CT, n = 5) nd those fed n HFD diet (HFD, n = 5). After 12 wk, ll mice were scrificed, nd liver histology, serum ALT levels, nd protein expression in the liver were exmined. Body weight, ALT mesurement, nd histologicl scores were evluted in 5 mice per group. Protein expression ws evluted in 3 mice per group. The body weight of the HFD mice ws significntly higher thn tht of the CT mice (Figure 1A). The ALT level ws significntly higher in the HFD mice thn in the CT mice (Figure 1A; 77 ± 1 IU/mL vs 25 ± 1.4 IU/mL). Histologicl nlysis reveled the ccumultion of lipid droplets in the liver (Figure 1B nd C) nd n increse in the number of bllooned heptocytes (Figure 1C) in HFD mice s compred to CT mice. These findings confirmed tht the HFD mice in the present study were comptible with NASH model. To confirm impirment of the utophgic process in this model, we evluted utophgic mrker protein expression in the liver by immunoblotting nlysis. When protein ws decomposed through utophgy, p62 ws induced, nd both p62 nd LC3-Ⅱ were subsequently degrded vi the utophgic process. The expression of p62 in the livers of the HFD mice ws higher thn in those of CT mice (Figure 1D). However, the expression of both LC3-I nd LC3-Ⅱ ws lso incresed in the HFD mice (Figure 1D). Expression of the utophgy inhibitor ws significntly higher in the HFD mice thn in the CT mice (Figure 1D). These findings indicted tht utophgy ws impired in the HFD mice. Additionlly, we evluted JNK phosphoryltion, which is key meditor of heptocyte lipopoptosis. Protein expression of phosphorylted JNK ws incresed in the HFD mice s compred to the CT mice. induces cell deth in dose- nd time-dependent mnner To investigte the detiled mechnism of the impired utophgic process, we employed the mouse heptocyte cell line AML12 in n in vitro study. A cell prolifertion ssy reveled tht induced cell deth in dose- nd time-dependent mnner, confirming cytotoxicity to AML12 cells (Figure 2A nd C). JNK phosphoryltion, ER stress, nd cleved cspse-3 were incresed by in both dose- nd timedependent mnner (Figure 2B nd D). Therefore, we considered tht induced poptosis in AML12 cells. inhibits the utophgic process in AML12 cells nd induces expression Next, we evluted utophgy-relted protein expression in -treted AML12 cells. induced p62 expression fter 10 h of incubtion with 800 µmol/l of in dose- nd time-dependent mnner (Figure 2B nd D). The incresed p62 level indicted induction of utophgy. If the utophgic process would progress normlly, LC3-Ⅱ would be expected to decrese with the decomposition of p62-ssocited protein ggregtes. However, LC3-Ⅱ expression peked t 6 h of incubtion with 800 µmol/l of. These findings indicted tht induced utophgy, but tht the utophgic process ws interrupted round 6 h of incubtion. Since LC3-Ⅱ expression ws further decresed t 10 h of incubtion (Figure 2D), utophgy progressed with longer incubtion times. In the in vitro model, 600 µmol/l of nd 4 h of incubtion with incresed expression, while 6512 July 28, 2016 Volume 22 Issue 28

5 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis A 50 BW P < ALT 40 P < Pre Cont. HFD 0 Cont. HFD B Cont. HFD C 2.5 D 2.0 p-jnk /b-ctin NAS score LC3-I LC3-II p62 Arbitrry unit (rtio) Stetosis Inflmmtion Cont.Bllooning Stetosis Inflmmtion HFD Bllooning b-ctin Cont. HFD Cont. HFD Figure 1 Mice fed high-ft diet show stetosis nd inflmmtion of the liver, nd impirment of the utophgic process. A: The body weights of mice t the strt of feeding or t 12 wk fter feeding with norml chow (Cont.) or high-ft diet (HFD) re presented in the left pnel. The serum ALT levels fter 12 wk of feeding re shown in the right pnel; B: Histology of the liver of control nd HFD mice is shown in the left nd the right pnel, respectively (Hemtoxylin nd Eosin stining); C: Non-lcoholic stetoheptitis ctivity scores (NAS) of control nd HFD mice; D: Immunoblotting nlyses of phosphorylted JNK, p62, LC3,, nd b-ctin. Protein smples were prepred from the liver tissue of ech of the control nd HFD mice. All of the bove experiments were repeted three times nd representtive results re shown. The quntittive dt re presented s the mens ± SD. 10 h of incubtion with 800 µmol/l of decresed expression in AML12 cells (Figure 2B nd D). Considering the progress of poptosis t higher dose nd longer incubtion time of, expression would be negtively relted with poptotic signls (Figure 2B nd D). -induced expression inhibits utophgy, resulting in enhnced lipotoxicity in AML12 cells To check whether -induced expression ws ssocited with lipopoptosis, we knocked down in AML12 cells using sirna (si). After incubtion with 800 µmol/l of for 4 h, the number 6513 July 28, 2016 Volume 22 Issue 28

6 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis A C Rtio of bsorbnce Rtio of bsorbnce mmol/l h B CHOP D CHOP p-jnk p-jnk Cleved Cspse 3 Cleved Cspse 3 LC3-I LC3-II LC3-I LC3-II p62 p62 b-ctin b-ctin mmol/l h E F CHOP Rtio of bsorbnce p62 LC3-I LC3-II CT CT sicont. si b-ctin sicont. si Figure 2 Tretment with plmitte induces poptosis in dose- nd time-dependent mnner, nd initites but impirs the utophgic process in AML12 cells. A nd B: AML12 cells were incubted with t the indicted concentrtions for 10 h. Untreted AML12 cells were used s the control. A: cytotoxicity s evluted by cell prolifertion ssy. Living cells re presented s the rtio of bsorbnce of cells treted with indicted conditions to tht of untreted AML12 cells; B: Immunoblotting nlyses of CHOP, phosphorylted JNK, cleved Cspse-3, LC3, p62,, nd ctin. Whole cell lystes were prepred from AML12 cells incubted t the indicted concentrtions for 10 h; C nd D: AML12 cells were incubted with 800 mmol/l of for the indicted periods. Untreted AML12 cells (0 h) were used s the control; C: cytotoxicity s evluted by cell prolifertion ssy. Living cells re presented s the rtio of bsorbnce t the indicted incubtion time to the bsorbnce t 0 h; D: Whole cell lyste ws prepred from the AML12 cells with 800 mmol/l for the indicted incubtion times; E nd F: AML12 cells were incubted with 800 mmol/l for 4 h fter trnsfection with the indicted sirna: control sirna (sicont) or sirna (si); E: cytotoxicity s evluted by cell prolifertion ssy. Living cells re presented s the rtio of bsorbnce in AML12 cells with 800 mmol/l for 10 h () to tht in AML12 control (CT) cells; F: Whole cell lyste ws prepred from AML12 cells treted with 800 mmol/l with the indicted sirna. All of the bove experiments were repeted three times nd representtive results re shown. The quntittive dt re presented s the men ± SD; P < July 28, 2016 Volume 22 Issue 28

7 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis A 2.0 B Rtio of bsorbnce CHOP b-ctin CT CT CT CT Control + Sp z-lehd-fmk + QVD-OPh SP z-lehd-fmk QVD-OPh C 2.0 D 9 /b-ctin Rtio of bsorbnce Arbitrry unit (rtio) z-lehd-fmk mmol/l Cont. Cont. Cont. Cont. SP z-lehd-fmk QVD-OPh Figure 3 c-jun N-terminl kinse inhibitor, Cspse-9 inhibitor, nd Pn-Cspse inhibitor meliorte plmitte-induced cell deth, nd Cspse-9 inhibition ttenutes the decrese of protein. A: AML12 cells were incubted with 800 mmol/l for 10 h in the presence or bsence of either 30 mmol/l SP600125, 20 mmol/l LEHD-fmk, or 20 mmol/l QVD-OPh. Untreted AML12 cells were used s the control. cytotoxicity ws evluted by cell prolifertion ssy. Living cells re presented s the rtio of the bsorbnce t the indicted conditions to tht of AML12 without tretment; B: Whole cell lystes were prepred from AML12 cells treted with (800 mmol/l) for 10 h in the presence or bsence of either 30 mmol/l SP600125, 20 mmol/l z-lehd-fmk, or 20 mmol/l QVD- OPh. Immunoblot nlysis of CHOP nd. b-ctin ws used s the loding control; C: The size of the AML12 cells fter tretment (800 mmol/l) for 10 h ws compred to tht of untreted AML12 cells using the Imge J softwre progrm. z-lehd-fmk nd/or si were used for Cspse-9 inhibition or knockdown. The difference in cell size under ech tretment condition is presented s the rtio to the cell size of the respective controls. All of the bove experiments were repeted three times nd representtive results re shown. The quntittive dt re presented s the men ± SD; P < 5. of vible cells ws significntly higher in sitreted thn in control sirna (sicont)-treted cells (Figure 2E). Expression of both p62 nd LC3-Ⅱ ws lower in si- thn in sicont-treted cells. Furthermore, -induced expression of the ER stressrelted trnscription fctor CHOP ws lower in silenced thn in control cells (Figure 2F). These results indicted tht -induced expression inhibited utophgy, nd tht enhnced lipotoxicity vi the impirment of utophgy. -induced expression is medited by JNK signling To evlute the mechnism of -induced expression, we used phrmcologicl inhibitors of lipopoptosis-relted molecules: JNK inhibitor (SP600125), cspse-9 inhibitor (Z-LEHD-FMK), nd pn-cspse inhibitor (QVD-OPh). All of the inhibitors meliorted lipotoxicity in the AML12 cells (Figure 3A). Interestingly, SP decresed expression in response to in comprison to the control (Figures 3B nd 4A). The results confirmed tht induced expression nd indicted tht expression ws medited by JNK signling in both AML12 cells nd the humn heptom cell line HepG2 (Figures 3B, 4A, 4C nd 5). JNK inhibition meliorted -induced c-jun phosphoryltion nd decresed -induced protein expression of both p62 nd LC3-Ⅱ (Figure 4A). Immunohistochemicl nlysis reveled tht p62 expression ws diffuse in the cytoplsm of AML12 cells tht were treted with, nd ws repressed by SP inhibitor (Figure 4B). p62 mrna expression in cells treted with nd SP ws higher thn tht in cells treted with SP lone, while it ws lower thn in cells treted with lone, s indicted by 6515 July 28, 2016 Volume 22 Issue 28

8 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis A p-c-jun p62 LC3-I LC3-II b-ctin SP mmol/l B CT CT + SP SP C p62/ctin rtio 2.0 /ctin rtio CT CT CT CT SP SP Figure 4 Plmitte induces expression of both p62 nd, nd -induced expression is medited by c-jun N-terminl kinse phosphoryltion. A: Whole cell lystes were prepred from AML12 cells treted with (400 or 800 mmol/l) for 4 h in the presence or bsence of SP Immunoblotting nlyses were performed for p62, LC3, nd. b-ctin ws used s the loding control; B: AML12 cells were incubted with 800 mmol/l for 6516 July 28, 2016 Volume 22 Issue 28

9 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis 4 h in the presence or bsence of 30 mmol/l SP p62 ntibody ws used s the primry ntibody, nd Alex Fluor 488-lbeled secondry ntibody ws used for detecting p62 ntibody. Smples were visulized using n EVOS microscope; C: Totl RNA ws prepred from AML12 cells treted with (800 mmol/l) for 4 h in the presence or bsence of 30 mmol/l SP Vehicle-treted cells were used s the control (CT). Both p62 nd mrna were quntified by RT-qPCR, normlized to Actin, nd expressed s the fold-chnge vs control cells without SP All of the bove experiments were repeted three times nd representtive results re shown. The quntittive dt re presented s the men ± SD; P < 5 vs control. p-c-jun Actin SP inhibition nd/or knockdown. The size of treted AML12 cells ws significntly lrger in the z-lehd-fmk group thn in the -lone group (Figure 3C). In contrst, knockdown meliorted heptocyte enlrgement in the -treted z-lehd-fmk group (Figure 3C). In ddition, cspse-9 inhibition induced ccumultion of CHOP protein (Figure 3B) mmol/l Figure 5 -induced expression ws medited by c-jun N-terminl kinse phosphoryltion. Whole cell lystes were prepred from HepG2 cells treted with (400 or 800 mmol/l) for 4 h in the presence or bsence of SP Immunoblotting nlyses were performed for p-c-jun nd. Actin ws used s the loding control. RT-qPCR (Figure 4C). These dt indicted tht p62 expression ws medited by both JNK-independent nd JNK-dependent signls. When -induced expression ws inhibited by sirna or JNK inhibitor, both -induced LC3-Ⅱ expression nd p62 expression decresed (Figures 2F nd 4A). -induced expression decreses with progression of lipopoptosis, which might be ffected by cspse-9 ctivtion We observed tht expression decresed t the protein level under the condition of lipotoxicity (Figure 2B nd D). Becuse poptosis ws correlted with utophgy, we hypothesized tht the lipopoptosis signling would ffect expression. When inhibitors of lipopoptosis were used in AML12 cells tht were treted with, Z-LEHD-fmk mintined greter mount of protein thn both AML12 with lone nd AML12 with other inhibitors (Figure 3B). Becuse the pn-cspse inhibitor QVD-OPh did not meliorte the decresed expression during lipopoptosis, the cuse of the decresed expression ws upstrem of the executioner cspses (cspse-3 nd -7). We considered tht cspse-9 ctivtion ws ssocited with inhibition of expression during lipopoptosis. Cspse-9 inhibition induces ER stress ccumultion nd heptocyte enlrgement, which is required for expression Both cspse-9 deletion nd JNK phosphoryltion hve been previously reported to be ssocited with heptocyte bllooning [22]. Therefore, we suspected tht lso ffects heptocyte morphology. To test this hypothesis, the effect of lipotoxicity on cell size ws evluted under the conditions of cspse-9 DISCUSSION NASH is chrcterized by both inflmmtion nd fibrosis in the liver. Lipotoxic insults led to persistent heptocyte poptosis, resulting in fibrosis in the liver. During these processes, ER stresses, such s the unfolding of proteins, ccumulte in the heptocytes s result of poptotic insults [23,24]. Autophgy cn inhibit the ccumultion of these insults by decomposing the ggregted proteins nd removing the dmged orgnelles [11,25]. Therefore, utophgy cn be considered not only process of energy supply but lso survivl mechnism tht protects ginst lipopoptosis. When the utophgic process is interrupted, insults ccumulte nd dmged cells re led to lipopoptosis (Figure 2B, D nd F). JNK phosphoryltion is key signl of heptocyte lipopoptosis [5,9,21]. Previous studies hve shown tht JNK-dependent pthwy leds to inflmmtion nd fibrosis in the NASH liver [26], nd tht JNK phosphoryltion is ssocited with heptocyte bllooning, which is hllmrk of NASH [22]. The present study demonstrted tht JNK phosphoryltion ws involved in the induction of lipopoptosis s well s in the inhibition of utophgy vi expression, reveling new role of JNK signling in heptocyte lipopoptosis. When ws knocked down, ER stress-induced lipopoptosis ws decresed. Impired utophgy vi induced the ccumultion of ER stress, consequently enhncing lipotoxicity. Indeed, expression ppered before poptosis (Figure 2B, D nd F). These dt suggest tht impirment of utophgy occurs before lipopoptosis. Thus, decresing the expression of could be n importnt erly intervention to prevent NASH development. As lipopoptosis progressed, expression decresed (Figure 2B nd D). The pn-cspse inhibitor did not meliorte the decrese of protein (Figure 3B). In contrst, cspse-9 inhibitor Z-LEHDfmk ttenuted the decrese of expression (Figure 3B). Thus, we speculte tht cspse-9 might decompose. Interestingly, cspse-9 hs been suspected to be key molecule in heptocyte 6517 July 28, 2016 Volume 22 Issue 28

10 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis bllooning. Levels of cspse-9 were found to be lower in bllooned thn in norml heptocytes in the NASH liver [22]. Furthermore, cspse-9-knockdown Huh-7 cells showed biologicl similrities with bllooned heptocytes, such s intrcellulr lipid ccumultion, ccumultion of ER stress, nd lipotoxic insult-induced sonic hedgehog signling vi JNK phosphoryltion [22]. tretment upon cspse-9 inhibition in AML12 induced the ccumultion of CHOP protein (Figure 3B). In contrst, recent report demonstrted tht pncspse inhibitor improved inflmmtion nd fibrosis in the liver, nd decresed the number of bllooned heptocytes in HFD mice [27]. These dt indicte tht cspse-9 might ply n importnt role in the formtion of bllooned heptocytes. According to the present study, cspse-9 inhibition hs cytologicl effects reminiscent of heptocyte bllooning, such s heptocyte enlrgement nd ccumultion of ER stress. Interestingly, knockdown of countercted the cspse-9 inhibition-induced heptocyte enlrgement. These dt indicte tht expression nd cspse-9 inhibition re required for the enlrgement of heptocytes. We hypothesize tht cspse-9, JNK, nd re key molecules for heptocyte bllooning in the NASH liver. The present study reveled number of significnt findings: (1) utophgy is interrupted in both in vivo nd in vitro NASH models; (2) -induced lipopoptosis inhibits utophgy by expression vi JNK phosphoryltion; (3) expression ppers prior to poptosis nd enhnces toxicity in the heptocytes; (4) Cspse-9 decreses t the protein level during lipopoptosis; nd (5) Cspse-9 inhibition with expression induces heptocyte enlrgement. We conclude tht -induced JNK phosphoryltion directly induced poptosis nd indirectly enhnced poptosis vi the inhibition of utophgy by expression, nd tht, cspse-9, nd JNK re key molecules for lipotoxic insult-induced heptocyte bllooning. ACKNOWLEDGMENTS We thnk Asko Wtnbe for western blotting nd Koko Motodte for providing excellent secretril support. COMMENTS Bckground Lipopoptosis in heptocytes leds to infiltrtion of immune cells, ctivtion of heptic stellte cells, nd genertion of collgen fibers in the liver. Thus, prevention of heptocyte poptosis is considered to be therpeutic strtegy for non-lcoholic stetoheptitis (NASH). As poptosis nd utophgy negtively interct with ech other, we focused this study on the protein, negtive regultor of utophgy, in the NASH liver. Bckground Bllooned heptocytes, which re well-known hllmrk of NASH disese severity, ccumulte toxic insult. Heptocytes in which cspse-9 ws knocked down with lipotoxicity demonstrted number of similrities with bllooned heptocytes, such s ccumultion of protein degrdtion mchinery or secretion of fibrogenic signl. The interction between exceeded poptosis, impired utophgy, nd bllooning of heptocytes hs never been elucidted. The detiled interction between these signls my be therpeutic trget of the NASH liver. Innovtions nd brekthroughs This is the first report to show tht is overexpressed in heptocyte lipopoptosis, enhnces lipotoxicity, cspse-9 decomposes protein, nd cspse-9 inhibition leds to bllooning of heptocytes. Although interctions between poptosis nd utophgy hve been previously reported elsewhere, the present study demonstrtes the role of ech of nd cspse-9 during heptocyte lipopoptosis. Applictions is induced during lipopoptosis by c-jun N-terminl kinse phosphoryltion nd enhnces poptosis vi utophgy inhibition. Since cspse-9 inhibition nd overexpression led to heptocyte bllooning, my be ssocited with bllooning in heptocytes. As the prevlence of bllooned heptocytes correltes with disese severity in the NASH liver, control of hs potentil for NASH therpy. Terminology Lipopoptosis is term for lipotoxicity-induced poptosis. Lipopoptosis in heptocytes induces the infiltrtion of immune cells into the NASH liver, which leds to the genertion of collgen fibers. Thus, lipopoptosis is considered leding cuse of NASH development. Peer-review This mnuscript contins fscinting nd novel dt for understnding of pthophysiology of NASH. REFERENCES 1 Rinell ME, Snyl AJ. NAFLD in 2014: Genetics, dignostics nd therpeutic dvnces in NAFLD. Nt Rev Gstroenterol Heptol 2015; 12: [PMID: DOI: / nrgstro ] 2 Yeh MM, Brunt EM. Pthologicl fetures of ftty liver disese. Gstroenterology 2014; 147: [PMID: DOI: /j.gstro ] 3 Wong RJ, Cheung R, Ahmed A. Nonlcoholic stetoheptitis is the most rpidly growing indiction for liver trnsplnttion in ptients with heptocellulr crcinom in the U.S. Heptology 2014; 59: [PMID: ] 4 Guiccirdi ME, Mlhi H, Mott JL, Gores GJ. Apoptosis nd necrosis in the liver. Compr Physiol 2013; 3: [PMID: DOI: /cphy.c120020] 5 Mlhi H, Bronk SF, Werneburg NW, Gores GJ. 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11 Suzuki A et l. JNK phosphoryltion nd in lipopoptosis jpgi ] 10 González-Rodríguez A, Myorl R, Agr N, Vldecntos MP, Prdo V, Miquilen-Colin ME, Vrgs-Cstrillón J, Lo Icono O, Corzzri M, Fimi GM, Picentini M, Muntné J, Boscá L, Grcí-Monzón C, Mrtín-Snz P, Vlverde ÁM. Impired utophgic flux is ssocited with incresed endoplsmic reticulum stress during the development of NAFLD. Cell Deth Dis 2014; 5: e1179 [PMID: DOI: /cddis ] 11 Jing P, Mizushim N. Autophgy nd humn diseses. Cell Res 2014; 24: [PMID: DOI: /cr ] 12 Amrvdi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evn GI, Thoms-Tikhonenko A, Thompson CB. Autophgy inhibition enhnces therpy-induced poptosis in Myc-induced model of lymphom. J Clin Invest 2007; 117: [PMID: DOI: /JCI28833] 13 Del Bello B, Toscno M, Moretti D, Mellro E. Cispltininduced poptosis inhibits utophgy, which cts s pro-survivl mechnism in humn melnom cells. PLoS One 2013; 8: e57236 [PMID: DOI: /journl.pone ] 14 Mtsung K, Sitoh T, Tbt K, Omori H, Stoh T, Kurotori N, Mejim I, Shirhm-Nod K, Ichimur T, Isobe T, Akir S, Nod T, Yoshimori T. Two Beclin 1-binding proteins, Atg14L nd, reciproclly regulte utophgy t different stges. Nt Cell Biol 2009; 11: [PMID: DOI: / ncb1846] 15 Cldwell S, Ikur Y, Dis D, Isomoto K, Ybu A, Moskluk C, Prmoonjgo P, Simmons W, Scruggs H, Rosenbum N, Wilkinson T, Toms P, Argo CK, Al-Osimi AM, Redick JA. Heptocellulr bllooning in NASH. J Heptol 2010; 53: [PMID: DOI: /j.jhep ] 16 Mtteoni CA, Younossi ZM, Grmlich T, Bopri N, Liu YC, McCullough AJ. Nonlcoholic ftty liver disese: spectrum of clinicl nd pthologicl severity. Gstroenterology 1999; 116: [PMID: DOI: /S (99) ] 17 Guy CD, Suzuki A, Abdelmlek MF, Burchette JL, Diehl AM. Tretment response in the PIVENS tril is ssocited with decresed Hedgehog pthwy ctivity. Heptology 2015; 61: [PMID: DOI: /hep.27235] 18 Brunt EM, Tinikos DG. Histopthology of nonlcoholic ftty liver disese. World J Gstroenterol 2010; 16: [PMID: DOI: /wjg.v16.i ] 19 Ztloukl K, French SW, Stumptner C, Strnd P, Hrd M, Toivol DM, Cdrin M, Omry MB. From Mllory to Mllory- Denk bodies: wht, how nd why? Exp Cell Res 2007; 313: [PMID: DOI: /j.yexcr ] 20 Kleiner DE, Brunt EM, Vn Ntt M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unlp-Arid A, Yeh M, McCullough AJ, Snyl AJ. Design nd vlidtion of histologicl scoring system for nonlcoholic ftty liver disese. Heptology 2005; 41: [PMID: DOI: / hep.20701] 21 Kkisk K, Cznve SC, Fings CD, Guiccirdi ME, Bronk SF, Werneburg NW, Mott JL, Gores GJ. Mechnisms of lysophosphtidylcholine-induced heptocyte lipopoptosis. Am J Physiol Gstrointest Liver Physiol 2012; 302: G77-G84 [PMID: DOI: /jpgi ] 22 Kkisk K, Cznve SC, Werneburg NW, Rzumilv N, Mertens JC, Bronk SF, Gores GJ. A hedgehog survivl pthwy in unded lipotoxic heptocytes. J Heptol 2012; 57: [PMID: DOI: /j.jhep ] 23 Akzw Y, Cznve S, Mott JL, Elmi N, Bronk SF, Kohno S, Chrlton MR, Gores GJ. Plmitolete ttenutes plmitte-induced Bim nd PUMA up-regultion nd heptocyte lipopoptosis. J Heptol 2010; 52: [PMID: DOI: / j.jhep.20103] 24 Cznve SC, Mott JL, Elmi NA, Bronk SF, Werneburg NW, Akzw Y, Khrmn A, Grrison SP, Zmbetti GP, Chrlton MR, Gores GJ. JNK1-dependent PUMA expression contributes to heptocyte lipopoptosis. J Biol Chem 2009; 284: [PMID: DOI: /jbc.M ] 25 Yoshii SR, Mizushim N. Autophgy mchinery in the context of mmmlin mitophgy. Biochim Biophys Act 2015; 1853: [PMID: DOI: /j.bbmcr ] 26 Seki E, Brenner DA, Krin M. A liver full of JNK: signling in regultion of cell function nd disese pthogenesis, nd clinicl pproches. Gstroenterology 2012; 143: [PMID: DOI: /j.gstro ] 27 Brreyro FJ, Holod S, Finocchietto PV, Cmino AM, Aquino JB, Avgnin A, Crrers MC, Poderoso JJ, Gores GJ. The pn-cspse inhibitor Emricsn (IDN-6556) decreses liver injury nd fibrosis in murine model of non-lcoholic stetoheptitis. Liver Int 2015; 35: [PMID: DOI: /liv.12570] P- Reviewer: Kucer O S- Editor: Qi Y L- Editor: A E- Editor: M S 6519 July 28, 2016 Volume 22 Issue 28

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