Basic Studies

DLPC and SAMe combined prevent leptin-stimulated TIMP-1 production in LX-2human hepatic stellate cells by inhibitingH2O2-mediated signal transduction

Qi Cao, Ki M. Mak and CharlesS. LieberAlcohol Research and Treatment Center, Bronx

Veterans Affairs Medical Center and Mount Sinai

School of Medicine, Bronx, NY, USA

Cao Q, Mak KM, Lieber CS. DLPC and SAMe combined prevent leptin-stimulated TIMP-1 production in LX-2 human hepatic stellate cells byinhibiting H2O2-mediated signal transduction.Liver International 2006: 26: 221–231. r Blackwell Munksgaard 2005

Abstract: Background/Aims: Both dilinoleoylphosphatidylcholine (DLPC)and S-adenosylmethionine (SAMe) have antioxidant properties andantifibrogenic actions. Because H2O2 mediates signal transduction-stimulating liver fibrogenesis, we investigated whether DLPC and SAMeattenuate the production of tissue inhibitor of metalloproteinase (TIMP)-1 byinhibiting H2O2 formation. Methods: LX-2 human hepatic stellate cells weretreated with leptin with or without DLPC, SAMe or various inhibitors.Results: Leptin-stimulated TIMP-1 mRNA and its protein were diminishedby DLPC or SAMe alone, and the response was fully prevented by thecombination of DLPC and SAMe. H2O2 was increased while glutathione wasdecreased; these changes were prevented by AG490, suggesting a Januskinases (JAK)-mediated process. Up-regulation of leptin receptor andactivation of JAK1 and 2 were not affected by DLPC1SAMe, whereasphosphorylation of ERK1/2 and p38 was blocked by DLPC1SAMe orcatalase, suggesting an H2O2-dependent mechanism. These treatments alsosuppressed leptin-stimulated TIMP-1 promoter activity and decreased TIMP-1 mRNA stability, contributing to TIMP-1 mRNA down-regulation.PD098059, an ERK1/2 inhibitor, suppressed TIMP-1 promoter activity,whereas SB203580, a p38 inhibitor, decreased TIMP-1 message stability; bothresulted in a partial reduction of TIMP-1 mRNA. Conclusion: As decreasedTIMP-1 production may enhance collagen deposition, the combinedadministration of DLPC1SAMe should be considered for the prevention ofH2O2-mediated signaling and the resulting fibrosis.

Keywords: catalase – ERK1/2 – Janus kinases –

p38 – reduced glutathione – TIMP-1 mRNA

stability – TIMP-1 promoter activity

Charles S. Lieber, MD, MACP; Alcohol & Nutri-

tion Research Center, Veterans Affairs Medical

Center, 130 West Kingsbridge Road, Bronx, NY

10468, USA.

Tel: 1 1 718 741 4244

Fax: 1 1 718 733 6257

e-mail: LIEBERCS@AOL.COM

Received 25 March 2005,

accepted 6 September 2005

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Dilinoleoylphosphatidylcholine (DLPC) is themajor compound (43–50%) of polyenylphospha-tidylcholine (PPC) extracted from soybeans (1). Itis responsible for many of the beneficial effects ofPPC against liver injury, including apoptosis ofhepatocytes (2), cytochrome P450IIE1 induction(3), mitochondrial respiratory dysfunction (4),TNF-a generation by Kupffer cells (5, 6), andoxidative stress in hepatoma cells (7). In culturedhepatic stellate cells (HSCs), the principal cellsthat mediate liver fibrogenesis (8), DLPC wasshown to inhibit their proliferation stimulated bythe platelet-derived growth factor (9) and tostimulate their collagenase activity (1). More

relevant to the present study is the finding thatDLPC down-regulates transforming growth fac-tor-b-stimulated a1(I) procollagen mRNA ex-pression in rat HSCs through inhibition ofH2O2-dependent p38 mitogen-activated proteinkinase (MAPK) signaling pathway (10), suggest-ing a link between DLPCs antioxidant propertiesand its antifibrogenic actions.S-adenosylmethionine (SAMe) is synthesized

from methionine catalyzed by methionine adeno-syltransferase (MAT) (11, 12). It is an ultimateprecursor of reduced glutathione (GSH) in thetranssulfuration pathway and the methyl donorfor most biological transmethylation reactions.

Liver International 2006: 26: 221–231 Copyright r Blackwell Munksgaard 2005

DOI: 10.1111/j.1478-3231.2005.01204.x

221

GSH is a major antioxidant against liver in-jury (12) and the oxidative stress-mediated deple-tion of hepatic GSH is a hallmark of liverinjury associated with intrahepatic cholestasis(13) or caused by ethanol in humans (14–16),baboons (17), and rats (15, 18) or by hepato-toxins in animal models (19–21). It is agreedthat the beneficial effects of SAMe on hepaticinjuries are because of its capacity to replenishcellular GSH levels. However, little is knownabout SAMe antifibrogenic effects. In onestudy, it was observed that SAMe administrationresulted in the attenuation of liver fibrosis in-duced by CCl4 in rats (20). The reduction offibrosis is accompanied by a restoration ofhepatic GSH level and a reduction of lipidperoxidation, suggesting that the antioxidant ac-tions of SAMe are responsible for its antifibro-genic effects. Thus far there are no studiesexamining the effects of SAMe on oxidant-mediated signaling pathways associated with he-patic fibrogenesis.A major determinant of liver fibrosis is an

inhibition of collagen degradation by interstitialcollagenase because of increased activity of thetissue inhibitor of metalloproteinase (TIMP)-1(22). We previously showed that activated HSCs(LX-2 human HSCs and culture-activated ratHSCs) synthesize and secrete TIMP-1 in responseto leptin (23), a peptide hormone with fibrogenicaction (24–26). The process is mediated, in part,by the JAK/STAT (Janus kinase/Signal transdu-cer and activator of transcription) pathways viathe leptin receptor long form (OB-RL). We alsofound that leptin stimulates H2O2 formation andthe effect appears to be linked to the activation ofJAK 1 and 2, because it is prevented by AG490,an inhibitor of JAKs. Increased H2O2 activatesextracellular signal-regulated kinases (ERK) 1/2and p38 MAPK pathways, which stimulateTIMP-1 production. Because reactive oxygenspecies (ROS), in particular H2O2, promote liverfibrogenesis (10), they are attractive targets ofantioxidant therapy.Accordingly, the present study evaluated the

prevention by DLPC, SAMe or their combina-tion of TIMP-1 production induced by leptin inHSCs. Their effects on leptin signaling throughJAKs and H2O2-dependent ERK1/2 and p38pathways leading to TIMP-1 mRNA expressionwere examined. We also assessed TIMP-1 pro-moter activity and its message stability. Theactions of DLPC and SAMe were studied inparallel with those of catalase, ERK1/2 and p38inhibitors. These were investigated in LX-2 cells,an immortalized human cell line, which retainsthe key features of activated HSCs (23, 27).

Materials and methods

Culture and treatment of HSCs

LX-2 human HSC line (27) was kindly providedby Dr. S. L. Friedman, Mount Sinai School ofMedicine, NY. The maintenance of LX-2 cells inDulbecco’s modified eagle medium (DMEM) con-taining 5% heat-inactivated fetal calf serum hasbeen described (23). At subconfluence, cells werewashed in serum-free DMEM and then incubatedin the same media containing leptin with or with-out DLPC, SAMe or various inhibitors for var-ious intervals of time. Additionally, cells wereserum starved in DMEM for 18h prior to thetreatment with leptin. Leptin (Sigma Chemicals,St. Louis, MO) was used at 75ng/ml, a concentra-tion that was shown before to stimulate maximallyTIMP-1 production (23). DLPC (Avanti PolarLipids, Alabaster, AL), dissolved in 0.05% bovinealbumin, was used at 2, 5, and 10mM, and SAMe(Sigma), dissolved in saline, at 3, 12, and 30mM(28–30). Inhibitors and their concentrations were50mM JAK inhibitor AG490 (Calbiochem, SanDiego, CA) (23, 31), 1000U/ml catalase (10)(Sigma), 30mM ERK1/2 inhibitor PD098059 (23,32) (Sigma), 20mM p38 inhibitor SB203580 (10,33) (Sigma) and 20mM SB202474, an inactiveanalog of SB203580 (23, 34) (Calbiochem, SanDiego, CA). Except for catalase, which was dis-solved in the culture media, other inhibitors weredissolved in dimethyl sulfoxide (DMSO) at aconcentration of 2.1mM. In these experiments,LX-2 cells were used at passages 20–30.

TIMP-l mRNA and protein assays

Expression of mRNA for the TIMP-1 in LX-2 cellswas evaluated by Northern blots as previouslydescribed (23). The levels of the mRNA werequantified by measuring the intensity of the bandson X-ray film by imaging densitometry. TIMP-1protein in the culture media of LX-2 cells wasquantified by ELISA, using the Quantikine HumanTIMP-1 ELISA kit according to the manufac-turer’s instruction (R&D System, Mineapolis,MN). Data are reported as ng/ml culture media.

Western blot analysis of leptin receptor long form (OB-RL)

LX-2 cells were treated with leptin or DLPC1SAMe or both for 24 h and protein lysates wereprepared for Western blot analysis of Tyr-1141-phosphorylated OB-RL expression, using 12%sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis (SDS-PAGE) (23). The primaryantibody was a goat anti-human phospho-OB-R(Tyr-1141) antibody (Santa Cruz Biotech, SantaCruz, CA), which reacts with the Tyr-1141-

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phosphorylated cytoplasmic domain at the ex-treme terminus of OB-RL (35). Signal intensitieswere quantified by imaging densitometry.

Immunoprecipitation assay for JAK phosphorylation

The level of JAK phosphorylation was deter-mined at 30min, the time at which maximalphosphorylation by leptin occurred in LX-2 cells(23). A 200-ml aliquot of the cell lysates wasincubated with a rabbit anti-p-JAK1 or p-JAK2antibody (Biosource, Camarillo, CA) and thenimmunoprecipitated with agarose hydrazidebeads as previously described (23). The immunecomplexes (20ml) were resolved by 12% SDS-PAGE. Equal protein loading was controlled byimmunoblotting of the corresponding nonpho-sphorylated JAK1 and JAK2, using the rabbitantibodies against the respective proteins (SantaCruz Biotech). Signal intensities were quantifiedby imaging densitometry.

Intracellular ROS and GSH determination

ROS and GSH were measured by stimulating LX-2cells with leptin in the presence or absence ofcatalase. Production of ROS was assessed by add-ing the probe 20,70-dichlorodihydrofluorescin diace-tate (DCFH-DA), obtained from molecular probes(Eugene, OR), to LX-2 culture at a final concentra-tion of 20mM and incubating for 30min in thedarkness following leptin treatment. In the cells, thenonfluorescent DCFH is oxidized to the fluorescent20,70-dichlorofluorescein (DCF) by ROS, mainlyperoxides, in the presence of peroxidases (36).DCF fluorescence was measured 1h after leptin,the time shown before at which the fluorescencepeaked in LX-2 cells (23). The fluorescence wasmeasured at 488nm for excitation and 525 foremission in a spectrofluorometer. GSH levels inLX-2 cells were measured using the Cayman’s GSHassay kit according to the manufacturer’s instruc-tion. Data are reported as nmol/mg protein.

ERKI/2 and p38 MAPK phosphorylation assays

The levels of ERK1/2 and p38 phosphorylationby leptin were determined at 2 h, the time shownto be associated with maximal phosphorylation inLX-2 cells (23). Phosphorylation was analyzed byWestern blots, using the components provided inthe PhosphoPlus p38 MAPK and ERKl/2MAPK antibody kits (Cell Signaling Technology,Beverly, MA) as previously described (23).ERK1/2 phosphorylation was detected using arabbit phospho-ERK1/2 (Thr-202/Tyr-204) anti-body and that of p38 was assayed using a rabbitphospho-p38 (Thr-180/Tyr-182) antibody. Im-

munoblotting of total (phosphorylated and non-phosphorylated) ERK1/2 and p38 was used ascontrol for equal protein loading.

Transfection of TIMP-l promoter and chloramphenicolacetyltransferase (CAT) assay

The activity of the TIMP-1 promoter was assessedusing the CAT reporter plasmid (pBLCAT3) con-taining nucleotides � 102 to 96 (minimal promoter)of the human TIMP-1 gene (37), which was kindlyprovided by Dr. D. A. Mann of SouthamptonGeneral Hospital, Southampton, UK. Transienttransfection of LX-2 cells was performed, as pre-viously described (23), with the LipofectAminet kitas per the manufacturer’s protocol (Invitrogen,Carlsbad, CA) using 320ng of the reporter plasmidDNA or the promoterless pBLCAT3. Transfectedcells were treated with leptin in the absence orpresence of catalase, SB203580, PD098059, orDLPC1SAMe for 24h. CAT activity was assayedusing the CAT ELISA kit and following themanufacturer’s protocol (Roche Mol Biochem,Indianapolis, IN). The sensitivity of the assay was� 50pg/ml. Data are reported as ng/mg protein.

TIMP-l mRNA stability determination

To assess whether p38, ERK1/2, catalase andDLPC1SAMe affect TIMP-l mRNA stability atthe posttranscriptional level, LX-2 cells were trea-ted with leptin for 24h to induce TIMP-l mRNA.This was followed by actinomycin D (10mg/ml)treatment for 20min to block the transcription(23). The culture medium was changed and freshmedium containing SB203580, PD098059, cata-lase, or DLPC1SAMe was added. After 2, 8, and12h of incubation, total RNA from LX-2 cells wasisolated for Northern blot analysis of TIMP-lmRNA levels, and the decay time course in theabsence or presence of the inhibitors was analyzed.

Statistics

Data are reported as means� SEM. The signifi-cance of difference between means was analyzedusing analysis of variance followed by Student–Newman Keuls tests. Po0.05 was considered tobe significant.

Results

Up-regulation of TIMP-1 mRNA by leptin: effect of serumand inhibition by different concentrations of DLPC orSAMe

Figure 1A shows that the basal level of TIMP-1mRNA in serum-starved LX-2 cells was compar-

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DLPC and SAMe prevent H2O2 and TIMP-1

able with that in serum-supplemented cells. ThemRNA levels after leptin were equivalent in LX-2cells with or without serum starvation. Theseresults rule out the possibility that leptin stimu-lates TIMP-1 mRNA by modulating the action ofserum-derived factors. Subsequently, the experi-ments described in this study were carried outwith LX-2 cells incubated in 5% FCS- supple-mented culture media prior to the treatment withleptin (in serum-free media).Leptin at 75 ng/ml up-regulated TIMP-1

mRNA 3.9-fold in LX-2 cells. DLPC decreasedTIMP-1 mRNA in a concentration manner, with

the greatest effect (41%) seen at 10mM (Fig. 1B).Of the three doses of SAMe tested (2, 12, 30M),the 12M one produced the greatest inhibition(38%) of TIMP-1 mRNA expression (Fig. 1C).Bovine albumin (0.05%), a vehicle control ofDLPC, had no effect on TIMP-1 mRNA expres-sion (data not shown). Accordingly, DLPC at10M and SAMe at 12M were used in the experi-ments described below.

Inhibition of leptin-induced TIMP-1 production by DLPC,SAMe, and their combination

Figure 2 shows that up-regulation of TIMP-1mRNA (3.9-fold) by leptin (A) was accompaniedby an increase in TIMP-1 protein concentration(3.2-fold) in the culture media (B) of LX-2 cells.Whereas DLPC (10mM) or SAMe (12mM) atte-nuated the rise of mRNA equally by 41% andthat of protein by 38% and 31%, respectively,DLPC and SAMe combined fully prevented bothchanges. The combination of DLPC and SAMewas at least two times more effective (Po0.01)than DLPC or SAMe alone in the prevention ofTIMP-1 production induced by leptin.

TIMP-1

mRNA

Fold: 3.9 1 3.7 1 2.9 2.31

β-actin

Leptin: – + – + – + – +

– + – + – + – +

DLPC (µM): 2 5 10

Fold:

Leptin:

SAMe (µM): 3 12 30

TIMP-1

mRNA

β-actin

0.9 kb

0.9 kb

TIMP-1 mRNA

β-actin

Serum-supplemented: + – + –Serum-starved: – + – +

Leptin: – – + +

Fold: 1 .9

0.9 kb

Effect of serum3.93.9

Inhibition by DLPC1

3.9 1 3.9 1 2.4 2.611Inhibition by SAMe

(A)

(B)

(C)

Fig. 1. Leptin stimulation of tissue inhibitor of metalloprotei-nase (TIMP)-1 mRNA. (A) Effect of serum: LX-2 cells wereincubated in 5% fetal calf serum-supplemented or serum-freeDulbecco’s modified eagle medium (DMEM) for 18 h prior tothe treatment with leptin (in serum-free media). (B) Inhibitionby dilinoleoylphosphatidylcholine (DLPC) and (C) cnhibitionby S-adenosylmethionine (SAMe): cells were cultured in 5%serum-supplemented media, washed in serum-free media, andtreated with increasing concentrations of DLPC or SAMe, asindicated, in the presence of leptin (75 ng/ml) or in its absence inserum-free DMEM. TIMP-1 mRNA was analyzed by Northernblot after 24 h of incubation. Values of the band intensity areexpressed, after normalization to b-actin, as fold change relativeto control (no leptin, DLPC, and SAMe) assigned a value of 1.The numbers above the blots refer to the means of threeindividual analyses.

TIMP-1 mRNA

Leptin: – + – + – + – +

– – + + – – + +

– – – – + + + +

DLPC (10µM):

SAMe (12µM):

0

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***

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***

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Fig. 2. Inhibition of tissue inhibitor of metalloproteinase(TIMP)-1 production by dilinoleoylphosphatidylcholine(DLPC), S-adenosylmethionine (SAMe), or their combination.LX-2 cells were incubated with leptin in the presence or absenceof DLPC (10mM) or SAMe (12mM), or DPLC (10mM)1SAMe(12mM) for 24 h. (A) Histograms summarizing the data of threeindividual analyses of TIMP-1 mRNA abundance by Northernblot. Values are expressed as fold change relative to control (noleptin, DLPC and SAMe), assigned a value of 1. (B) TIMP-1protein concentration in the culture media as quantified byELISA. nnnPo0.001 vs. leptin without DLPC and SAMe.

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Effects of DLPC and SAMe on leptin-induced expressionof OB-RL and on phosphorylation of JAKs

In HSC, the leptin-stimulated TIMP-1 produc-tion is associated with an increased expression ofOB-RL, accompanied by the stimulation of JAKphosphorylation (23). Therefore, we evaluatedwhether DLPC and SAMe affect the expressionof these proteins.Leptin treatment increased the level of Tyr-

1141-phosphorylated OB-RL (Fig. 3A) and thatof p-JAK1 and 2, but not of JAK3 (Fig. 3B).

Both increases were not altered by DLPC andSAMe combined.

Effects of DLPC, SAMe, and catalase on intracellularROS and GSH levels after leptin

Figures 4A and B show that ROS formation wassignificantly increased while cellular GSH wasdecreased (45%) by leptin after 1 h of treatment;these changes were partially diminished by DLPCor SAMe. By contrast, the combination of DLPCand SAMe normalized the levels of ROS andGSH to the values of control cells. The effectexerted by DLPC and SAMe combined wasfound to be statistically more significant thanthat exerted by DLPC or SAMe alone.Addition of catalase to the culture abolished

the increase in DCF fluorescence after leptin,demonstrating that H2O2 is largely responsiblefor the ROS increase. Catalase treatment alsoresulted in a normalization of GSH to the levelof untreated cells, suggesting an involvement ofH2O2 in the consumption of GSH. Treatment ofLX-2 cells for 1 h with the JAK inhibitor AG490

p-JAK 1

p-JAK 2

p-JAK 3

JAK1

JAK2

JAK3

JAK phosphorylation

GAPDH

Tyr-1141 phosphorylated OB-RL

Fold:

Leptin: – + – ++DLPC + SAMe: – – +

Leptin: – + – ++DLPC + SAMe: – – +

204 kDa _

122 kDa _

1 2.1 1.1 2.1

Fold: 1 4.5 1 4.6

Fold: 1 4.8 1 4.8

Fold: 1 1.0 1 1.0

(A)

(B)

Fig. 3. Increased expression of leptin receptor long form (OB-RL) and phosphorylation of Janus kinases (JAKs) after leptinare not affected by dilinoleoylphosphatidylcholine (DLPC) andSAMe. LX-2 cells were incubated with leptin or DLPC1S-adenosylmethionine (SAMe), or either of the two for 24 h.Detection of Tyr-1141-phosphorylated OB-RL was performedby Western blot, using an anti-human phospho-OB-RL (Tyr-1141) antibody. Phosphorylation of JAK1, 2, and 3 wasdetermined by Western blot after 30min of incubation, usingthe respective p-JAK polyclonal antibodies. Equal proteinloading was controlled by immunoblotting of the correspondingnonphosphorylated JAKs.

ROS formation

DC

F fl

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SAMe:

Catalase:

***

***

+ + + + +– – – – –– + – + –– + – + –

– – + + –– – + + –

– – – – +– – – – +

Fig. 4. Effects of dilinoleoylphosphatidylcholine (DLPC), S-adenosylmethionine (SAMe), and catalase on reactive oxygenspecies (ROS) and glutathione (GSH) levels after leptin. LX-2cells were treated with DLPC (10 mM), SAMe (12 mM), or bothin the presence or absence of leptin for 1 h. Additional cells weretreated with catalase (1000U/ml) with or without leptin. (A)Intracellular ROS formation was detected with the probedichlorodihydrofluorescin (DCFH) as described in the Materi-als and methods section. Increased dichlorofluorescein (DCF)fluorescence was proportional to elevated levels of ROS. Resultsare presented as % of control (no leptin, DLPC, SAMe, andcatalase) assigned 100%. (B) Cellular GSH levels were measuredwith Cayman’s assay kit. nPo0.05, nnPo0.01 and nnnPo0.001vs. leptin (without DLPC, SAMe and catalase).

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completely blocked the increased levels of ROScaused by leptin (data not shown), in accordancewith an involvement of JAK1 and 2 in leptinstimulation of ROS formation as previously re-ported (23).

Effects of ERK1/2 inhibitor PD098059, p38 inhibitorSB203580, catalase, and DLPC1SAMe on H2O2-mediated ERK1/2 and p38 MAPK phosphorylation

The effects of PD098059, SB203580, and catalaseon MAPK phosphorylation were studied in par-allel with those of DLPC1SAMe. After 2 h ofleptin treatment, the level of phosphorylatedERK1/2 was increased 3.5-fold (Fig. 5A) andthat of p38, 3.9-fold (Fig. 5B). PD098059 andSB203580 abrogated the basal and leptin-stimu-lated phosphorylation of ERK1/2 and p38, re-spectively, in accordance with the actions of theseinhibitors. SB202474 (an inactive analog ofSB203580) had no effect on p38 phosphorylation.Catalase prevented the phosphorylation of bothERK1/2 and p38, consistent with an H2O2-

mediated process. DLPC1SAMe, like catalase,fully inhibited ERK1/2 and p38 phosphorylation,suggesting that DLPC and SAMe inhibit MAPKphosphorylation by a mechanism similar to thatof catalase.

Figures 5A and B also illustrate ERK1/2 andp38 phosphorylation in serum supplementationand starvation conditions. The basal levels ofphosphorylated ERK1/2 and p38 were equivalentin serum-supplemented and serum-starved LX-2cells. No difference in ERK1/2 and p38 phos-phorylation in response to leptin was observed.These results ensure that leptin stimulation ofMAPK is not related to the modulation of serum-derived factors.

Inhibition by catalase, ERK1/2 inhibitor PD098059, andp38 inhibitor SB203580 of leptin up-regulation ofTIMP-1 mRNA

Catalase fully prevented the 3.6-fold increase ofTIMP-1 mRNA induced by leptin, determinedafter 24 h of treatment (Fig. 6A). Both PD098059

ERK1/2 phophorylation

Leptin: – + – + – + – +

– – – – + + – –

– – – – – – + +

– – + + – – – –

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SB203580:

Catalase:

p-p38

Total p38

p38 phosphorylation

Fold:

Leptin: – – + +DLPC+SAMe: – – + +

Fold:

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Fold:

p-ERK1/2

Total ERK1/2

Leptin: – – ++– + +–DLPC +SAMe:

Fold:

Serum-supplemented: + –Serum-starved: – – + +

Leptin: – – +

Serum-supplemented: + + – –Serum-starved: – – + +

Leptin: – + – +

Fold:Fold:

p-ERK1/2

Total ERK1/2

p-p38

Total p38

p-ERK1/2

Total ERK1/2

p-p38

Total p38

1 3.5 1 1 0 0

1 1 13.8

1 1 3.83.81 3.9 1.1 3.9

–+

+

1

1 3.9 1 1

3.9 .9 .9 1 3.9 .1 .1

(A) (B)

Fig. 5. Increased ERK1/2 and p38 MAPK phosphorylation by leptin and its inhibition by catalase, ERK1/2 inhibitor PD098059, p38inhibitor SB203580, and dilinoleoylphosphatidylcholine (DLPC)1SAMe. LX-2 cells were cultured in 5% serum-supplementedDulbecco’s modified eagle medium (DMEM), washed in serum-free media, and then incubated for 2 h with leptin in the presence ofcatalase, PD098059, SB203580, or DLPC1SAMe, or in their absence. Additional cells were cultured in serum-free DMEM for 18 hprior to leptin treatment. The phosphorylated ERK1/2 (A) and p38 (B) protein contents were analyzed by Western blot usingpolyclonal anti-phospho-ERK1/2 and anti-phospho-p38 antibodies as primary antibodies, respectively. The intensity of the bands onthe blots was normalized to that of total ERK1/2 or p38, detected by nonphosphorylated ERK1/2 or p38 antibody, and values arepresented as fold change relative to controls (without leptin and inhibitors) assigned a value of 1. The numbers above theimmunoblots refer to the mean values of three individual analyses.

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and SB203580 halved the increase of the mRNA(Fig. 6B and C). In the absence of leptin, catalase,PD098059, and SB203580 had no effect on thebasal mRNA level. These results demonstratethat TIMP-1 mRNA up-regulation by leptin ismediated, at least in part, by H2O2 throughERK1/2 and p38 signaling pathways. DMSO, avehicle control for D098059 and SB203580, hadno effect on TIMP-1 mRNA levels, whether inthe presence or absence of leptin (data notshown).

Effects of ERK1/2 inhibitor PD098059, p38 inhibitorSB203580, catalase, and DLPC1SAMe on leptin-stimulated TIMP-1 promoter activity

TIMP-1 promoter activity was stimulated 2.5-fold by leptin, which was fully prevented byPD098059, catalase, or DLPC1SAMe (Fig. 7),implicating an involvement of ERK1/2 signalingand H2O2 in the transcriptional regulation ofTIMP-1 gene expression. SB203580 had no effecton leptin-stimulated TIMP-1 promoter activity,suggesting that p38 is not involved in the tran-scriptional regulation of TIMP-1 gene. Treatmentwith these inhibitors alone had no effect on thebasal promoter activity (data not shown).

Effects of p38 inhibitor SB203580, ERK1/2 inhibitorPD098059, catalase, and DLPC1SAMe on TIMP-1mRNA stability

Figure 8 shows that SB203580, catalase, orDLPC1SAMe treatment resulted in an increased

decay of TIMP-1 mRNA with an approximatehalf-life (t1/2) of 4 h compared with a t1/2 of 10 hin the absence of the inhibitors. PD098059 hadno effect on the rate of the mRNA decay.These results suggest that p38 mediates leptin-induced TIMP-1 mRNA expression through astabilization of the mRNA and that DLPC1SAMe, like catalase, affect TIMP-1 mRNAexpression by decreasing the stability of themessage.

0

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+–

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Leptin: – + – + – +– – + + – –– – – – + +

SB202474:SB203580:

TIMP-1 mRNA

β-actin

TIMP-1 mRNA

β-actin

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# #

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catalase SB203580 PD098059

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P-1

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6

(A) (B) (C)

Fig. 6. Inhibition of leptin-induced tissue inhibitor of metalloproteinase (TIMP)-1 mRNA by catalase, ERK1/2 inhibitor PD098059,and p38 inhibitor SB203580. LX-2 cells were treated with leptin alone or with the addition of catalase, PD098059, or SB203580, andTIMP-1 mRNA expression was analyzed by Northern blot after 24 h of incubation. (A) Catalase reduced the increased TIMP-1mRNA induced by leptin to the control level (no leptin and catalase). (B) and (C) PD098059 and SB203580 decreased the mRNA levelby one-half. SB202474, an inactive analog of the p38 inhibitor SB203580, had no effect on TIMP-1 mRNA expression. Upper panelsare typical Northern blots, and lower panels are histograms of data of three individual analyses. Values are expressed as fold changerelative to the control (no leptin and inhibitors) assigned a value of 1. nnnPo0.001 vs control; ##Po0.01 and ###Po0.001 vs leptinwithout inhibitors)

TIM

P-1

prom

oter

act

ivity

(ng/

mg

prot

ein,

mea

n+/–

SEM

, n=

3)Empty

plasmidLeptin

PD098059Leptin Leptin

SB203580Leptin

CatalaseLeptin

DLPC +SAMe

***

******

******

0

2

4

6

8

10

Minimalpromoter

Fig. 7. Stimulation of tissue inhibitor of metalloproteinase(TIMP)-1 promoter activity and its inhibition by ERK1/2inhibitor PD098059, catalase, and dilinoleoylphosphatidylcho-line (DLPC)1SAMe, but not by p38 inhibitor SB203580. LX-2cells were transiently transfected with the human TIMP-1mini-mal promoter contained in the pBLCAT reporter plasmid orwith the empty vector as described in the Materials andMethods section. After 24 h of leptin treatment in the presenceof inhibitors, LX-2 cell extracts were prepared for quantificationof TIMP-1 promoter activity, using a CAT ELISA kit.nnnPo0.001 vs leptin without inhibitors.

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DLPC and SAMe prevent H2O2 and TIMP-1

Discussion

This study revealed that the combination ofDLPC and SAMe fully prevents the up-regula-tion of TIMP-I mRNA and increase in TIMP-1protein induced by leptin in LX-2 human HSC.The protection afforded by DLPC and SAMecombined is at least two times more effective thanthat by DLPC or SAMe alone. The prevention ofTIMP-1 production is associated with the inhibi-tion of H2O2-dependent ERK1/2 and p38 signaltransduction, TIMP-1 promoter activity, andTIMP-1 message stability. The findings are sche-matically represented in Fig. 9.We selected DLPC at 10mM and SAMe at

12mM for the combination treatment of LX-2cells. This is based on the observations thatDLPC or SAMe alone attenuated TIMP-1mRNA expression stimulated by leptin in a con-centration manner, with a maximal effect of 41%reduction at 10mM of DLPC or at 12mM ofSAMe. In combination, DLPC and SAMe fullyprevented TIMP-1 mRNA expression as well asits protein secreted into the culture media. Theaddition of DLPC at 10mM to the culture med-ium is relevant, because it is near the range ofconcentrations in the blood (15–27mM), as mea-sured in healthy volunteers given PPC (unpub-

lished observation). Furthermore, it representsjust a 10% increment measured in the livers ofbaboons fed DLPC as PPC (1). The use of SAMeat 12mM is also appropriate, because it is withinthe physiological concentrations in the extracel-lular space of the liver (o0.1mM) compared withpharmacological concentrations at 1mM orhigher (38). Uptake of SAMe from the culturemedium by LX-2 cells is expected, as SAMe atphysiological concentrations rapidly crosses thecell membranes and is converted to GSH, asshown in studies with cultured hepatocytes (28–30). The intracellular concentration of SAMe inrat hepatocytes has been reported to be 35 (30) to60mM (39), but there is no corresponding data inHSCs. It will be of future interest to measure theintracellular level of SAMe in HSC and to deter-mine whether these cells possess MAT, the keyenzyme in the conversion of methionine toSAMe. The localization of nonliver specific

0 2 4 6 8 10 12

100

50

0

TIM

P-1m

RN

A r

emai

ning

(%

, n=

3)

Time (hr)

t ½=4 hr t½=10 hr

LeptinLeptin + SB203580Leptin + catalase

Leptin + PD098059Leptin + DLPC+SAMe

Fig. 8. p38 inhibitor SB203580, catalase, and dilinoleoylpho-sphatidylcholine (DLPC)1S-adenosylmethionine (SAMe), butnot ERK1/2 inhibitor PD098059, decreased leptin-inducedtissue inhibitor of metalloproteinase (TIMP)-1 message stabi-lity. LX-2 cells were treated with leptin for 24 h to induce TIMP-1 mRNA expression, followed by actinomycin D treatment for20min to block the transcription, as described in the Materialsand Methods section. TIMP-1 mRNA stability in the presenceor absence of the inhibitors is expressed as the percent of themRNA remaining relative to the level at time zero at the start ofactinomycin D treatment.

Leptin

ROS (H2O2)

p-p38p-ERK1/2

PD098059 SB203580

Tyr-1141

TIMP-1 mRNA

TIMP-1 promoter

Stabilization

TIMP-1 protein

–102 +96

DLPC + SAMeCatalase

AG490p-JAK1

p-JAK2

OB-RL

Fig. 9. Schematic representation of tissue inhibitor of metallo-proteinase (TIMP)-1 induction by leptin and its inhibition bydilinoleoylphosphatidylcholine (DLPC) and S-adenosylmethio-nine (SAMe). The effects of AG490, catalase, ERK1/2 inhibitorPD098059, and p38 inhibitor SB203580 are also illustrated.Leptin triggers OB-RL signaling, phosphorylating Janus kinases(JAK)1 and JAK2, that, in turn, stimulate reactive oxygenspecies (ROS) formation, mainly H2O2, and the response isinhibited by AG490 (which blocks JAK activation). IncreasedH2O2 production activates ERK1/2 and p38 signaling path-ways. ERK1/2 stimulates TIMP-1 promoter activity with up-regulated TIMP-1 mRNA expression, while p38 stabilizesTIMP-1 message with enhanced TIMP-1 expression.DLPC1SAMe and catalase prevent H2O2 generation stimu-lated by leptin via JAKs, resulting in a blockade of ERK1/2 andp38 phosphorylation, suppression of TIMP-1 promoter activity,and decrease of TIMP-1 mRNA stability. These lead to adiminished TIMP-1 protein secretion into the culture media.

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MAT2 to rat HSCs, as observed by Simizu-Saitoet al. (40) suggested that SAMe synthesis frommethionine may occur in these cells.Leptin has been shown to be a fibrogenic factor

in liver fibrogenesis (24–26). As in a previouslystudy (23), we show here that TIMP-1 productionin LX-2 cells by leptin stimulation is associatedwith increased expression of OB-RL and activa-tion of JAK 1 and 2 that, in turn, stimulate theproduction of H2O2. The increased H2O2 is pre-vented by AG490, linking activation of JAKs tothe generation of the peroxide. In the presentstudy, we found that DLPC and SAMe combinedhad no effect on the up-regulation of the leptinreceptor or on the increased phosphorylation ofJAK1 and 2 after leptin, while they inhibited thegeneration of H2O2 and blocked its signalingthrough ERK1/2 and p38, consistent with thespecificity in their antioxidant properties.DLPC and SAMe combined were two to three

times more effective than DLPC or SAMe alone inthe prevention of H2O2 generation. Like catalase,which scavanges H2O2, DLPC1SAMe completelyblocked ERK1/2 and p38 activation phosphoryla-tion, resulting in a total suppression of TIMP-1mRNA expression and its protein production (seeFigs 2 and 6). The effect afforded by the combina-tion of DLPC and SAMe in the prevention ofTIMP-1 production doubled that by DLPC orSAMe alone. This is likely attributed to thesuppression of TIMP-1 promoter activity elicitedby leptin in conjunction with the decrease ofTIMP-1 message stability (see Figs 7 and 8). Bycomparison, the ERK1/2 inhibitor PD098059,which inhibited TIMP-1 promoter activity to thesame extent as DLPC1SAMe or catalase but hadno effect on the stability of TIMP-1 message,halved the level of TIMP-1 mRNA (see Fig. 6B).On the other hand, SB203580, a p38 inhibitor,which had no effect on TIMP-1 promoter activitybut destabilized TIMP-1 mRNA, was found todiminish TIMP-1 mRNA level to the same extentas PD098059 (see Fig. 6C). These results suggestthat a concomitant inhibition of TIMP-1 promo-ter activity at the transcriptional level and adecrease in its stability at the posttranscriptionallevel are required for a complete prevention ofTIMP-1 gene expression. In this context, DLPCand SAMe or catalase, which exhibit antioxidantproperties against H2O2 generation and its signaltransduction through ERK1/2 and p38 pathways,are more effective than the ERK1/2 inhibitorPD098059 or p38 inhibitor SB203580, which lackantioxidant actions, in the prevention of TIMP-1mRNA expression.In addition to the stimulation of ROS forma-

tion, mainly H2O2, which mediates cell signaling,

leptin treatment also resulted in a 45% decreasein cellular GSH level compared to untreated cells,reflecting a consumption of GSH in response tooxidative stress. DLPC alone replenished GSHlevel by 36%, and SAMe alone by 48%. Bycontrast, DLPC and SAMe combined normalizedthe level of GSH to that of untreated cells. As acompensatory mechanism for GSH consumptionbecause of oxidative stress, SAMe was utilized viathe transsulfuration pathway to replenish thecellular GSH (11, 12), which scavenges ROS,thereby correcting, at least partially, the leptin-induced oxidative stress in LX-2 cells. However, acomplete normalization of the oxidative stressrequires the presence of DLPC as well.Our data implicate that DLPC acts synergisti-

cally with SAMe in the replenishment of GSHthat was depleted in response to leptin treatment.DLPC (and its parent compound PPC) have highbioavailability and capacity for incorporatinginto cell membranes (41). As such, they couldplay a role in the repair of oxidative damage ofphosphatidylcholines (PCs), the backbone of cellmembranes. PCs are produced by stepwisemethylations of phosphatidylethanolamine cata-lyzed by the enzyme phosphatidylethanolaminetransferase (PEMT) (42). This reaction, which isactive in the liver (43), requires a considerableamount of SAMe, which provides the methylgroups to phosphatidylethanolamine to formPCs (44). For each mole of PC formed, 3mol ofSAMe are needed. Therefore, one could antici-pate that DLPC, by providing PCs, spares theutilization of SAMe for PC synthesis and therebycontributes to the restoration of the intracellularSAMe pool, allowing an unabated synthesis ofGSH with correction of the oxidative damageinduced by leptin. This hypothesis is strengthenedby the finding of Aleynik and Lieber (45) thatPPC administration in vivo restored the depletionof hepatic SAMe in response to oxidative stressafter ethanol feeding of rats. Furthermore, it wasfound that SAMe addition to cultured hepato-cytes attenuated the decrease of PC synthesisinduced by cytokines, in accordance with theutilization of SAMe for PC synthesis (29). Inview of the observation that DLPC in vitroprotected the oxidation of low-density lipopro-teins by free radicals (46), an additional mechan-ism can be considered by which DLPC couldprovide a ‘trap’ for free radicals, thereby attenu-ating their adverse effects.We conclude that both DLPC and SAMe have

antioxidant properties and antifibrogenic actionsin LX-2 HSC. The prevention of TIMP-1 pro-duction by DLPC1SAMe is due, at least in part,to the inhibition of the oxidant H2O2 and its

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signal transduction. Since DLPC and SAMecombined are more effective than DLPC orSAMe alone, and since decreased TIMP-1 pro-duction may enhance collagen deposition, thecombination of DLPC and SAMe, two innocu-ous complementary neutraceuticals, should beconsidered for the prevention of ROS formationand the resulting fibrosis. Admittedly, this studydoes not yet address other aspects of fibrogenesisin HSC, in particular collagen and matrix metal-loproteinase expression, or the efficacy of DLPCand SAMe on in vivo fibrogenesis, but nonethe-less, the approach with LX-2 cells offers a startingpoint for the advancement of our understandingof the link between the antioxidant and antifibro-genic actions of DLPC and SAMe in liver disease.

Acknowledgements

This study was supported, in part, by NIH grants AA11115,AT001583, the Department of Veterans Affairs, the Kings-bridge Research Foundation, and the Christopher D. SmithersFoundation. This study was presented in abstract form at the55th annual AASLD meeting in Boston, MA.

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