ligands of peroxisome proliferator-activated receptor-γinhibits proliferation and mcp-1 expression...

Ligands of Peroxisome Proliferator–Activated Receptor ggModulate Profibrogenic and Proinflammatory Actions inHepatic Stellate Cells

FABIO MARRA,* EVA EFSEN,* ROBERTO G. ROMANELLI,* ALESSANDRA CALIGIURI,*SABRINA PASTACALDI,* GIACOMO BATIGNANI,‡ ANDREA BONACCHI,* ROBERTO CAPORALE,§

GIACOMO LAFFI,* MASSIMO PINZANI,* and PAOLO GENTILINI**Dipartimento di Medicina Interna and ‡Dipartimento di Fisiopatologia Clinica, Universita di Firenze, Florence, Italy; and §Divisione diEmatologia, Azienda Ospedaliera Careggi, Florence, Italy

Background & Aims: Proliferation and migration of he-patic stellate cells (HSCs) and expression of chemokinesare involved in the pathogenesis of liver inflammationand fibrogenesis. Peroxisome proliferator–activated re-ceptor (PPAR)-g is a receptor transcription factor thatcontrols growth and differentiation in different tissues.We explored the effects of PPAR-g agonists on thebiological actions of cultured human HSCs. Methods:HSCs were isolated from normal human liver tissue andused in their myofibroblast-like phenotype or immedi-ately after isolation. Activation of PPAR-g was inducedwith 15-deoxy-D12,14-prostaglandin J2 or with troglita-zone. Results: PPAR-g agonists dose-dependently inhib-ited HSC proliferation and chemotaxis induced by plate-let-derived growth factor. This effect was independent ofchanges in postreceptor signaling or expression of c-fosand c-myc and was associated with inhibition of cellcycle progression beyond the G1 phase. Activation ofPPAR-g also resulted in a complete inhibition of theexpression of monocyte chemotactic protein 1 at thegene and protein levels. Comparison of quiescent andculture-activated HSCs revealed a marked decrease inPPAR-g expression in activated cells. Conclusions: Acti-vation of PPAR-g modulates profibrogenic and proin-flammatory actions in HSCs. Reduced PPAR-g expres-sion may contribute to confer an activated phenotype toHSCs.

Hepatic stellate cells (HSCs) represent the key cellu-lar elements in the liver wound healing process and

development of hepatic fibrosis.1 The ability of HSCs tomodulate liver tissue repair is dependent on a processknown as activation.1,2 Upon liver injury, HSCs acquirethe ability to proliferate and migrate toward the dam-aged areas and increase the production of extracellularmatrix components. In addition, activated HSCs regulatethe recruitment of inflammatory cells via secretion ofchemotactic factors, including chemokines, and immu-nomodulatory cytokines such as interleukin (IL)-10.3

These properties of the activated or myofibroblast-likephenotype of HSCs contribute to the morphologic andfunctional changes observed during chronic liver dam-age. The molecular mechanisms regulating the transitiontoward myofibroblast-like cells are only partially under-stood. Because the activation process is associated withde novo expression of specific genes, attention has fo-cused on molecules capable of regulating gene transcrip-tion. Generation of lipid peroxidation products fromdamaged hepatocytes has been suggested to promoteHSC activation via transcription factors such as c-myband nuclear factor–kB (NF-kB).4 More recently, it hasbeen reported that Zf9, a Kruppel-like transcriptionfactor, is rapidly induced during the process of HSCactivation in vivo and in vitro and transactivates thepromoters of genes up-regulated during fibrogenesis,including type I collagen, transforming growth factor b,and its receptors.5,6 In addition, it is known that HSCactivation is associated with loss of retinoids,7,8 a groupof molecules with potent effects on gene transcriptionand cell differentiation.

Peroxisome proliferator–activated receptors (PPARs)are a family of ligand-activated nuclear transcriptionfactors belonging to the nuclear hormone receptor super-family.9 Three mammalian subtypes have been identi-fied, referred to as PPAR-a, -b (or -d), and -g, which areencoded by separate genes.10 Transcriptional regulationby PPAR occurs through binding to specific regulatory

Abbreviations used in this paper: 15d-PGJ2, 15-deoxy-D12,14-prosta-glandin J2; ERK, extracellular signal–regulated kinase; HSC, hepaticstellate cell; IFN, interferon; IL, interleukin; MCP-1, monocyte chemo-tactic protein 1; NF-kB, nuclear factor–kB; PDGF, platelet-derivedgrowth factor; PPAR-g, peroxisome proliferator–activated receptor g;RXR, retinoid X receptor; SDS-PAGE, sodium dodecyl sulfate–polyac-rylamide gel electrophoresis; TNF, tumor necrosis factor.

© 2000 by the American Gastroenterological Association0016-5085/00/$10.00

doi:10.1053/gast.2000.9365

GASTROENTEROLOGY 2000;119:466–478

elements located in noncoding regions of the gene. Bind-ing to the regulatory element requires the formation of aheterodimeric complex comprised of a PPAR and theretinoid X receptor (RXR), another member of the samefamily of transcription factors.11 The 3 PPAR subtypesdiffer in terms of ligand specificity. Agonists of PPAR-ginclude oxidative metabolites of polyunsaturated fattyacids,12 and prostaglandins (PG) of the J series, including15-deoxy-D12,14-PGJ2 (15d-PGJ2), which is by far themost potent activator.13,14 Although the precise enzy-matic pathway leading to 15d-PGJ2 is not completelyunderstood, it has been shown that these compounds areproduced in intact cells and organisms15 and are likely torepresent physiologic ligands for PPAR-g. PPAR-g isalso bound and activated by antidiabetic drugs of thethiazolidinedione group, such as troglitazone, and bysome nonsteroidal anti-inflammatory drugs.16,17

Activation of PPAR-g has been shown to play aleading role in the process of adipocyte differentiationand glucose metabolism, and thiazolidinedione ana-logues are used as antidiabetic drugs in the clinicalpractice.18,19 Studies show that this transcription factorregulates neoplastic cell growth and modulates monocyteactivation and differentiation.20–23 Because PPAR-g hasthe ability to control gene transcription and cellulardifferentiation, we evaluated whether PPAR-g agonistscould modulate the biological actions that characterizethe activated phenotype of HSCs. We report that treat-ment of cultured human HSCs with ligands of PPAR-ginhibits cell proliferation, migration, and chemokineexpression, 3 actions relevant to the process of liverwound healing and fibrogenesis.

Materials and MethodsReagents

15d-PGJ2 was purchased from Cayman Chemical Co.(Ann Arbor, MI). Aliquots of the original solution in ethylacetate were dried under a nitrogen stream and resuspended indimethyl sulfoxide. Troglitazone was kindly provided by Dr.Toshihiko Hashimoto (Sankyo Co. Ltd., Tokyo, Japan) anddissolved in dimethyl sulfoxide. The final concentration ofdimethyl sulfoxide was maintained equal to 0.1% (vol, vol) inall experimental conditions. Monoclonal, agarose-conjugatedantiphosphotyrosine antibodies were purchased from Calbio-chem (La Jolla, CA). Phosphospecific antibodies against extra-cellular signal–regulated kinase (ERK) and p38MAPK were pur-chased from New England Biolabs (Beverly, MA). Polyclonalantibodies directed against ERK, p38MAPK, and PPAR-g werepurchased from Santa Cruz Biotechnology (Santa Cruz, CA),and monoclonal antiphosphotyrosine antibodies (clone 4G10)from Upstate Biotechnology Inc. (Lake Placid, NY). The rab-bit antiserum against baboon monocyte chemotactic protein

(MCP)-1 (100% cross-reacting with human MCP-1) was akind gift of Dr. Anthony J. Valente (University of TexasHealth Science Center at San Antonio, TX). The luciferasereporter plasmid under the control of PPAR-g (ACO-PPRE)was generously provided by Dr. Brian Seed (MassachusettsGeneral Hospital, Boston, MA). Phosphatidylinositol wasfrom Sigma Chemical Co. (St. Louis, MO). Protein A–Sepha-rose was purchased from Pharmacia (Uppsala, Sweden).[g-32P]Adenosine triphosphatase (3000 Ci/mmol) and [methyl-3H]thymidine were from New England Nuclear (Milan, Italy).Human recombinant platelet-derived growth factor (PDGF)-BB, IL-1a, tumor necrosis factor (TNF)-a, and interferon(IFN)-g were from Peprotech (London, England). All otherreagents were of analytical grade.

Isolation and Culture of HSCs

Human HSCs were isolated from wedge sections ofliver tissue unsuitable for transplantation by collagenase/pro-nase digestion and centrifugation on stractan gradients. Pro-cedures used for cell isolation and characterization have beendescribed extensively.24 Purity of the cell was .95%. Unlessindicated otherwise, all the experiments were conducted oncells cultured on uncoated plastic dishes (passage 3–6), show-ing an activated or myofibroblast-like phenotype. To assess thelevels of PPAR-g in the activation process, HSCs were washedand the pellet was frozen in liquid nitrogen and stored at280°C until lysis and quantified for protein concentration asindicated later. Aliquots of freshly isolated cells (4 3 106 cells)were seeded onto 100-mm petri dishes and cultured in com-plete medium for 3 days or until the second passage. Cellularproteins were prepared and quantified as described later.

Measurement of DNA Synthesis

Confluent HSCs in 24-well dishes were washed withphosphate-buffered saline (PBS) and incubated in serum-freemedium for 48 hours. The cells were incubated with PPAR-gligands for 15 minutes, and then with different mitogens foran additional 24 hours. DNA synthesis was measured as theincorporation of [3H]thymidine, as described previously.25

Cell Migration Assay

Confluent HSCs were serum-starved for 48 hours andthen exposed to PPAR-g ligands for 15 minutes. The cellswere then washed, trypsinized, and resuspended in serum-freemedium containing 1% albumin at a concentration of 3 3 105

cells/mL. Chemotaxis was measured in modified Boyden cham-bers equipped with 8-mm-pore filters (Poretics, Livermore,CA) coated with rat tail collagen (Collaborative BiomedicalProducts, Bedford, MA) as previously described.26 At the endof the incubation, the filters were fixed, stained with Giemsa(Merck, Darmstadt, Germany), mounted, and viewed at 4503magnification. Data are expressed as the average of cell countsobtained in 10 randomly chosen high-power fields.

August 2000 PPAR–g AND HEPATIC STELLATE CELLS 467

Analysis of MCP-1 Secretion

Confluent HSCs in 24-well plates were deprived ofserum for 24 hours. After replacement of the medium withfresh serum-free medium, the cells were exposed to PPAR-gagonists for 15 minutes and then to IL-1, TNF, or IFN-g for24 hours. At the end of the incubation, the medium wascollected and stored at 220°C until assaying. MCP-1 secretionin the conditioned medium was measured using Western blotanalysis as previously described.27 Briefly, 50–100 mL of con-ditioned medium was dried, resuspended in Laemmli buffer,28

separated by 15% sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE), and electroblotted on a polyvi-nylidene-difluoride membrane. The membranes were blockedovernight at 4°C with 2% bovine serum albumin in 0.1%PBS–Tween, and then sequentially incubated at room temper-ature with an antiserum against baboon MCP-1 (1:1000) andwith a horseradish peroxidase–conjugated secondary antibody.Detection was performed using chemiluminescence accord-ing to the manufacturer’s protocol (Amersham, ArlingtonHeights, IL). This technique has been shown to detect as littleas 1 ng of human MCP-1 and has been previously validatedusing a commercially available enzyme-linked immunosorbentassay kit.27

Preparation of Cell Lysates

Confluent, serum-starved HSCs were treated with theappropriate conditions, quickly placed on ice, and washed withice-cold PBS. The monolayer was lysed in RIPA (radioimmu-noprecipitation assay) buffer (20 mmol/L Tris-HCl [pH 7.4],150 mmol/L NaCl, 5 mmol/L EDTA, 1% Nonidet P-40, 1mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride,and 0.05% [wt/vol] aprotinin). Insoluble proteins were dis-carded by high-speed centrifugation at 4°C. Protein concen-tration in the supernatant was measured in triplicate using acommercially available assay (Pierce, Rockford, IL).

ERK Assay

ERK was immunoprecipitated from 25 mg of total celllysate using polyclonal anti-ERK antibodies and proteinA–Sepharose. After washing, the immunobeads were incu-bated in a buffer containing 10 mmol/L HEPES (pH 7.4), 10mmol/L MgCl2, 0.5 mmol/L dithiothreitol, 0.5 mmol/LNa3VO4, 25 mmol/L adenosine triphosphatase, 1 mCi [g-32P]-adenosine triphosphatase, and 0.4 mg/mL myelin basic proteinfor 30 minutes at 30°C. At the end of the incubation, thereaction was stopped by addition of Laemmli buffer and run on15% SDS-PAGE. After electrophoresis, the gel was dried andautoradiographed.

Phosphatidylinositol 3-Kinase Assay

This assay was performed after immunoprecipitationwith antiphosphotyrosine antibodies, as described previously.26,29

Radioactive lipids were separated by thin-layer chromatogra-phy, using chloroform/methanol/30% ammonium hydroxide/water (46/41/5/8). After drying, the plates were autoradio-

graphed. The radioactive spots were then scraped and countedin a b-counter.

Western Blot Analysis of Cellular Proteins

Equal amounts of total cellular proteins were separatedby SDS-PAGE and analyzed by Western blot as described forthe MCP-1 assay. Immunoblot analysis of platelet-derivedgrowth factor (PDGF) receptor tyrosine phosphorylation wasconducted after immunoprecipitation. Briefly, 150 mg of totalcellular proteins was incubated with anti–PDGF-b receptorantibodies and protein A–Sepharose for 2 hours at 4°C. Theimmunobeads were washed twice in lysis buffer and once in 20mmol/L Tris-HCl (pH 7.4) and 1 mmol/L Na3VO4, resus-pended in Laemmli buffer, and analyzed by Western blot asdescribed earlier.

Analysis of Cell Cycle

The cell cycle was analyzed using a previously de-scribed technique with minor modifications.30 SubconfluentHSCs (60%–70% density) were serum-deprived and preincu-bated with 5 mmol/L 15d-PGJ2 or its vehicle for 15 minutes,and then exposed to 50 ng/mL PDGF for 20 hours. At the endof incubation, cells were harvested using PBS/EDTA andwashed twice with PBS. Cells were suspended in a solutioncontaining 50 mg/mL propidium iodide, 0.02% NonidetP-40, and 0.5 mg/mL ribonuclease A in PBS. Samples wereincubated in the dark at room temperature for 30 minutes andstored at 4°C until analysis. Cell fluorescence was measured byFACScan (BD, Franklin Lakes, NJ) and analyzed by the Mod-Fit LT 2.0 software (Verity Software House, Topsham, ME) todetermine the distribution of cells in the various phases of thecell cycle.

Preparation of Nuclear Extracts and GelMobility Shift Assay

Nuclear extracts were prepared according to Andrewand Faller.31 Gel mobility shift analysis was performed usingAP-1 or NF-kB consensus oligonucleotides (Promega, Madi-son, WI), as described previously.32 Briefly, 5–10 mg of nu-clear extracts was incubated for 30 minutes at room temper-ature in a buffer containing 35 mmol/L HEPES, pH 7.8, 0.5mmol/L EDTA, 0.5 mmol/L dithiothreitol, 10% glycerol, 10mg/mL polydI-dC, 0.28 mmol/L spermidine, and 50,000–100,000 cpm of 32P-labeled oligonucleotide probe. The DNA-protein complexes were separated by PAGE in 0.53 Tris-borate-EDTA. At the end of the run, the gel was dried andautoradiographed.

Northern Blot Analysis

Isolation of total RNA and Northern blot analysis wereperformed using previously described methods.33 After trans-fer, the blots were sequentially hybridized with radiolabeledcomplementary DNA probes encoding for c-fos, c-myc, MCP-1,and the ribosomal protein 36B4 (control gene).

468 MARRA ET AL. GASTROENTEROLOGY Vol. 119, No. 2

Analysis of Transcriptional Activity UsingReporter Plasmids

HSCs were seeded in 6-well dishes at a density of300,000 cells/well and grown in complete medium for 24hours. The cells were then washed and transfected with 2.5 mgof the reporter plasmid ACO-PPRE and 1.5 mg of a b-galac-tosidase expression vector using 4 mL of Lipofectamine (LifeTechnologies, Rockville, MD). The ACO-PPRE reporter plas-mid contains 3 copies of PPAR response element from thepromoter of rat acyl coenzyme A oxidase.23 After 5 hours,complete medium was added overnight and then replaced withfresh complete medium for 12 hours. The cells were thenserum-starved overnight and incubated with 15d-PGJ2 ordimethyl sulfoxide for an additional 24 hours. At the end ofthe incubation, the cells were lysed using 13 reporter lysisbuffer (Promega) and the protein concentration was measured.Luciferase activity was measured in a buffer containing 25mmol/L Gly-Gly buffer, 5 mmol/L adenosine triphosphatase,15 mmol/L MgSO4, and 0.6 mmol/L coenzyme A. Light wasmeasured for 10 seconds immediately after addition of lucif-erin (0.2 mmol/L). b-Galactosidase activity was measured us-ing a commercially available kit (Promega).

Data Analysis

Unless otherwise indicated, all data are representativeof at least 3 experiments with similar results. Statistical anal-ysis was performed by the Student t test or by 1-way analysisof variance, when appropriate. P values of 0.05 were consideredsignificant.

Results

PPAR-g Ligands Inhibit Proliferation andMigration of HSCs

We first investigated whether PPAR-g agonistscould modulate DNA synthesis of cultured human HSCsas an index of cell proliferation. As expected, incubationof serum-starved HSCs with PDGF induced a severalfoldincrease in the uptake of [3H]thymidine (Figure 1A).Preincubation of the cells with increasing concentrationsof the PPAR-g agonists 15d-PGJ2 or troglitazone re-sulted in a marked and dose-dependent inhibition ofDNA synthesis. At concentrations as high as 5 mmol/L15d-PGJ2, or 10 mmol/L troglitazone, the stimulation ofDNA synthesis by PDGF was virtually eliminated. Toconfirm the inhibitory effects of PPAR-g agonists on cellproliferation, we also tested the effects of 15d-PGJ2 andtroglitazone on DNA synthesis in response to fetal bo-vine serum (Figure 1B). In this case also, the markedincrease in cell proliferation was inhibited by PPAR-gligands in a dose-dependent fashion, although the effectswere less evident than in cells treated with PDGF. We

also tested the effects of 15d-PGJ2 on the proliferativeresponse induced by other mitogens, such as epidermalgrowth factor and thrombin.25,34 The stimulation ofthymidine uptake was less pronounced than in responseto fetal bovine serum, but exposure to the PPAR-gagonist blocked cell proliferation (Figure 1C). These data

Figure 1. PPAR-g ligands inhibit proliferation of HSCs. Serum-starvedHSCs were preincubated with increasing concentrations of 15d-PGJ2

or troglitazone for 15 minutes, before exposure to (A) 10 ng/mL PDGFor (B) 10% fetal bovine serum. (C) HSCs were preincubated with 5mmol/L 15d-PGJ2 or its vehicle before exposure to 100 ng/mL epi-dermal growth factor (EGF) or 5 U/mL thrombin (THR). Fetal bovineserum (10%) was used as a positive control. After 24 hours, DNAsynthesis was measured as the incorporation of [3H]thymidine.Mean 6 SD of a representative experiment.


indicate that exposure of HSCs to PPAR-g ligands in-hibits HSC proliferation independently of the mitogenused.

Because chemotaxis represents an important character-istic of cells involved in the wound healing response inseveral tissues, we tested the effects of 15d-PGJ2 andtroglitazone on PDGF-induced migration of HSCs.PDGF stimulated an 8-fold induction of HSC migration(Figure 2), as reported previously.32 Incubation with15d-PGJ2 or troglitazone resulted in a marked inhibitionof HSC chemotaxis. Remarkably, low concentrations ofPPAR-g ligands resulted in ,75% reduction of cellmigration, indicating that this parameter is very sensi-tive to the effects of these compounds.

Effects of PPAR-g Ligands on PDGF’sSignaling, Proto-oncogene Activation,and Cell Cycle

We next investigated whether the observed effectsof PPAR-g ligands could be mediated by changes inPDGF-dependent postreceptor signaling or proto-onco-gene activation. Although PDGF activates several intra-cellular pathways,35 we focused on the signaling inter-mediates that have been shown to be necessary forPDGF-induced mitogenic or motogenic action inHSCs.27,32 Autophosphorylation on tyrosine residues im-mediately follows the binding of PDGF dimers to thecognate receptors. Exposure of serum-starved HSCs toPDGF resulted in the appearance of an evident increasein receptor tyrosine phosphorylation, as evaluated byimmunoprecipitation followed by immunoblot analysis(Figure 3A). Preincubation with PPAR-g ligands, atconcentrations that markedly reduce DNA synthesis orcell migration, did not inhibit PDGF-receptor phosphor-ylation. Activation of phosphatidylinositol 3-kinase (PI3-K) is mediated by the recruitment of this enzyme bythe activated PDGF receptor,36 and inhibitors of PI 3-Kactivity completely block PDGF-induced proliferationand chemotaxis.27 However, the increase in PI 3-K ac-tivity induced by PDGF (Figure 3B) was unchanged incells treated with troglitazone or 15d-PGJ2, indicatingthat the inhibition of cell proliferation and chemotaxiscaused by PPAR-g ligands is independent of any inter-ference with PI 3-K activation.

We reported recently that in PDGF-stimulated HSCs,activation of ERK is necessary for mitogenesis and con-tributes to chemotaxis.32 In addition, troglitazone has

Figure 2. PPAR-g ligands inhibit HSC chemotaxis. Serum-starvedHSCs were preincubated with increasing concentrations of 15d-PGJ2

or troglitazone for 15 minutes. The cells were then trypsinized, andcell migration to 10 ng/mL PDGF was measured in modified Boydenchambers. Results are expressed as the mean number of cells (6SD)migrated to the underside of the filter in a representative experiment.

Figure 3. Effects of PPAR-g ligands on PDGF-induced tyrosine phosphorylation and PI 3-K activation. (A) Serum-starved HSCs were preincubatedwith 3 mmol/L troglitazone or with different concentrations of 15d-PGJ2 before addition of 10 ng/mL PDGF for 10 minutes. Total cell lysates wereimmunoprecipitated with anti–PDGF-b receptor antibodies, separated by SDS-PAGE, and blotted with antiphosphotyrosine antibodies. Migrationof the molecular-weight marker is shown on the left. (B) Serum-starved HSCs were preincubated with different concentrations of troglitazone or15d-PGJ2 before addition of 10 ng/mL PDGF for 10 minutes. Analysis of PI 3-K activity was performed on antiphosphotyrosine immunoprecipi-tates as described in Materials and Methods. Migration of 3-OH–phosphorylated phosphatidylinositol is shown on the left.


been shown to interfere with the ERK pathway in vas-cular smooth muscle cells.37,38 To assess whether inhibi-tion of ERK could contribute to the effects of PPAR-gligands on HSCs, we analyzed the activation of ERK incells exposed to PDGF in the presence or absence oftroglitazone or 15d-PGJ2. ERK activation is associatedwith phosphorylation of specific threonine and tyrosineresidues by the upstream kinase MEK. We found thatneither of the 2 PPAR-g ligands affected ERK activationwhen we used antibodies specifically recognizing thephosphorylated form of ERK (Figure 4A). To confirmand extend this observation, we immunoprecipitatedERK from total cell lysates and measured ERK activityin an immune complex kinase assay using myelin basicprotein as a substrate (Figure 4B). Also in this case, theevident increase induced by incubation with PDGF was

unaffected by pretreatment with the 2 PPAR-g ligands.Together, these experiments show that agonists ofPPAR-g modulate PDGF action without interferingwith postreceptor signaling pathways known to be in-volved in mitogenesis or chemotaxis.

Activation of proto-oncogenes such as c-fos or c-mychas been shown to be implicated in the mitogenic sig-naling of several growth factors including PDGF.39,40

Expression of c-fos is mediated, at least in part, bymolecules located downstream of ERK and has beenshown to be inhibited by troglitazone in smooth musclecells. In HSCs, PDGF markedly increased the expressionof c-fos, but neither 15d-PGJ2 or troglitazone affected thesteady-state messenger RNA (mRNA) levels for thisproto-oncogene (Figure 5A). Similarly, exposure of HSCsto PDGF increased c-myc expression, but PPAR-g li-

Figure 4. Effects of PPAR-g ligands on ERK phosphorylation and activation. (A) Serum-starved HSCs were preincubated with 3 mmol/Ltroglitazone or 5 mmol/L 15d-PGJ2 before addition of 10 ng/mL PDGF for 10 minutes. Total cell lysate (40 mg) was separated by SDS-PAGE andsequentially immunoblotted with antibodies specifically recognizing the phosphorylated form of ERK (top) or directed against ERK (bottom).Migration of the molecular-weight marker is shown on the left. (B) Serum-starved HSCs were preincubated with 10 mmol/L troglitazone or5 mmol/L 15d-PGJ2 before addition of 10 ng/mL PDGF for 10 minutes. Total cell lysate (25 mg) was immunoprecipitated with anti-ERK antibodies,and ERK activity was measured as the ability to phosphorylate myelin basic protein as described in Materials and Methods.

Figure 5. Effects of PPAR-g ligands on proto-oncogene expression in HSCs. (A) Serum-starved HSCs were preincubated with 3 mmol/Ltroglitazone or 5 mmol/L 15d-PGJ2 before addition of 10 ng/mL PDGF for the indicated time points. Total RNA (15 mg) was sequentially analyzedby Northern blotting with probes encoding for c-fos and the ribosomal protein 36B4 (control gene). (B) Serum-starved HSCs were preincubatedwith 3 mmol/L troglitazone before addition of 10 ng/mL PDGF for 60 minutes. Total RNA (15 mg) was sequentially analyzed by Northern blottingwith probes encoding for c-myc and the ribosomal protein 36B4.


gands did not inhibit this effect (Figure 5B, and data notshown). These results show that PPAR-g acts down-stream of early nuclear signaling and proto-oncogeneactivation mediated by PDGF.

To better define the level of action of PPAR-g agonistson PDGF-induced mitogenesis, we analyzed the cellcycle in HSCs in the presence or absence of 15d-PGJ2

(Figure 6). As expected, exposure to PDGF was associ-ated with a marked decrease in the percentage of cells inthe G0/G1 phase, together with an increase in the num-ber of cells in the S phase. Addition of the PPAR-gagonist before PDGF reduced the number of cells in theS phase, in agreement with the effects on [3H]thymidineuptake, and the percentage of cells in the G0/G1 phasewas similar to the percentage observed in untreated cells.Thus, addition of 15d-PGJ2 inhibits PDGF-induced pro-gression of the cell cycle beyond the G1 phase.

PPAR-g Ligands Inhibit Expression andSecretion of MCP-1

Activated HSCs acquire the ability to modulatethe recruitment and activation of inflammatory cells. Theproinflammatory role of HSCs is well exemplified by theexpression of MCP-1, a potent chemoattractant formonocytes and T lymphocytes, which is secreted at highlevels by HSCs in their myofibroblast-like phenotype.3

Secretion of MCP-1 in HSC-conditioned medium wasdetectable by immunoblotting and appeared as 2 strongbands and a weaker band because of differences in gly-cosylation.3 MCP-1 secretion is stimulated by severalproinflammatory cytokines, including IL-1, TNF-a, andIFN-g (Figure 7).3 Exposure of HSCs to PPAR-g ligandsbefore the addition of the cytokines resulted in a dose-dependent reduction of the amount of immunoreactiveMCP-1 detectable in the conditioned medium (Figure7A, B, and C). Whereas 0.5–1 mmol/L PGJ2 generallyresulted in an inhibition of approximately 50%, as eval-uated by densitometric analysis, concentrations of 5mmol/L 15d-PGJ2 or 10 mmol/L troglitazone virtuallyabolished the cytokine-mediated induction of MCP-1secretion.

We have previously shown that the up-regulation ofMCP-1 secretion in response to proinflammatory cyto-kines in HSCs is accompanied by an increase in MCP-1gene expression. Accordingly, IL-1, TNF, and IFN-gincreased the abundance of MCP-1 mRNA (Figure 8Aand 8B). The expression levels of MCP-1 in response toall 3 agonists were reduced by 15d-PGJ2 (Figure 8A).Troglitazone had similar effects, although the inhibitionof MCP-1 mRNA expression was somehow less impres-sive (Figure 8B, and data not shown).

Figure 6. Effects of PPAR-g ligands on cell cycle. Serum-starved HSCswere preincubated with 5 mmol/L 15d-PGJ2 or its vehicle and eitherleft untreated or incubated with 50 ng/mL PDGF for 20 hours asindicated. At the end of the incubation, the cells were detached andcell cycle analysis was performed as described in Materials andMethods. Data show the percentage of cells in each phase of the cellcycle in a representative experiment.

Figure 7. PPAR-g ligands inhibitcytokine-induced secretion ofMCP-1. Serum-starved HSCswere preincubated with the indi-cated concentrations of 15d-PGJ2 or with troglitazone (B, 10mmol/L; C, 3 mmol/L) before ad-dition of IL-1 (A, 4 ng/mL), TNF-a(B, 100 ng/mL), or IFN-g (C,1000 U/mL) for 24 hours.Aliquots of the conditioned me-dium were separated by SDS-PAGE and immunoblotted withanti–MCP-1 antibodies. Migra-tion of the molecular-weightmarker is shown on the left.


Inhibition of MCP-1 Occurs Independentlyof Changes in the Activity of p38MAPK or inthe Activation of NF-kB or AP-1

The intracellular signaling pathways leading fromreceptor activation to MCP-1 expression are only par-tially known. Activation of p38MAPK, a member of thestress-activated protein kinase family, has been shown tobe necessary for induction of many inflammation-relatedgenes, including MCP-1.41 As described earlier for ERK,activation of p38MAPK requires phosphorylation on thre-onine and tyrosine residues by a dual-specificity kinase,and the activation of the molecule may be detected usingphosphospecific antibodies. Exposure of HSCs to TNF-aresulted in increased phosphorylation of p38MAPK (Figure9). However, neither 15d-PGJ2 or troglitazone, at con-centrations that markedly reduce MCP-1 expression andsecretion, modified the activation of this kinase.

The inhibition of monocyte activation by PPAR-gligands has been shown to be associated with reducedtranscriptional activity of NF-kB or AP-1–driven pro-

Figure 8. Inhibition of MCP-1 gene expression byPPAR-g ligands. (A) Serum-starved HSCs were pre-incubated with 5 mmol/L 15d-PGJ2 for 15 minutesbefore addition of IL-1 (4 ng/mL), TNF-a (100 ng/mL), or IFN-g (1000 U/mL) for 4 hours. (B) Serum-starved HSCs were preincubated with 10 mmol/Ltroglitazone for 15 minutes before addition of 100ng/mL TNF-a for 4 hours. Total RNA (15 mg) wassequentially analyzed by Northern blotting withprobes encoding for MCP-1 and for the ribosomalprotein 36B4.

Figure 9. Effects of PPAR-g ligands on phosphorylation of p38MAPK.Serum-starved HSCs were preincubated with 3 mmol/L troglitazone or5 mmol/L 15d-PGJ2 before addition of 100 ng/mL TNF-a for 15minutes. Total cell lysate (40 mg) was separated by SDS-PAGE andsequentially immunoblotted with antibodies specifically recognizingthe phosphorylated form of p38MAPK (top) or directed against p38MAPK

(bottom).


moters.22 Because NF-kB and AP-1 are also importantfor MCP-1 transcription, we analyzed the effects ofPPAR-g agonists on the activation of these factors. TNFincreased the DNA-binding activity present in HSCnuclear extracts to consensus oligonucleotides recogniz-ing NF-kB (Figure 10A) or AP-1 (Figure 10B), as shownby electrophoretic mobility shift assays. However, pre-incubation of HSCs with 5 mmol/L 15d-PGJ2 did notaffect the activation of these transcriptional regulators,indicating that the inhibitory effect of PPAR-g ligandson MCP-1 expression is independent of these pathways.

The Effects of PPAR-g Ligands on CulturedHSCs Are Not Caused by Cell Toxicity

To rule out that the actions of PPAR-g ligands onHSCs could be mediated by cell toxicity, we evaluatedthe effects of these compounds on cell viability. Asshown in Table 1, no differences in cell viability wereobserved using the highest concentration of PPAR-gligands and the longest time point (24 hours) used in thepresent study. In addition, when PPAR-g agonists wereadded to HSC cultures for 45 minutes, and subsequentlywithdrawn, the inhibitory effects on MCP-1 secretionwere partially reverted (data not shown), further confirm-ing the lack of toxic effects in this system. We alsoinvestigated whether PPAR-g exerts transcriptional ef-fects in cultured human HSCs. In cells transfected with

a luciferase reporter plasmid under the control ofPPAR-g (ACO-PPRE),23 exposure to 15d-PGJ2 induceda 2-fold, statistically significant increase in the activity ofthe reporter gene (Figure 11), indicating that PPAR-gagonists are transcriptionally active in HSCs.

Reduced Expression of PPAR-gin Activated HSCs

The levels of PPAR-g are markedly variable indifferent tissues and cultured cells. We evaluatedwhether the phenotypic changes observed in the processof HSC activation are accompanied by modifications inthe levels of PPAR-g. We compared cell lysates obtainedfrom HSCs immediately after isolation from normal livertissue, and, therefore, showing a quiescent phenotype,with those from cells cultured for 3 days or after 2passages in culture, when transition to a myofibroblast-like phenotype is complete (Figure 12). In freshly iso-lated HSCs a clearly detectable band of an apparentmolecular weight corresponding to that of PPAR-g was

Figure 10. Effects of PPAR-g ligands on activation of NF-kB and AP-1.Serum-starved HSCs were preincubated with 5 mmol/L 15d-PGJ2

before addition of 100 ng/mL TNF-a for 45 minutes. Total nuclearextract (5 mg) was used in gel mobility shift assays using consensusoligonucleotides for (A) NF-kB or (B) AP-1. Migration of the shiftedcomplex is indicated by arrows.

Table 1. Evaluation of the Toxic Effects of PPAR-g Agonistson Cultured HSCs

Living cells (% of total)

DMSO 96.7 6 1.715d-PGJ2 95.1 6 1.4Troglitazone 98.4 6 1.6

NOTE. Subconfluent HSCs were incubated with 0.1% dimethylsulfox-ide (vehicle), 5 mmol/L 15d-PGJ2, or 10 mmol/L troglitazone for 24hours. At the end of incubation, floating cells were collected andpooled with adherent cells after trypsinization. The number of alivecells was measured by trypan blue exclusion; results are expressedas mean 6 SEM (n 5 3). No significant differences among the 3groups were observed by 1-way ANOVA.

Figure 11. 15d-PGJ2 activates PPAR-g–induced transcription in HSCs.Subconfluent HSCs were transfected with a reporter plasmid underthe control of PPAR-g and an expression vector for b-galactosidase, asdescribed in Materials and Methods. The cells were then incubatedwith 15d-PGJ2 or its vehicle for 24 hours, and the levels of luciferaseand b-galactosidase activity were determined. The data representluciferase activity (arbitrary units) normalized for b-galactosidase ac-tivity and protein concentration. Mean 6 SEM of 3 experiments. *P ,0.05.


observed. Culture of HSCs for 3 days led to a markedreduction (;60% by densitometric analysis) of PPAR-gexpression, which became barely detectable after cellsubculture, a condition associated with a fully activatedphenotype, as shown by expression of a–smooth muscleactin (Figure 12).

Discussion

Understanding the molecular mechanisms thatregulate the biology of HSCs has a major relevance forthe pathophysiology of liver fibrosis.2,42 The data of thepresent study indicate that activation of PPAR-g, amember of the nuclear hormone–receptor superfamily,modulates different biological actions of HSCs that con-tribute to the process of liver inflammation and fibro-genesis. Exposure of HSCs to 2 well-established ligandsof PPAR-g, the endogenously produced prostanoid 15d-PGJ2 and troglitazone, resulted in complete inhibition ofHSC proliferation, migration, and expression of the che-mokine MCP-1, 3 biological actions associated with theactivated phenotype. We also found a striking decreasein the expression of PPAR-g in activated vs. quiescentHSCs, as established using freshly isolated and culture-activated human HSCs. Interestingly, PPAR-g levels

were reduced as early as after 3 days in culture, when theexpression of a–smooth muscle actin is not yet evident.The decrease in PPAR-g at the early stages of HSCactivation and the fact that PPAR-g ligands inhibit atleast some of the characteristics associated with the ac-tivated phenotype of HSCs suggest that PPAR-g may beinvolved in the maintenance of a quiescent phenotype.Along these lines, the ability of PPAR-g to regulatedifferentiation and phenotypic modulation has beenshown in several systems, as in the cases of the transitionof preadipocytes to adipocytes,19 differentiation of mono-cytes to macrophages and foam cells,43 or reversal of thetransformed phenotype in cancer cells.20,21

The molecular mechanisms responsible for the effectsof PPAR-g agonists are still controversial. In rat smoothmuscle cells, troglitazone interfered with the Ras/ERKpathway at the level of ERK activation or of c-fos expres-sion, depending on the agonist used.37,38 Therefore, in-hibition of the ERK cascade has been suggested tomediate the inhibition of proliferation and migration inthese cells.37,38 In contrast, the results of this study showthat neither troglitazone nor 15d-PGJ2 inhibits ERKphosphorylation or activation at concentrations thatcompletely block proliferation and migration of HSCs.In addition, the PDGF-induced increase in c-fos expres-sion, which is at least partly dependent on ERK activa-tion, was unchanged in HSCs exposed to PPAR-g ago-nists. We also explored other pathways that have beenshown to contribute to transduce PDGF mitogenic ormotogenic signals. However, neither activation of PI 3-Knor expression of c-myc was affected by PPAR-g ligands.These findings indicate that in HSCs the effects ofPPAR-g activators on cell proliferation and migrationoccur downstream of the activation of postreceptor-sig-naling pathways and of proto-oncogene expression. Thishypothesis is supported by the observation that the in-hibition of HSC proliferation was observed irrespective ofthe agonist used because PPAR-g agonists blocked mi-togenesis also in response to epidermal growth factor andthrombin. In addition, analysis of the cell cycle in HSCsexposed to PPAR-g agonists showed the inability toproceed beyond the G1 phase when stimulated withPDGF. In adipocytes, where PPAR-g agonists induceterminal differentiation and withdrawal from the cellcycle, PPAR-g bypasses the requirement for Rb hyper-phosphorylation,44 and the growth-inhibitory effect ofPPAR-g on fibroblasts is mediated by the inhibition ofE2F/DP DNA-binding activity.45 These data in othersystems are in keeping with an action downstream ofERK activation or c-fos expression and with a block inthe G1 phase of the cell cycle.

Figure 12. Reduced expression of PPAR-g in activated HSCs. Totalcell lysate (40 mg) from freshly isolated human HSCs (lane 1), fromHSCs cultured on plastic for 3 days (lane 2), or after subculture (lane3) was analyzed by immunoblotting using an antibody directed againstPPAR-g (top panel). The membrane was stripped and reblotted with anantibody against a–smooth muscle actin (middle panel). The lowerpanel shows Ponceau red staining of the membrane indicating com-parable protein loading. Migration of the molecular-weight marker isindicated on the left.


The ability of 15d-PGJ2 and troglitazone to block theproduction of MCP-1 in HSCs indicates that PPAR-gmay interfere with the expression of members of thechemokine family. We focused our attention on MCP-1,as a paradigm chemokine, in consideration of its emerg-ing importance in the regulation of leukocyte traffickingwithin the liver. MCP-1 expression is directly related tothe number of monocytes infiltrating the portal tractduring chronic hepatitis,46 and its expression precedesleukocyte recruitment in a rodent model of liver dam-age.47 Expression of MCP-1, and of other chemokines, ismarkedly increased in activated HSCs and may be in-duced by several soluble mediators, including proinflam-matory cytokines.3 Exposure of HSCs to PPAR-g ligandsresulted in complete inhibition of MCP-1 gene andprotein expression, indicating that this class of com-pounds modulates a key mechanism leading to hepaticinflammation. Interestingly, recent data indicate thatmonocyte activation may be inhibited by PPAR-g li-gands.22,23 The ability of these compounds to modulatemonocyte gene expression is associated with inhibition oftranscription driven by NF-kB and AP-1 in reporterassays. Because NF-kB and AP-1 play an important rolein the regulation of transcription of the MCP-1 gene,48,49

we investigated whether activation of these transcrip-tional regulators was affected by PPAR-g ligands. How-ever, the DNA-binding activity of both NF-kB andAP-1 was not changed in cells exposed to effective con-centrations of a PPAR-g agonist. This is in agreementwith data by Peraldi et al.,50 who showed that PPAR-gagonists block insulin resistance induced by TNF-a butdo not affect the ability of this cytokine to activateNF-kB. PPAR-g may modulate transcription at a leveldownstream of the interaction of transcription factorswith their responsive elements. Alternatively, the inhib-itory effect on chemokine expression may be the result ofthe effect on specific regulatory sequences in the MCP-1gene. This latter hypothesis is indirectly supported bythe observation that PPAR-g inhibits MCP-1 expressionelicited by 3 different agonists: IL-1, TNF, and IFN-g.Although IL-1 and TNF are believed to activate MCP-1transcription through partially overlapping pathways, ithas been recently shown that IFN-g–induced transcrip-tion of MCP-1 occurs through specific regulatory ele-ments in the 59-flanking region.51 These data suggestthat the action of PPAR-g is mediated by a specific effecton the MCP-1 gene independent of the activating cyto-kine used.

An intriguing aspect of the present results is related tothe interaction between PPAR-g and the retinoids. Theretinoids activate RXR and the retinoid acid receptor,

which are members of the same family of nuclear hor-mone receptors that include PPAR-g, and PPAR-g re-quires RXR to form a transcriptionally active het-erodimer.11 The HSCs play an important role in thestorage and metabolism of retinoids, and it has beenextensively shown that acquisition of the activated phe-notype is associated with loss of retinoid content.7,8

Treatment of HSCs with retinoids inhibits several bio-logical actions that accompany the activated phenotype,such as cell proliferation or expression of extracellularmatrix.52 A recent study showed that exposure of HSC to9-cis retinoic acid, a metabolite that selectively activatesRXR, results in the reduction of cell proliferation.53

Moreover, the mRNA levels for RXR-a were decreasedin HSC isolated from bile duct–ligated rats.54 Thesefindings are particularly interesting because ligands ofRXR have been shown to exert biological actions similarto those elicited by PPAR-g agonists, and the simulta-neous addition of the 2 types of ligands is associated witha synergistic effect.55 These data, together with our re-sults, suggest that vitamin A metabolites and PPAR-gligands may cooperate in maintaining a quiescent HSCphenotype in the normal liver.

The identification of PPAR-g as a novel modulator ofHSC biology raises a number of questions on its possibleregulatory mechanisms. In quiescent HSCs, where highexpression is observed, the molecules responsible forPPAR-g activation need to be identified. Besides PG ofthe J series, other endogenous activators of PPAR-g arebeing discovered, including oxidative metabolites of li-noleic acid.12 It will be important to establish if these orother compounds are synthesized in quiescent cells andwhether their abundance decreases along with the acti-vation process. Recently, rat liver myofibroblasts havebeen characterized as another matrix-producing cell par-tially distinct from activated HSCs.56 Although thesecells have not yet been characterized in the human liver,it will be relevant to understand whether they exhibitdifferent levels of PPAR-g or are differentially modu-lated by agonists of this transcription factor. An impor-tant consequence of our present data is the potentialapplication of PPAR-g agonists in the treatment of liverfibrosis. Most drugs capable of blocking the biologicalactions of HSCs in vitro may not be used in vivo unlessa delivery system is developed to selectively target thesecells. On the other hand, thiazolidinediones have beenapproved for use in type II diabetes and are generally welltolerated. The in vitro data obtained in this study andthe relative safety of thiazolidinediones call for in vivostudies aimed at establishing whether interference with


the PPAR-g pathway is beneficial for the treatment ofliver fibrosis.

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Received August 26, 1999. Accepted March 3, 2000.Address requests for reprints to: Fabio Marra, M.D., Ph.D., Diparti-

mento di Medicina Interna, Viale Morgagni, 85, I-50134 Florence, Italy.e-mail: [email protected]; fax: (39) 055-417-123.

Supported by a MURST grant (project: molecular and cellular biologyof hepatic fibrosis) and by the Italian Liver Foundation.

The authors thank Wanda Delogu and Chiara Sali for skillful tech-nical help and Drs. T. Hashimoto, A. J. Valente, and B. Seed forproviding some of the reagents used in this study.


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