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  • TLR4 Enhances TGF-! Signaling and Hepatic Fibrosis

    Ekihiro Seki1,2, Samuele De Minicis1,2, Christoph H. Österreicher1,2, Johannes Kluwe1,

    Yosuke Osawa1, David A. Brenner2, and Robert F. Schwabe1

    SUPPLEMENTARY METHODS

    Mice and Fibrosis induction

    CCl4 (Sigma-Aldrich, diluted 1:3 in corn oil) or vehicle (corn-oil) was administered by

    gavage at a dose of 1 µl/g body weight every five days for a total of eight injections.

    TAA-induced liver fibrosis was induced by administering TAA (Sigma-Aldrich) at a dose

    of 300 mg/L in drinking water for 20 weeks. Some mice were infected with adenoviruses

    expressing dnBAMBI or empty Adshuttle control virus (1x109 pfu/ml in PBS i.v./mouse)

    48 hours before BDL. Some mice received liposomal clodronate (200 µl i.v.) one day

    before BDL to deplete KCs. Liposomal clodronate was prepared as described 1.

    Microarray

    Six hours after isolation, quiescent HSCs were treated with E.coli 055:B5 LPS (Sigma) at

    a concentration of 100 ng/ml or vehicle (endotoxin-free H2O) for six hours (n=3 HSC

    isolations). RNA was extracted by a combination of Trizol (Invitrogen) and RNeasy

    columns (Qiagen). Microarray analysis was performed using mouse genome 430 2.0

    array gene chips (Affymetrix) according to the manufacturer’s instructions. Briefly, first

    strand and second strand synthesis were performed using the one-cycle cDNA synthesis

    kit (Affymetrix) followed by cleanup of double-stranded cDNA using cDNA spin columns

    (Affymetrix). Biotin-labeled cRNA was generated using one cycle IVT labeling kit

    followed by cRNA cleanup using cRNA spin columns (both Affymetrix). cRNA

    fragmentation which was confirmed by agarose gel electrophoresis. After hybridization to

    gene chip 430, scanning with a genechip Scanner 3000 7G (Affymetrix) data analysis

    were performed using Genespring GX 7.3 (Agilent). Analysis was performed as paired

    analysis comparing untreated and LPS-treated HSCs from the same isolation. Genes

    that displayed a significant (p

  • Reverse transcription PCR and real time quantitative PCR

    RNA was isolated from mouse HSCs by the RNeasy kit, DNAse treated and was reverse

    transcribed. PCR for BAMBI was performed using primers 5’-atggatcgccactccagctac-3’

    and 5’-tcatatgaattccagctttccatgac-3’ at 95°C (45s), 60°C (45s) and 72°C (90s) for 33

    cycles. Actin PCR was performed using 5’-GACGATATCGCTGCGCTG-3’ and 5’-

    GTACGACCAGAGGCATACAGG-3’ for 27 cycles. Real time quantitative PCR was

    performed for 40 cycles of 15 seconds at 95°C and 60 seconds at 60° C using an ABI

    7000 sequence detection system (Applied Biosystems) and primer-probe sets from ABI.

    Quantification was performed by comparing the Ct values of each sample to a standard

    curve and normalization to 18s or !-actin. Values are expressed as fold induction in

    comparison to untreated or sham controls.

    Adenoviral construction and purification

    Adenoviruses expressing I"Bsr, GFP, beta-galactosidase and NF-"B-driven luciferase

    have been described previously and were amplified as previously described 2,3. Full-

    length BAMBI (cloned from murine HSC cDNA library by RT-PCR), N-terminally deleted

    dominant negative BAMBI 4 or BAMBI promoter-driven luciferase were constructed

    using the AdEasy system (ATCC). Briefly, dominant negative BAMBI 4 was excised from

    pcDNA 3.0 using KPNI and SmaI and ligated into CMV-Track and CMV-Shuttle using

    KPNI and EcoRV. Full length BAMBI was ligated into CMV-Track and CMV shuttle using

    HindIII and EcoRV. The human BAMBI promoter (-3382 to +82) 5,6 and luciferase were

    excised from PGL3 basic by KPNI and NcoI, and NcoI and SalI, respectively, and ligated

    into Shuttle plasmid using KPNI and SalI. Linearization, recombination and adenoviral

    expansion were performed as previously described 3.

    NF-"B, Smad and BAMBI reporter assays

    For NF-"B reporter assays, HSCs (4x105 cells/well) were co-infected with AdNF"Bluc 3

    and CMV-promoter driven Ad5LacZ 3 at at a multiplicity of infection (moi) of 100

    particles/cell 12 hours after isolation for 12 hours followed by stimulation with various

    concentrations of LPS for six hours. For TGF! reporter assays, HSCs were co-infected

    with AdCAGA-Luc 7 at an moi of 100 and Ad5Shuttle,AdGFP, AddnBAMBI or AdBAMBI

    12 hours after isolation using an moi of 300. 24 hours after isolation, cells were

    pretreated with LPS (100 ng/ml) for 16 hours followed by treatment with recombinant

    TGF!1 (R&D systems) at 300 pg/ml for six hours. For BAMBI reporter assays, HSCs

  • were co-infected with AdBAMBI-Luc and Ad5LacZ at an moi of 100 12 hours after

    isolation. 18 hours after isolation, cells were treated with LPS (100 ng/ml) for 24 hours.

    Luciferase activity was measured in a multiwell platereader (Fluostar Optima) and

    normalized to !-galactosidase as described 3.

    Measurement of hepatic collagen and hyaluronan content

    Hydroxyproline content was measured as previously described 8. Hepatic collagen

    content was analyzed by Sirius red staining of paraffin-embedded sections 8. The Sirius

    red positive area was measured in at least six low power (40x) fields on each slide and

    quantified using NIH imaging software. To determine the hepatic hyaluronan content,

    liver was homogenized in 10mM Tris-Hcl (pH 7.4), 1mM EDTA, 150mM NaCl. Serum

    and hepatic hyaluronan content were measured by ELISA (Echelon Biosciences Inc) as

    suggested by the manufacturer.

    Cell migration and cell adhesion assay

    Cell migration assay was analyzed by the QCM Chemotaxis Cell Migration Assay (5 µm

    pore size, Chemicon International). The lower chamber was filled with conditioned

    medium from HSCs stimulated with LPS (100 ng/ml) or vehicle for 48 hours. KCs from

    either TLR4-wild type or TLR4-mutant mice were placed into the upper chamber (105

    cells/well) and incubated at 37°C for eight hours. Adhesion of KCs to HSCs was

    determined cell adhesion assay as previously described 9. Briefly, HSCs were isolated

    from TLR4 wild-type mice and incubated with LPS (100 ng/ml) or vehicle for 24 hours.

    KCs were isolated from either TLR4-wild type or TLR4-mutant mice and labeled by

    CellTracker Green CMFDA (Molecular Probes) and placed on HSCs for one hour. HSCs

    were then gently washed and adherent KCs were counted in ten randomly chosen high-

    power fields.

    SUPPLEMENTARY REFERENCES

    1. Van Rooijen, N. & Sanders, A. Liposome mediated depletion of macrophages:

    mechanism of action, preparation of liposomes and applications. J Immunol

    Methods 174, 83-93 (1994).

    2. Iimuro, Y. et al. NFkappaB prevents apoptosis and liver dysfunction during liver

    regeneration. J Clin Invest 101, 802-11 (1998).

    3. Schwabe, R.F. & Sakurai, H. IKKbeta phosphorylates p65 at S468 in

    transactivaton domain 2. Faseb J 19, 1758-60 (2005).

  • 4. Onichtchouk, D. et al. Silencing of TGF-beta signalling by the pseudoreceptor

    BAMBI. Nature 401, 480-5 (1999).

    5. Sasaki, T., Sasahira, T., Shimura, H., Ikeda, S. & Kuniyasu, H. Effect of Nma on

    growth inhibition by TGF-betaa in human gastric carcinoma cell lines. Oncol Rep

    11, 1219-23 (2004).

    6. Sekiya, T. et al. Identification of BMP and activin membrane-bound inhibitor

    (BAMBI), an inhibitor of transforming growth factor-beta signaling, as a target of

    the beta-catenin pathway in colorectal tumor cells. J Biol Chem 279, 6840-6

    (2004).

    7. Dooley, S. et al. Smad7 prevents activation of hepatic stellate cells and liver

    fibrosis in rats. Gastroenterology 125, 178-91 (2003).

    8. Bataller, R. et al. NADPH oxidase signal transduces angiotensin II in hepatic

    stellate cells and is critical in hepatic fibrosis. J Clin Invest 112, 1383-94 (2003).

    9. Hellerbrand, Wang, S.C., Tsukamoto, H., Brenner, D.A. & Rippe, R.A.

    Expression of intracellular adhesion molecule 1 by activated hepatic stellate cells.

    Hepatology 24, 670-6 (1996).

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