Ablation of the Decorin Gene Enhances Experimental Hepatic
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Laboratory Investigation (2011) 91, 439–451 & 2011 USCAP, Inc All rights reserved 0023-6837/11 $32.00 Ablation of the decorin gene enhances experimental hepatic fibrosis and impairs hepatic healing in mice Korne´lia Baghy1, Katalin Dezso+ 1, Vikto´ria La´szlo´ 1, Alexandra Fulla´r1,Ba´lint Pe´terfia1,Sa´ndor Paku1, Pe´ter Nagy1, Zsuzsa Schaff 2, Renato V Iozzo3 and Ilona Kovalszky1 Accumulation of connective tissue is a typical feature of chronic liver diseases. Decorin, a small leucine-rich proteoglycan, regulates collagen fibrillogenesis during development, and by directly blocking the bioactivity of transforming growth factor-b1 (TGFb1), it exerts a protective effect against fibrosis. However, no in vivo investigations on the role of decorin in liver have been performed before. In this study we used decorin-null (DcnÀ/À) mice to establish the role of decorin in experimental liver fibrosis and repair. Not only the extent of experimentally induced liver fibrosis was more severe in DcnÀ/À animals, but also the healing process was significantly delayed vis-a`-vis wild-type mice. Collagen I, III, and IV mRNA levels in DcnÀ/À livers were higher than those of wild-type livers only in the first 2 months, but no difference was observed after 4 months of fibrosis induction, suggesting that the elevation of these proteins reflects a specific impairment of their degradation. Gelatinase assays confirmed this hypothesis as we found decreased MMP-2 and MMP-9 activity and higher expression of TIMP-1 and PAI-1 mRNA in DcnÀ/À livers. In contrast, at the end of the recovery phase increased production rather than impaired degradation was found to be responsible for the excessive connective tissue deposition in livers of DcnÀ/À mice. Higher expression of TGFb1-inducible early responsive gene in decorin-null livers indicated enhanced bioactivity of TGFb1 known to upregulate TIMP-1 and PAI-1 as well. Moreover, two main axes of TGFb1-evoked signaling pathways were affected by decorin deficiency, namely the Erk1/2 and Smad3 were activated in DcnÀ/À samples, whereas no significant difference in phospho-Smad2 was observed between mice with different genotypes. Collectively, our results indicate that the lack of decorin favors the development of hepatic fibrosis and attenuates its subsequent healing process at least in part by affecting the bioactivity of TGFb1. Laboratory Investigation (2011) 91, 439–451; doi:10.1038/labinvest.2010.172; published online 18 October 2010 KEYWORDS: decorin; Erk1/2; liver fibrosis; MMP-2; MMP-9; TGFb1 Accumulation of connective tissue is a typical feature of pro- and antifibrotic mechanisms failed to uncover curative several chronic diseases.1–3 The development of fibrosis is the approaches to combat this debilitating disease. Among fac- result of imbalance between the synthesis and degradation tors implicated in the induction and maintenance of excess of extracellular matrix proteins.4 Liver is especially prone matrix production, transforming growth factor-b1(TGFb1) to fibrotic remodeling5 because it is extremely susceptible to occupies a central position.14–18 This peptide growth factor is chronic and acute viral injury, toxic insults such as ethanol, capable of activating fibroblast and stellate cells, inhibiting and storage and autoimmune diseases.6–8 Without eradica- their apoptosis and forcing them to synthesize excess amount tion of the etiological agent, liver fibrosis normally progresses of matrix proteins.17 This implies that inactivation of TGFb1 to cirrhosis and destroys the normal architecture of the liver could be a strategic approach in the management of liver culminating with parenchymal and vascular decompensation fibrosis.19 of the organ. Furthermore, hepatic cirrhosis increases the Decorin, a small leucine-rich proteoglycan (SLRP)20–22 can probability of development of hepatocellular carcinoma.9,10 block the proliferation of cells that are dependent on TGFb1for In spite of recent progress, human liver cirrhosis is their in vitro growth.23 This observation has been exploited in practically still incurable.11–13 Our better understanding of various in vivo models of experimental glomerulonephritis 11st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary; 22nd Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary and 3Department of Pathology, Anatomy, and Cell Biology and The Cancer Cell Biology and Signaling Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA Correspondence: Dr I Kovalszky, MD, PhD, DSc, 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Ulloi ut 26, Budapest 1085, Hungary. E-mail: [email protected] Received 9 March 2010; revised 23 July 2010; accepted 18 August 2010 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 91 March 2011 439 Decorin and hepatic fibrosis K Baghy et al and nephrosclerosis, as well as in scar formation models.24,25 with hematoxylin and eosin or picrosirius red (PS), or pro- However, there is no conclusive information on the role of cessed further for immunohistochemistry. Stained sections decorin in the fibrotic remodeling of the liver.26 Decorin, as a were used for histological diagnosis and PS-stained sections regulator of matrix assembly targets not only TGFb1, but it is were used for morphometric analysis. involved in the maturation of collagen fibrils as well.27–29 Furthermore, it serves as ligand for growth factor receptors Cell Cultures, Decorin Gene Silencing, and TGFb1 modulating signals initiated on EGFR, IGFR, and Met re- Treatment ceptors.30–37 A central question is whether decorin-evoked We used human hepatic stellate LX2 cells (a kind gift from modulation of signal transduction is directly implicated in Dr Scott Friedman) that were spontaneously immortalized by fibrogenesis or is acting indirectly by controlling cell proliferation. continuous growth in low serum.38 The cells were cultured The main goal of this research was to determine whether in a humidified atmosphere containing 5% CO2 at 371Cin decorin could have a protective role in liver fibrosis or Dulbecco’s modified Eagle’s medium (DMEM) supple- whether the accumulation of this proteoglycan observed in mented with 10% fetal bovine serum (FBS), 100 U/ml peni- human cirrhotic liver samples would be just due to a by- cillin and 100 mg/ml streptomycin (Sigma, St Louis, MO, stander effect. Furthermore, we wanted to determine whether USA). Routinely, 2 Â 105 cells were seeded in six-well culture the hypothesized role for decorin could be attributed to plates and transfected with Silencer Select Pre-Designed modulation of the TGFb signaling pathway in the liver. In siRNA specific for decorin (siRNA ID: s223389; Applied this study, we used an experimental animal model of liver Biosystems, Welterstadt, Germany) using SiPORT NeoFX fibrosis based on decorin-null and wild-type animals sharing Transfection Agent (Applied Biosystems) according to the the same genetic background. Our results provide evidence manufacturer’s instructions. Control cells were transfected that decorin has a protective role in liver fibrogenesis insofar with Silencer Select negative control siRNA (siRNA ID: as the absence of decorin correlates with enhanced hepatic 4390843). After 24 h the medium was replaced with DMEM fibrosis and attenuation of its subsequent healing process. supplemented with 0.2% FBS alone or in the presence of 2 ng/ml human recombinant TGFb1 (cat. no. PHP143; AbD Serotec, Oxford, UK) for 48 h. MATERIALS AND METHODS Decorin-Null Mice Morphometry, Fluorescence Immunostaining, and All animal experiments were performed under an Quantitative Real-Time RT-PCR institutionally approved protocol. Decorin-deficient mice were Picrosirius-red-stained sections were analyzed by using the 29 generated as previously described. In brief, the inactivation Cue-2 program (Olympus, Japan). At least five fields were of the decorin gene was achieved by targeted disruption of analyzed in each section. Frozen sections of the liver and cells the exon 2 inserting a PGK-Neo cassette. Two male and two grown on coverslips in a 24-well plate (4 Â 104 cells per well) female C57Bl/6 mice heterozygous for decorin gene (Dcn þ /À), were fixed in ice-cold methanol, washed in phosphate-buf- which were backcrossed into C57Bl/6 background for nine fered saline (PBS), blocked with 5 (w/v%) BSA/PBS con- generations, were bred until homozygosity. The genotype of taining 10% non-immune serum of secondary antibody at the offspring was determined by PCR. Tail DNA was isolated 371C for 30 min. After washing, sections were incubated by using high-salt method. Subsequently three primers were with the primary antibody diluted in PBS containing 1 (w/v%) applied, sense and antisense specific for the exon 2, and one BSA at 371C for 1.5 h or at 41C for 16 h. Appropriate fluor- corresponding to the PGK-Neo cassette. PCR products were escent secondary antibodies were applied at room temperature analyzed by 2% agarose gel electrophoresis. for 30 min. Nuclei were stained with 40-60-diamidino-pheny- lindole (DAPI). Primary and secondary antibodies with their Thioacetamide Treatment appropriate dilutions are shown in Supplementary Table 1. For induction of hepatic fibrosis, we used 48 one-month-old Pictures were taken by Nikon Eclipse E600 microscope with male mice, all in a C57Bl/6 background. In all, 24 wild-type the help of Lucia Cytogenetics version 1.5.6 program. and 24 DcnÀ/À animals were exposed to thioacetamide For RT–PCR, total RNAs were isolated from frozen livers (TA) dissolved in drinking water (150 mg/l). To obtain fully and LX2 cells. After homogenization in liquid nitrogen developed hepatic fibrosis, we subjected the animals to TA TRIzol (Invitrogen, Carlsbad, CA, USA) method was used for treatment for 4 months followed by an additional 4 months RNA extraction. cDNAs were generated from 1 mg total RNA (recovery phase) in which the drug was withdrawn. Three by M-MLV Reverse Transcriptase kit (Invitrogen) following animals were killed at each time point at monthly intervals.