Comparative proteomic analysis of rat hepatic stellate cell activation: A comprehensive view and suppressed immune response

Authors

  • Juling Ji,

    Corresponding author
    1. Department of Pathology, Medical School of Nantong University, Nantong, China
    2. Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China
    • Juling Ji, Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou, 510632, P.R. China===

      Yuhua Ji, Department of Pathology, Medical School of Nantong University, Nantong, 226001, P.R. China===

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    • These authors contributed equally to this work.

  • Feng Yu,

    1. Department of Animal Biochemistry, College of Animal Science and Technology, Northwest A&F University, Yangling, China
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  • Qiuhong Ji,

    1. Department of Neurology, Affiliated Hospital of Nantong University, Nantong, China
    2. Key Laboratory of Neuroregeneration, Nantong University, Nantong, China
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  • Zhiyao Li,

    1. Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangdong, China
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  • Kuidong Wang,

    1. Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangdong, China
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  • Jinsheng Zhang,

    1. Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, China
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  • Jinbiao Lu,

    1. Department of Pathology, Medical School of Nantong University, Nantong, China
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  • Li Chen,

    1. Department of Pathology, Medical School of Nantong University, Nantong, China
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  • Qun E,

    1. Department of Pathology, Medical School of Nantong University, Nantong, China
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  • Yaoying Zeng,

    1. Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangdong, China
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  • Yuhua Ji

    Corresponding author
    1. Key Laboratory of Neuroregeneration, Nantong University, Nantong, China
    2. Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangdong, China
    • Juling Ji, Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou, 510632, P.R. China===

      Yuhua Ji, Department of Pathology, Medical School of Nantong University, Nantong, 226001, P.R. China===

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    • fax: (86)-513-85258669


  • Potential conflict of interest: Nothing to report.

  • fax: (86)-20-85227730

Abstract

Elucidation of the molecular events underlying hepatic stellate cell (HSC) activation is an essential step toward understanding the biological properties of HSC and clarifying the potential roles of HSCs in liver fibrosis and other liver diseases, including hepatocellular carcinoma. High-throughput comparative proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with online two-dimensional nanoscale liquid chromatography and tandem mass spectrometry (2D nano-LC-MS/MS) were performed on an in vitro HSC activation model to obtain a comprehensive view of the protein ensembles associated with HSC activation. In total, 2,417 proteins were confidently identified (false discovery rate <1%), of which 2,322 proteins were quantified. Compared with quiescent HSCs, 519 proteins showed significant differences in activated HSCs (≥3.0-fold). Bioinformatics analyses using Ingenuity Pathway Analysis revealed that the 319 up-regulated proteins represented multiple cellular functions closely associated with HSC activation, such as extracellular matrix synthesis and proliferation. In addition to the well-known markers for HSC activation, such as α-smooth muscle actin and collagen types 1 and 3, some novel proteins potentially associated with HSC activation were identified, while the 200 down-regulated proteins were primarily related to immune response and lipid metabolism. Most intriguingly, the top biological function, top network, and top canonical pathway of down-regulated proteins were all involved in immune responses. The expression and/or biological function of a set of proteins were properly validated, especially Bcl2-associated athanogene 2, BAG3, and B7H3.

Conclusion:

The present study provided the most comprehensive proteome profile of rat HSCs and some novel insights into HSC activation, especially the suppressed immune response. (HEPATOLOGY 2012;56:332–349)

Hepatic stellate cells (HSCs) are star-shaped cells resident in the space of Disse that play a major role in retinoid metabolism.1 Typically, 70%-80% of the retinoid compounds in the body are stored in HSC cytoplasmic lipid droplets. HSCs are also regarded as the principle cell type responsible for liver fibrosis. Following liver injury, HSCs are known to become “activated”: they lose their cytoplasmic retinoid lipid droplets and transdifferentiate into a fibrogenic and proliferative myofibroblast-like cell type. This leads to intrahepatic extracellular matrix (ECM) accumulation. Recently, this simple paradigm has become more complex, especially regarding the emerging roles of HSCs in intrahepatic immunoregulation.2, 3 Accumulating studies have led to a greater appreciation of the role of HSCs in many other human liver diseases in addition to liver fibrosis, such as hepatocellular carcinoma (HCC).1, 4 Elucidation of the molecular events occurring during HSC activation is an essential step toward a more comprehensive understanding of the biological role of HSCs in fibrosis and other liver diseases as well as the development of effective strategies for the diagnosis and treatment of such disorders.

Mass spectrometry–based proteomics is a revolutionary technology that can rapidly identify and accurately quantify thousands of proteins within a complex biological specimen. Comparative proteomic analysis can provide an overview of the dynamic changes during HSC activation and novel insights into this process. Long-term culture of primary HSCs in polystyrene dishes can recapitulate the features of activated HSCs and has been widely accepted as an in vitro model for HSC activation studies.5 Due to the difficulties of HSC isolation and the requirement of a large quantity of protein for two-dimensional gel electrophoresis–based comparative proteomic analysis, existing proteomic studies of HSC activation are very limited. To date, the only study that specifically characterized the proteome of quiescent and activated HSCs was accomplished by Kristensen et al. in 2000.6 Using two-dimensional gel electrophoresis and silver staining, they found 43 differentially expressed proteins in activated rat HSCs. Their study also presented the first HSC proteome, containing about 150 proteins. As new proteomics strategies have been developed, especially stable isotope labeling and multidimensional protein identification technology, it is now possible to track the changes of a thousand physiologically relevant proteins within a sample containing no more than 100 μg protein.7 This capability provides an opportunity to re-evaluate the molecular events that occur during HSC activation.

In the present study, to obtain an unbiased overview of HSC activation and gain more insight into the underlying molecular mechanism, we performed high-throughput quantitative proteomic analysis. By using an isobaric tag for relative and absolute quantitation (iTRAQ) combined with online two-dimensional nanoscale liquid chromatography and tandem mass spectrometry (2D nano-LC-MS/MS), we identified proteins differentially expressed during HSC activation in the well-established in vitro model. In total, 2,417 proteins were confidently identified (false discovery rate [FDR] <1%) and 2,322 proteins were quantified, of which 319 and 200 were up- or down-regulated (≥3.0-fold) during HSC activation, respectively. In addition to the well-documented markers of HSC activation, such as α-smooth muscle actin (α-SMA) and collagen type 1 (COL1A1, COL1A2) and 3 (COL3A1), the majority of the altered proteins were novel. Bioinformatics analysis and biological validation of these altered proteins not only expanded our understanding of the major characteristics of activated HSCs, but also cast new light on the role of HSCs in intrahepatic immunoregulation.

Abbreviations

2D nano-LC-MS/MS, two-dimensional nanoscale liquid chromatography and tandem mass spectrometry; α-SMA, α-smooth muscle actin; B2M, β2-microglobulin; BAG2, Bcl2-associated athanogene 2; CCl4, carbon tetrachloride; COL, collagen; CRBP1, cellular retinol-binding protein-1; ECM, extracellular matrix; EdU, 5-ethynyl-2′-deoxyuridine; ERK1/2, extracellular-regulated kinase 1/2; FDR, false discovery rate; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; Hsp, heat shock protein 70; IPA, Ingenuity Pathway Analysis; iTRAQ, isobaric tag for relative and absolute quantitation; LTA4H, leukotriene A4 hydrolase; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T cells; NTC, nontargeting control; RE, retinyl ester; siRNA, small interfering RNA; TGF-β, transforming growth factor-β.

Materials and Methods

Isolation and Culture of Rat HSCs.

Pathogen-free male Sprague-Dawley rats (body weight, ≈400 g) used for HSC isolation were cared for according to the Guide for the Care and Use of Laboratory Animals formulated by Fudan University. HSCs were isolated from rat livers by two-step digestion as described.8 Density gradient separation yielded 40-70 million cells per animal. Trypan blue dye exclusion test showed that the isolated HSCs were more than 98% viable. The purity of HSCs was determined by autofluorescence of vitamin A.

Rat Liver Fibrosis Model.

Liver fibrosis was induced in male Sprague-Dawley rats (body weight, 300-350 g) by intraperitoneal administration of carbon tetrachloride (CCl4) in olive oil according to a previous study.9

Immunofluorescent Staining.

Primary cultured HSCs grown on cover slips and cryostat sections (5 μm) of normal or cirrhotic rat liver specimens were fixed with 2% and 4% v/v paraformaldehyde/phosphate-buffered saline, respectively. The staining procedures were performed as described.8 The following primary antibodies were used: α-SMA (Sigma, St. Louis, MO), COL I (Calbiochem, La Jolla, CA), desmin (Calbiochem) and B7H3 (also known as CD276, Santa Cruz Biotechnology, Santa Cruz, CA). Fat droplets were stained with preheated Sudan III (Sigma) for 2 minutes at room temperature.

Comparative Proteomic Analysis Based on iTRAQ Labeling and 2D Nano-LC-MS/MS.

iTRAQ labeling and 2D nano-LC-MS/MS experiments were performed essentially as described.7 Digested peptides from quiescent and activated HSCs were labeled with 114 and 116 iTRAQ reagents, respectively. In the present study, proteins with 95% or greater confidence as determined by ProteinPilot Unused scores (≥1.3) were reported, and the corresponding FDR was less than 1%. Details are provided in the Supporting Information.

Bioinformatics Analysis of Differentially Expressed Proteins.

The bioinformatics analysis of the differentially expressed proteins was performed with Ingenuity Pathways Analysis (IPA) software (version 6.3, Ingenuity Systems, Redwood City, CA; http://www.ingenuity.com). Details are provided in the Supporting Information.

Western Blotting.

A total of 14 commercial antibodies were used for western blotting, including antibodies to B7H3, β2-microglobulin (B2M), biglycan, Bcl2-associated athanogene 2 (BAG2), BAG3, calponin-1, CD1d1, galectin-1, leukotriene A4 hydrolase (LTA4H), cellular retinol-binding protein-1 (CRBP1), RT1-A, RT1-Ba, transforming growth factor-β3 (TGF-β3) and β-actin. Except for RT1-A (BioLegend, San Diego, CA) and β-actin (Sigma), all antibodies were purchased from Santa Cruz Biotechnology. The RT1-A antibody was used for the detection of the RT1-EC2 A2q protein, which is the synonym of RT1- A2. β-actin served as a loading control.

Small Interfering RNA Transfection, Cell Proliferation, Adhesion, and Migration Assays.

According to the manufacturer's protocol, passage 2–activated HSCs8 were transfected with small interfering RNAs (siRNAs) targeting rat Bag2 and Bag3 (Sigma) by RNAiMAX (Invitrogen, Carlsbad, CA). Nontargeting control (NTC) siRNA was transfected simultaneously as a negative control. The effects of Bag2 and Bag3 knockdown on HSC proliferation, adhesion, and migration were measured by 5-ethynyl-2′-deoxyuridine (EdU) assay, cell adhesion assay, and a modified Boyden chamber assay, respectively. Details are provided in the Supporting Information.

Statistical Analysis.

Data are expressed as the mean ± SD. Pearson correlation coefficients and two-tailed test were used to assess the relationships of 116/114 ratios in three independent analyses, and comparisons between groups were made by use of one-way ANOVA with SPSS 10.0 software. In IPA analysis, the statistical data were generated by the software. Statistical significance was set at P < 0.05. Unless otherwise specified, all assays were performed in triplicate.

Results

Characterization of Primary HSCs.

According to the autofluorescence of vitamin A and Sudan III staining for lipid droplets, the purity of primary rat HSCs was about 95% in all isolations (Supporting Fig. 1). The purity of HSCs was further validated by immunostaining of desmin (Supporting Fig. 2A), a marker of rat HSCs that was expressed in almost all primary cultured HSCs. Compared with HSCs cultured for 2 days, two well-established markers of activated HSCs, α-SMA and COL I, were significantly up-regulated in HSCs cultured for 10 days (Supporting Fig. 2B-D). Thus, in the present study, HSCs maintained for no more than 2 days (D2 HSCs) are referred to as quiescent HSCs, and those maintained for 10 days (D10 HSCs) are referred to as activated HSCs.

iTRAQ Analysis of Quiescent and Activated HSCs.

To elucidate the molecular events occurring during HSC activation, quantitative proteomic analysis based on iTRAQ labeling was executed in the in vitro rat HSC activation model. As shown in Fig. 1 and Supporting Table 1, more than 1,700 proteins were identified in each of the three independent biological replicates (FDR <1%). Having merged the proteins under identical accession number and/or gene symbol, the total number of nonredundant proteins identified in the present study was 2,417 (Supporting Table 1). Among these proteins, 72.78% (1,759/2,417) were shared by at least two of the three experiments (Fig. 1A), demonstrating good reproducibility of protein identification. Detailed information about the identified proteins of the three independent biological replicates is provided in Supporting Table 1.

Figure 1.

(A) Venn diagram depicting the overlap of proteins identified in three independent iTRAQ experiments. Numbers in parentheses indicate the number of identified proteins for each sample. To examine the biological reproducibility, linear regression analyses were performed on ln-transformed 116/114 ratios (activated HSCs/quiescent HSCs) of three independent analyses. Pearson correlation coefficients between samples 1 and 2 (B), samples 2 and 3 (C), and samples 1 and 3 (D) are 0.9087, 0.9296, and 0.8943 respectively, P < 0.01.

Afterward, linear regression analyses were performed on ln-transformed 116/114 ratios of three independent experiments to examine the biological reproducibility. As illustrated in Fig. 1B-D, the Pearson correlation coefficients between samples 1 and 2, samples 2 and 3, and samples 1 and 3 were 0.9087, 0.9296, and 0.8943, respectively (P < 0.01), indicating good biological reproducibility of this in vitro HSC activation model.

To identify proteins that were either up- or down-regulated during HSC activation, the 116/114 ratios of proteins identified in three independent iTRAQ analyses were averaged (Supporting Table 1). The threshold values that were set for significant up- and down-regulated proteins in the present study were ≥3.0000 or ≤0.3333 (≥3.0-fold). Accordingly, 319 and 200 proteins were significantly up- and down-regulated, respectively, in activated HSCs, suggesting a drastic phenotypic alteration during HSC activation. The abbreviated lists of up- and down-regulated proteins are provided in Tables 1 and 2.

Table 1. List of Up-regulated Proteins During Rat HSC Activation
Accession NO.Gene Symbol and NameAccession NO.Gene Symbol and NameAccession NO.Gene Symbol and Name
  • This table contains the 319 proteins that displayed more than 3.0-fold up-regulation in activated rat HSCs in three independent experiments. The International Protein Index (IPI) accession number, gene symbol, and name of each protein are provided here. The proteins are listed in descending order according to their fold change, and the top 10 up-regulated proteins appear in bold. For detailed information about these proteins, refer to Supporting Table 1.

  • *

    Proteins reported by Kristensen et al.6

IPI00200134.1Asam Adipocyte adhesion moleculeIPI00851116.1Nt5e 5′ nucleotidase, ecto*IPI003641892Eif3j Eukaryotic translation initiation factor 3 subunit J
IPI00391995.2Tpm1 Isoform 2 of Tropomyosin alpha-1 chain*IPI00204078.1Nradd P75-like apoptosis-inducing death domain protein long isoformIPI00209789.1Fkbp3 FK506 binding protein 3, 25kDa
IPI00193981.3Bag2 Bag2 proteinIPI00210187.1Pdlim5 PDZ and LIM domain protein 5IPI00361798.2Igfbp7 Insulin-like growth factor binding protein 7
IPI00208118.1Caldi Non-muscle caldesmon*IPI00476899.1Eef1b2 Eukaryotic translation elongation factor 1 beta 2IPI00480766.1Acat2 Acetyl-CoA acetyltransferase, cytosolic
IPI00231196.5Tagln TransgelinIPI00203250.1Dpysl3 Isoform 2 of Dihydropyrimidinase-related protein 3IPI00324585.3Itga1 Integrin alpha-1
IPI00366944.2Col3a1 Collagen alpha-1(lll) chain*IPI00952140.1Nedd4 E3 ubiquitin-protein hgase NEDD4IPI00370411.2Fbln1 Putative uncharacterized protein Fbln1
IPI00198250.1Akap12 Isoform 2 of A-kinase anchor protein 12IPI00201858.4Pbxipi Pre-B-cell leukemia transcription factor-interacting protein 1IPI00364502.5Nfu1 NFU1 iron-sulfur cluster scaffold homolog precursor
IPI00208221.1Fkbp10 65kDa FK506-binding protein, isoform CRA_aIPI00363849.2Lamc1 laminin, gamma 1IPI00208306.1Tpt1 Translationally-controlled tumor protein*
IPI00204375.2Uchl1 Ubiquitin carboxyl terminal hydrolase isozyme L1IPI00195673.1Tubb6 Tubulin, beta 6IPI00371266.1Naca Nascent-polypeptide-associated complex alpha polypeptide (Predicted), isoform CRA_b
IPI00231784.3Dbn1 Isoform E1 of DrebrinIPI00202627 1Scrn1 Secernin-1IPI00206818.1Ifitm3 Interferon-inducible protein variant 10
IPI00365582.3Cad Putative uncharacterized protein CadIPI00869592.3Mylk Myosin light chain kinaseIPI00360771.3Ccdc6 Putative uncharacterized protein Ccdc6
IPI00361824.3Fam114a1 hypothetical proteinIPI00191681.1Itgb1 Integrin beta-1IPI00373505.2Tmod3 Tropomodulin 3
IPI00200757.1Fn1 Isoform 1 of FibronectinIPI00930907.1Sdf4 Isoform 1 of 45 kDa calcium-binding proteinIPI00882548.1Hdlbp High density lipoprotein binding protein
IPI00187731.4Tpm2 Isoform 2 of Tropomyosin beta chain*IPI00765344.2Sfrp1 Secreted frizzled-related protein 1IPI00957286.1Sorbs1 sorbin and SH3 domain containing 1
IPI00476991.1Ncam1 Neural cell adhesion molecule 1*IPI00393975.2Map4 Isoform 1 of Microtubule-associated protein 4IPI003391974.1Pkm2 Isoform M2 of Pyruvate kinase isozymes M1/M2*
IPI00188909.3Col1a1 Collagen alpha-1(l) chain*IPI00948019.1Fst1 Ab2-379IPI00958724.1Mcc mutated in colorectal cancers
IPI00188590.1Fkbp7 FK506 binding protein 7IPI00763873.3Itga11 Integrin, alpha 11IPI00400579.1Gaa Lysosomal alpha-glucosidase
IPI00203974.2Bag3 Bcl-2-interacting death suppressorIPI00326462.1Enpp3 Ectonucleotide pyrophosphatase/phosphodiesterase: family member 3IPI00200070.1Nucb2 Nucleobindin-2
IPI00231275.7Lgals1 Galectin-1*IPI00205566.1Cnn3 Calponin-3IPI00200353.1Ril PDZ and LIM domain protein 4
IPI00214905.3Tpm4 Tropomyosin alpha-4 chain*IPI00204748.1S100a4 Protein S100-A4*IPI00207037.3Eif4ebp1 Eukaryotic translation initiation factor 4E-binding protein 1
IPI00362131.3Cdh2 Cadherin-2IPI00392930.2Mprip ENSRNOP00000035264IPI00471525.2Eef1d Isoform 2 of Elongation factor 1-delta*
IPI00763060.2REVERSED LOC684327 rCG55860-likeIPI00205631.1Vcam1 Vascular cell adhesion protein 1IPI00199325.1Htrai Insulin-like growth factor binding protein 5 protease
IPI00957717.1Plod2 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 isoform 1IPI00366079.1LOC100362805 RCG43947IPI00372259.4Tpm3 Isoform 1 of Tropomyosin alpha-3 chain*
IPI00554194.1Calu Calumenin isoform b*IPI00204099.5Mcfd2 Multiple coagulation factor deficiency protein 2 homologIPI00365542.4Lamb1 Putative uncharacterized protein Lamb1
IPI00951503.1Hspb7 Heat shock protein beta-7IPI00957368.1LOC100360976 Peroxisomal biogenesis factor 19-like, partialIPI00560977.2Caprin1 Caprin-1
IPI00215465.1Cryab Alpha-crystallin B chainIPI00212270.7Gng12 Guanine nucleotide binding protein (G protein), gamma 12IPI00817070.1Myl6b;Myl6 myosin, light chain 6, alkali, smooth muscle and non-muscle*
IPI00207050.5Rcn3 Reticulocalbin 3, EF-hand calcium binding domainIPI00230921.9S100a10 Protein S100-A10IPI00210495.1Rad23b UV excision repair protein RAD23 homolog B
IPI00563816.2Flnc Putative uncharacterized protein FlncIPI00944216.1Plec Plectin isoform 1hijIPI00199980.1Psmb7 Proteasome subunit beta type-7
IPI00325151 1Cnn1 Calponin-1IPI00947746.1Pawr Putative uncharacterized protein PawrIPI00758461.1Sra1 Isoform 1 of Steroid receptor RNA activator 1
IPI00362160.1Tubb3 Tubulin beta-3 chainIPI00231107.5Ptms ParathymosinIPI00776551.1Cytsb Sperm antigen with calponin homology and coiled-coil domains 1
IPI00188921.1Col1a2 Collagen alpha-2(l) chain*IPI00952078.1Pls3 Putative uncharacterized protein Pls3IPI00567393.2Mtap Putative uncharacterized protein Mtap
IPI00231566.1Tpm1 Isoform 6 of Tropomyosin alpha-1chainIPI00957937.1LOC100362061 glutathione peroxidase 8-likeIPI00200145.1LOC100360522:Rplp1 60S acidic ribosomal protein P1
IPI00365982.2Ckap4 Putative uncharacterized protein Ckap4IPI00555171.3Tagln2 Transgelin-2IPI00207068.1Glg1 Golgi apparatus protein 1
IPI00388257.4Fbln2 Fibulin 2 isoform 1IPI00206056.1Fas Tumor necrosis factor receptor superfamily member 6IPI00207325.1Lepre1 Prolyl 3-hydroxylase 1
IPI00396852.2Nexn Isoform 2 of NexilinIPI00326759.6Rcn2 Reticulocalbin-2IPI00951440.1Tpd52I2 Tumor protein D52-like 2, isoform CRA_h
IPI00569885.2Hspb1 Heat shock protein beta-1*IPI00230937.5Pebp1 Phosphatidylethanolamine-binding protein 1*IPI00327705.5Dap Death-associated protein 1
IPI00949156.1Lmo7 Putative uncharacterized protein Lmo7IPI00230902.5Gyg1 Glycogenin-1IPI00734740.2LOC498555 Putative uncharacterized protein ENSRNOP00000054740
IPI00204206.1Tpm1 Tropomyosin 1 alpha chain isoform dIPI00896144.1Cav1 Caveolin-1 alpha isoformIPI00373045.1Eif4b Eukaryotic translation initiation factor 4B
IPI00195384.2RGD1559896 Similar to RIKENcDNA 2310022B05IPI00914765.1Zyx ZyxinIPI00951329.1Col14a1 Putative uncharacterized protein Col14a1
IPI00949017.1Csrp2 Csrp2 proteinIPI00766661.2LOC679129 Peroxisomal biogenesis factor 19-likeIPI00367437.6Rbm3 Putative RNA-binding protein 3
IPI00950740.1Krt14 Putative uncharacterized protein Krt14IPI00204483.1Serpine1 Plasminogen activator inhibitor 1*IPI00566460.2Notch2 Putative uncharacterized protein Notch2
IPI00777920.1Cttn Putative uncharacterized protein CttnIPI00858359.2Myl9 Myosin, light polypeptide 9, regulatoryIPI00949471.1Pin1 Putative uncharacterized protein Pin1
IPI00565165.2Lrrfip1 Putative uncharacterized protein Lrrfip1IPI00769110.2Rrbp1 Ribosome binding protein 1 isoform 3IPI00782070.2Serpinb6a Serine (Or cysteine) peptidase inhibitor, clade B, member 6a
IPI00189424.2Sparc Secreted protein acidic and rich in cysteine*IPI00203972.2Gys1 Glycogen [starch] synthase, muscleIPI00959247.1Sept11 Putative uncharacterized protein Sept11
IPI00191090.1Bgn BiglycanIPI00480840.2Cdv3 RCG25673, isoform CRA_dIPI00325146.6Anxa2 Isoform Short of Annexin A2
IPI00210945.7Tpm1 Tropomyosin 1 alpha chain isoform cIPI00201868.1Nenf NeudesinIPI00203443.3Ptgr1 Prostaglandin reductase 1
IPI00197129.1Acta2 Actin, alpha-smooth muscle actin*IPI00767054.2Nid1 Nidogen 1IPI00191454.1Fhl3 Putative uncharacterized protein Fhl3
IPI00211206.7Pdlim1 PDZ and LIM domain protein 1IPI00764713.3Kid Isoform A of Kinesin light chain 1IPI00211709.1Gpx8 Glutathione peroxidase
IPI00471669.1Gpc4 Glypican 4IPI00367746.3Setd7 RCG49977, isoform CRA_bIPI00565749.1Rai14 Isoform 1 of Ankycorbin
IPI00231825.5Rbp1 Retinol-binding protein 1*IPI00476709.1Vldlr Putative uncharacterized protein VldlrIPI00190848.1Triap1 RCG21156
IPI00187707.1Ppic Peptidyl-prolyl cis-trans isomeraseIPI00831721.1Gcsh H proteinIPI00337173.1Cd276 CD276 antigen (B7H3)
IPI00201261.2Nes Isoform 2 of NestinIPI00198667.7Clu ClusterinIPI00231434.6Fkbp1a Peptidyl-prolyl cis-trans isomerase FKBP1A
IPI00206193.1Fhl2 Four and a half LIM domains protein 2IPI00189471.1Lpl Lipoprotein lipaseIPI00369539.2Mfap4 Microfibrillar-associated protein 4
IPI00373011.3Limai LIM domain and actin binding 1IPI00951791.1Flna Filamin, alpha (Predicted), isoform CRA_aIPI00555287.3Sptbn1 Non-erythroid spectrin beta
IPI00555213.1Pea15a Astrocytic phosphoprotein PEA-15IPI00214192.1Sh3gl1 Endophilin-A2IPI00324451.4Ddb1 DNA damage-binding protein 1
IPI00188956.1Thy1 Thy-1 membrane glycoproteinIPI00209358.1Pdlim7 PDZ and LIM domain protein 7IPI00199861.1Dcn Decorin
IPI00192504.2Mrc2 C-type mannose receptor 2IPI00769176.2Tgfb1i1 Putative uncharacterized protein Tgfb1i1IPI00191112.1Ndufab1 Acyl carrier protein
IPI00554148.1S100a11 Protein S100-A11*IPI00958283.1LOC100366237 prefoldin subunit 4-likeIPI00480687.2Marcks Myristoylated alanine-rich C-kinase substrate
IPI00782515.1Akap2 Putative uncharacterized protein Akap2IPI00392468.5Cnpy4 Putative uncharacterized protein RGD1307636IPI00562248.1App App protein
IPI00194930.5Gpc1 Glypican-1IPI00949391.1Sec24d Sec24d proteinIPI00198887.1P4hb Protein disulfide-isomerase*
IPI00776882.1Calu Calumenin isoform a*IPI00951259.1Flnb Putative uncharacterized protein FlnbIPI00190287.1Prelp Prolargin
IPI00201608.5Col5a1 Collagen alpha-1(V) chainIPI00208280.3Ptgfrn Prostaglandin F2 receptor negative regulatorIPI00369330.1Ttc1 Tetratricopeptide repeat domain 1
IPI00192912.1Rcn1 Reticulocalbin 1 (Predicted), isoform CRA_a*IPI00950587.1Pdgfrb Beta-type platelet-derived growth factor receptorIPI00200257.1Cdh13T-cadherin
IPI00201300.2Ptrf Polymerase I and transcript release factorIPI00768308.2LOC687057 calponin 2-likeIPI00200898 3Slc9a3r1 Na(+)/H(+) exchange regulatory cofactor NHE-RF1
IPI00782742.2Map1a Putative uncharacterized protein Map1aIPI00372789.3Tnks1bp1 Putative uncharacterized protein Tnks1bp1IPI00782227.1Hook3 Hook homolog 3
IPI00210119.1Map6 Isoform 1 of Microtubule-associated protein 6IPI00763901.2Upk3b Uroplakin 3BIPI00563982.3Zc3h18 ENSRNOP00000048376
IPI00194999.1Tgfb3 Transforming growth factor beta-3IPI00957850.1Itga5 Putative uncharacterized protein Itga5IPI00370450.3Plxnb2 Putative uncharacterized protein Plxnb2
IPI00372009.3Map1b Microtubule-associated protein 1BIPI00564409.3RGD1309537 Myosin regulatory light chain RLC-A*IPI00213463.2Actn4 Alpha-actinin-4
IPI00215190.1Fkbp9 Peptidyl-prolyl cis-trans isomerase FKBP9IPI00358406.2Ctnna1 Catenin, alpha 1, isoform CRA_bIPI00188112.1Psph Phosphoserine phosphatase
IPI00365286.3Vcl Vinculin*IPI00369995.2Lrp1 Low density lipoprotein receptor-related protein 1IPI00207574.1Slit3 Slit homolog 3 protein
IPI00950560.1- Putative uncharacterized protein ENSRNOP00000058924IPI00421723.1Tbca Tubuhn-specific chaperone AIPI00200661.1Fasn Fatty acid synthase
IPI00388880.5Yap1 Putative uncharacterized protein Yap1IPI00215135.2II6st Ac1055IPI00211216.4Eif5a Eukaryotic translation initiation factor 5A-1*
IPI00231651.6Baspi Brain acid soluble protein 1IPI00326412.4Eno2 Gamma-enolaseIPI00193171.1Npc2 Niemann-Pick disease, type C2
IPI00204703.5Serpinh1 Serpin H1IPI00210351.2Ak1 Adenylate kinase isoenzyme 1IPI00203773.3Mrpl12 Putative uncharacterized protein Mrpl12
IPI00199867.2Emilin1 Putative uncharacterized protein Emilin1IPI00230927.1Cltb Isoform Non-brain of Clathrin light chain BIPI00208154.1Cd81 CD81 antigen
IPI00764966.2Ahnak2 similar to KIAA2019 proteinIPI00204818.2S100a6 Protein S100-A6*IPI00947644.1Sqrdl 36 kDa protein
IPI00607192.1P4ha2 Prolyl 4-hydroxylase subunit alpha-2IPI00231615.5Anxal Annexin A1IPI00190240.1Rps27a;LOC100363345 Ubiquitin-40S ribosomal protein S27a*
IPI00968512.1LOC290704 117 kDa proteinIPI00327185.3Nap1l1 Nucleosome assembly protein 1-like 1IPI00947893.1Ltbp2 192 kDa protein
IPI00231194.5Ddahl N(G),N(G)-dimethylarginine dimethylaminohydrolase 1*IPI00394021 5Lvrn Putative uncharacterized protein LvrnIPI00325912.1Ctnnb1 Catenin beta-1
IPI00199778.1RGD1305457 Isoform 1 of Inhibitor of nuclear factor kapp-B kinase-interacting proteinIPI009S0067.1Cdkn2b Cdkn2b proteinIPI00760137.1Sord Sorbitol dehydrogenase
IPI00763134.1RGD1564327 RGD1564327 proteinIPI00203158.3Stub1 STIP1 homology and U-Box containing protein 1IPI00845873.1Zranb2 Zinc finger, RAN-binding domain containing 2
IPI00195803.1Ugdh UDP-glucose 6-dehydrogenaseIPI00198796.1Rwddi RWD domain-containing protein 1IPI00325189.4Nme2 Nucleoside diphosphate kinase B
IPI00204984.1Bst1 ADP-ribosyl cyclase 2IPI00515802.1Pcolce Pcolce proteinIPI002013251Txndc17 Thioredoxin-like 5 (Predicted), isoform CRA_b
IPI00197074.3Dag1 Dystroglycan 1IPI00193547.2Pdcd5 RCG53732, isoform CRA_aIPI00957217.1Bin1 Putative uncharacterized protein Bin1
IPI00210111 1Rgc32 Response gene to complement 32 proteinIPI00213015.1Dctn2 Dynactin subunit 2IPI00198567.1Laspi LIM and SH3 domain protein 1
IPI00210360.3Hspg2 394 kDa proteinIPI00231690.5Csrp1 Cysteine and glycine-rich protein 1IPI00366399.3Eny2 RCG59696, isoform CRA_e
IPI00209863.2P4ha1 Prolyl 4-hydroxylase subunit alpha-1*IPI00870042.1Tjp1 Tight junction protein 1IPI00766955.1LOC687820 Coiled-coil domain-containing protein 6-like
IPI00567305.1- Putative uncharacterized protein ENSRNOP00000039900IPI00951644.1FdpsAc2-125IPI00779594.2Aldh1l2 Putative uncharacterized protein Aldh1l2
IPI00422076.1Thbs1 Thrombospondin 1IPI00190701.5Apoe Apolipoprotein EIPI00212651.1Timm13 Mitochondrial import inner membrane translocase subunit Tim13
IPI00194087.3Actc1 Actin, alpha cardiac muscle 1IPI00211448.2Ehd2 EH domain-containing protein 2IPI00361686.5C1qbp Complement component 1 Q subcomponent-binding protein, mitochondrial
IPI00201548.1Carhsp1 Calcium-regulated heat stable protein 1IPI00208184.1Fam136a Protein FAM136AIPI00209283.3Vapb Vesicle-associated membrane protein-associated protein B
IPI00207199.3Ctgf Connective tissue growth factorIPI00210783.4Fam25a hypothetical protein LOC684972IPI00555327.1Clec10a Macrophage galactose N-acetyl-galactosamine specific lectin 1
IPI00327143.1Alpl Alkaline phosphatase, tissue-nonspecific isozymeIPI00765234.2Ktn1 Kinectin 1IPI00367715.3Flrt2 RCG20814
IPI00949586.1Myh10 Myosin-10IPI00197553.1Npm1 Isoform B23.1 of Nucleophosmin*IPI00951274.1Dync1i2 Putative uncharacterized protein Dync1i2
IPI00209574.3Leprel2 Leprel2 proteinIPI00388209.2Prkcsh Protein kinase C substrate 80K-H (Predicted) isoform, CRA_bIPI00766527.1LOC683470 growth arrest specific 1-like
IPI00766972.1Mxra7 Matrix-remodelling associated 7-likeIPI00197216.3M6prbp1 Mannose-6-phosphate receptor binding protein 1-likeIPI00959549.1Tp53bp1 212 kDa protein
IPI00201034.5Cdh3 Cadherin 3IPI00360916.3Gcc2 Putative uncharacterized protein Gcc2IPI00952449.1Arfip1 Putative uncharacterized protein Arfip1
IPI00896761.2LOC683788 FascinIPI00197711.1Ldha L-lactate dehydrogenase A chainIPI00195719.1Olr1 Oxidized low-density lipoprotein receptor 1
IPI00627074.2Cd99 CD99 antigenIPI00949745.1Ece1 Endothelin converting enzyme 1IPI00958129.1Col12a1 Putative uncharacterized protein Col12a1
IPI00327697.4Dpepi Dipeptidase 1IPI00200920.1Khsrp Far upstream element-binding protein 2IPI00360246.2Reep5 Receptor expression-enhancing protein 5
IPI00958032.1LOC100363622;LOC100363145 rCG42396-like isoform 2IPI00421366.1RGD1305481 LRRGT00030IPI00392935.3Myl6b RCG42490, isoform CRA_f
IPI00555275.1LOC100365629;LOC100362623 MetallothioneinIPI00363265.3Hspa9 Stress-70 protein, mitochondrial*IPI00215294.1Ddah2 N(G),N(G)-dimethylargimne dimethylaminohydrolase 2
IPI00207480.3Crtap Cartilage-associated protein, isoform CRAb*IPI00471890.1F3 Tissue factorIPI00766695.2Golga4 golgi autoantigen, golgin subfamily a, 4
IPI00952031.1Mfge8 Putative uncharacterized protein Mfge8IPI00568756.1Epb4 113 Putative uncharacterized protein Epb4.1l3IPI00957678.1LOC100364427 ribosomal protein S12-like*
IPI00959858.1Vcan VersicanIPI00779937.3LOC100361890 ubiquitin-conjugating enzyme E2H-like isoform 1IPI00869493.2Sdf2 Stromal cell derived factor 2
IPI00371230.2Tmemi 19 Transmembrane protein 119IPI00231660.5S100g Protein S100-GIPI00950239.1Bsg 30 kDa protein
IPI00373753.5Ptk7 102 kDa protein    
Table 2. List of Down-regulated Proteins During Rat HSC Activation
Accession NO.Gene Symbol and NameAccession NO.Gene Symbol and NameAccession NO.Gene Symbol and Name
  • This table contains the 200 proteins that displayed more than 3.0-fold down-regulation in activated rat HSCs in three independent experiments. The International Protein Index (IPI) accession number, gene symbol, and name of each protein are provided here. The proteins are listed in ascending order according to their fold change, and the top 10 down-regulated proteins appear in bold. For detailed information about these proteins, refer to Supporting Table 1.

  • *

    Proteins reported by Kristensen et al.6

IPI00208422.2Dpp4 Dipeptidyl peptidase 4IPI00768591.1Pstpip2 Proline-serine-threonine phosphatase interacting protein 1-likeIPI00370714.1Umps Uridine monophosphate synthetase
IPI00464895.1LOC298116 Rat alpha-2u-globulinIPI00464672.1Abi3 ABI gene family, member 3, isoform CRA bIPI00914737.1.Tmtc3 Transmembrane and tetratricopeptide repeat containing i (Predicted), isoform CRA_a
IPI00372776.3Dnajc 10 DnaJ homolog subfamily C member 10IPI00327398.1Enpep Isoform 1 of Glutamyl aminopeptidaseIPI00373076.1Atp6v1a ATPase, H+ transporting, lysosomal V1 subunit A
IPI00212697.1Napsa napsin A aspartic peptidaseIPI00231200.5Por NADPH--cytochrome P450 reductaseIPI00421601.3Asah1 Acid ceramidase
IPI00192301.2Gpx1 Glutathione peroxidase 1IPI00766273.1LOC684828 Histone cluster 1, H1d-likeIPI00464668.1Irgm Immunity-related GTPase family M protein
IPI00194804.1Gzma Granzyme AIPI00210444.5Hmgcs2 Hydroxymethylglutaryl-CoA synthase, mitochondrialIPI00193108.1Pycard Apoptosis-associated speck-like protein
IPI00231262.7S100a9 Protein S100-A9IPI00373492.2Lcp1 Lymphocyte cytosolic protein 1IPI00366190.4Lmnb2 Putative uncharacterized protein Lmnb2
IPI00210644.1Cps1 Carbamoyl-phosphate synthase, mitochondrial*IPI00365967.3Gzmm Granzyme MIPI00197770.1Aldh2 Aldehyde dehydrogenase, mitochondrial*
IPI00392753.3LOC298109 RCG32004IPI00767601.1LOC100366216 Nuclear antigen Sp100-likeIPI00367063.1Cndp1 Beta-Ala-His dipeptidase
IPI00785608.2Mup5 Alpha-2u globulinIPI00369397.5H2afx Histone H2AIPI00326972.6Ces3 Carboxylesterase 3*
IPI00213847.3Grn Granulins isoform aIPI00213828.1LOC619574 Uncharacterized protein C17orf62 homologIPI00421874.4Vdad Voltage-dependent anion-selective channel protein 1
IPI00230979.1Nampt Nicotinamide phosphoribosyltransferaseIPI00829505.1Lap3 Isoform 2 of Cytosol aminopeptidaseIPI00367815 1MGC108823 Similar to interferon-inducible GTPase
IPI00369234.3Igtp Ac2-233IPI00551812.1Atp5b ATP synthase subunit beta, mitochondrialIPI00370711.3Epx Putative uncharacterized protein Epx
IPI00207390.9Anxa3 Annexin A3*IPI00766882.1Rbmxrt RNA binding motif protein, X-linked-likeIPI00782366.1Anxa7 Putative uncharacterized protein Anxa7
IPI00230874.10Blvra Biliverdin reductase AIPI00948721.1Acaa2 42 kDa proteinIPI00210920.11Got2 Aspartate aminotransferase, mitochondrial
IPI00231742.5Cat CatalaseIPI00204359.1B2m Beta-2-microglobulinIPI00210280.11Comt Isoform 1 of Catechol O-methyltransferase*
IPI00364591.2Plbdi Putative phospholipase B-like 1IPI00562255.2Ctla2a Similar to ctla-2-beta protein (141 AA) (Predicted) isoform CRA_aIPI00214434.3Rab11a Ras-related protein Rab-11A
IPI00364616.2RGD1309676 Uncharacterized protein C10orf58 homologIPI00190499.5Tpp1 Tripeptidyl-peptidase 1IPI00204763.4/Arhgap25 Similar to Rho-GTPase-activating protein 25
IPI00195423.1Ugt2b37 UDP-glucuronosyltransferase 2B37IPI00949858.1Ctsc Putative uncharacterized protein CtscIPI00782093.2Prexi Putative uncharacterized protein Prexi
IPI00203054.2Acsf2 Acyl-CoA synthetase family member 2, mitochondrialIPI00959089.1Fth1 Ferritin (Fragment)IPI00195123.1Atp5o ATP synthase subunit O, mitochondrial
IPI00767419.2Siae Putative uncharacterized protein SiaeIPI00382226.1Fam65bAb2-162IPI00198327.2Vdac2 Voltage-dependent anion-selective channel protein 2
IPI00567836.2Ptgs1 Ptgs1 proteinIPI00199695.3Serpinf2 Serine (Or cysteine) peptidase inhibitor, clade F member 2IPI00205157.1Hadh Hydroxyacyl-coenzyme A dehydrogenase, mitochondria
IPI00206626.1Hmox1 Heme oxygenase 1IPI00325975.3Vamp8 Vesicle-associated membrane protein 8IPI00210692.11Casp7 Caspase-7
IPI00760118.1Hsd11b1 Isoform 11-HSD1B of Corticosteroid 11-beta-dehydrogenase isozyme 1IPI00763589.2LOC684681 histone cluster 1, H1c-likeIPI00361732.44Diaph2 Diaphanous homolog 2
IPI00360317.1Cd180 Putative uncharacterized protein Cd180IPI00950371.1Stat4 88 kDa proteinIPI00198237.11Psmb10 Proteasome subunit beta type-10
IPI00554206.3Ugt2b UDP glycosyltransferase 2 family, polypeptide BIPI00949517.1Actr3 Actin-related protein 3IPI00196648 55Stx7 Syntaxin-7
IPI00958846.1LOC683399 Igk protein-like isoform 2IPI00388462.4- Putative uncharacterized protein 687510IPI00209184.3Cd1d1 Antigen-presenting glycoprotein CD1d
IPI00895603.1Pld4 Phospholipase D4IPI00948996.1- Putative uncharacterized protein Lpcat2IPI00198080.1Pcyox1 Prenylcysteine oxidase
IPI00421898.1Ifi47 Ifi47 proteinIPI00211970.5Psme2;LOC100364216 Proteasome activator complex subunit 2*IPI00948302.1Atp5c1 ATP synthase gamma chain
IPI00207947.6Lta4h Leukotriene A4 hydrotaseIPI00422051.2RT1-Ba MHC class II antigen RT1.B alpha chainIPI00951116.1Hbb 16 kDa protein
IPI00948229.1Gbp2 Putative uncharacterized protein ENSRNOP00000022899IPI00569009.1RT1-Bb RT1 class II histocompatibility antigen, B-1 beta chain precursorIPI00515816.1Lgmm Legumain
IPI00231191.7Glrxi Glutaredoxin-1IPI00200806.3Ada Adenosine deaminaseIPI00373418.3Dbt dihydrolipoamide branched chain transacylase E2
IPI00882532.1Afmid ArylformamidaseIPI00944815.1Psmb9 Proteasome subunit beta type-9IPI00370158.3Rac2 Ras-related C3 botulinum toxin substrate 2
IPI00209291.1Esyt1 Extended synaptotagmin-1IPI00480679.4Krt18 Keratin, type I cytoskeletal 18IPI00191737.6Alb Serum albumin
IPI00371870.1Smpdl3a Acid sphingomyelinase-like phosphodiesterase 3aIPI00781895.1Fyb Similar to FYN binding protein (Predicted), isoform CRA_bIPI00206092.1Akr1b8 Aldose reductase-like protein
IPI00209690.1Ephxi Epoxide hydrolase 1IPI00193049.1Sult1a1 Sulfotransferase 1A1*IPI00564976 3Fgd2 Putative uncharacterized protein Fgd2
IPI00969321.1RT1-EC2 A2qIPI00231418.5Lmnbi Lamin-B1IPI00198897 1Ndufa6 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6
IPI00911233.2Tapbp TAP-binding proteinIPI00734729.1Tcirgi V-H+ATPase subunit a3IPI00360541 4tgb2 Integrin beta 2
IPI00958096.1LOC100360950 GF20391-like isoform 1IPI00359383.3LOC100359680;Lpcat2 Lysophosphatidylcholine acyltransferase 2IPI00394488.2Gbas Glioblastoma amplified sequence
IPI00476115.1RT1-Db1 RT1 class II histocompatibility antigen, D-1 beti chainIPI00382229.1Stat1;Stat4Ab2-131IPI00957241.1Vdac3 Putative uncharacterized protein Vdac3
IPI00958624.1Stab2 Stabilin 2IPI009589621Hist1h4b;Hist1h4m histone cluster 1, H4mIPI00327781 1Cyp2c11 Cytochrome P450 2C11
IPI00231248.5Hpgds Hematopoietic prostaglandin D synthaseIPI00207663.2Ctsz Cathepsin ZIPI00210228.5Ctss Cathepsin S
IPI00758468.2Pgcp Plasma glutamate carboxypeptidaseIPI00368211.2Ifit2 Interferon-induced protein with tetratricopeptide repeats 2IPI00193485 2Idh2 Isocitrate dehydrogenase [NADP], mitochondrial
IPI00324309.4Cybb Endothelial type gp91-phoxIPI00471577.1Uqcrc1 Cytochrome b-c1 complex subunit 1, mitochondrialIPI00213457 1Atp6v1c1 V-type proton ATPase subunit C 1
IPI00372391.2F5AC2-120IPI00202580.3Cyp2d3 Cytochrome P450 2D3IPI00562259.1Slc25a3 Phosphate carrier protein, mitochondrial
IPI00555265.1Nnt Nicotinamide nucleotide transhydrogenaseIPI00559910.1Rbmxrtl Heterogeneous nuclear ribonucleoprotein GIPI00361887.1LOC685909 Histone H2A
IPI00208382.1Ppt1 Palmitoyl-protein thioesterase 1IPI00948938.1Mrc1 Putative uncharacterized protein Mrc1IPI00780674.1Ugt1a7c UDP-glucuronosyltransferase 1-7
IPI00371511.4Creg1 Cellular repressor of E1A-stimulated genes 1IPI00214461.11ll 1rn lnterleukin-1 receptor antagonist proteinIPI00231370.7S100a8 Protein S100-A8
IPI00212635.2Igf2 insulin-like growth factor 2 precursorIPI00212219.1Ssb Lupus La protein homologIPI00362072.2Actr2 Actin-related protein 2
IPI00372986.5Vsig4 V-set and immunoglobulin domain containing 4IPI00471794.1Npl N-acetylneuraminate lyaseIPI00876603 1Hnrpm Heterogeneous nuclear ribonucleoprotein M isoform a
IPI00199448.4Lgals3bp Galectin-3-bmding proteinIPI00948998.1Naga Putative uncharacterized protein NagaIPI00205519.5Uggti UDP-glucose:glycoprotein glucosyltransferase 1
IPI004762957Ahcy AdenosylhomocysteinaseIPI00777022.2Fcgr2b Low affinity immunoglobulin gamma Fc region receptor IIIPI00196107.1Atp5f1 ATP synthase subunit b, mitochondrial
IPI00210038.1Lipa Lysosomal acid lipase/cholesteryl ester hydrolaseIPI00198717.8Mdh1 Malate dehydrogenase, cytoplasmic*IPI00231780.5H1f0 Histone H1.0
IPI00464497.1LOC305806 Similar to glutaredoxin 1IPI00950779.1Nckap1l Putative uncharacterized protein Nckap1lIPI00947965 1Lrmp 25 kDa protein
IPI00392676.4Blvrb Biliverdin reductase BIPI00366405.2Fam49b Similar to 0910001A06Rik protein (Predicted), isoform CRA_aIPI00470288.4Ckb Creatine kinase B-type*
IPI00201891.1Hk3 Hexokinase-3IPI00421899.1Cndp2 Cytosolic non-specific dipeptidaseIPI00208568 3Plek Pleckstrin
IPI00368206.1Ifit3 Interferon-induced protein with tetratricopeptide repeats 3IPI00203406.1Osbpl1a Oxysterol-binding protein-related protein 1IPI00200466.3Slc25a5 ADP/ATP translocase 2
IPI00210901.1Cd38 ADP-ribosyl cyclase 1IPI00192876.1Mx1 Interferon-induced GTP-binding protein Mx1IPI00764346.2REVERSED - 48 kDa protein
IPI00365035.2Adfp Adipose differentiation related proteinIPI00188924.4Uqcrc2 Cytochrome b-d complex subunit 2, mitochondrialIPI00213667.1LOC286987 Hemiferrin
IPI00230788.6Car3 Carbonic anhydrase 3IPI00954695.1RGD1309586 RCG20177IPI00358463.1Arhgdib Rho, GDP dissociation inhibitor (GDI) beta*
IPI00959683.1Hist2h3c2;Hist1h3f;Hist2h3c;LOC100364555;LOC684762 Histone H3IPI00231949.5Cd14 Monocyte differentiation antigen CD14IPI00565330 1Acaa1 Putative uncharacterized protein Acaa1
IPI00369645.2Cd68 Cd68 moleculeIPI00949639.1Pik3r1 ProteinIPI00327330.2Cd2ap CD2-associated protein
IPI00190377.2Taldo1 TransaldolaseIPI00760125.1Ndel1 Isoform 2 of Nuclear distribution protein nudE-like 1IPI00194341.5Lgals3 Galectin-3
IPI00903439.1RT1-EC2A2bIPI00951515.1Paox RCG47968, isoform CRA_aIPI00189981.1F2 Prothrombin (Fragment)
IPI00951700.1Cd163 Putative uncharacterized protein Cd163IPI00327644.5Lyn Isoform LYN A of Tyrosine-protein kinase LynIPI00365613.2Snx6 Sorting nexin 6
IPI003914422Stom Putative uncharacterized protein StomIPI00371634.1Bcap31 B-cell receptor-associated protein 31IPI00194222.1Cox4i1 Cytochrome c oxidase subunit 4 isoform 1, mitochondrial
IPI00776581.2Gusb Beta-alucuronidaseIPI00896162.1Fermt3 Fermt3 protein  

Confirmation of Previous Findings.

As demonstrated in Table 1, most of the top 10 up-regulated proteins have been shown to be involved in HSC activation and typify the characteristics associated with HSC activation. These proteins include COL1A1, a well-known marker of HSC activation, as well as tropomyosin alpha-1, nonmuscle caldesmon, and transgelin, implicating myogenic differentiation of activated HSCs.1 In addition, the other well-known markers of HSC activation, α-SMA, COL3A1, TGF-β3, and beta-type platelet-derived growth factor receptor, were also overexpressed in activated HSCs (Table 1 and Supporting Table 1). Furthermore, the list obtained in the present study covered over 85% (126/147) of the proteins in Kristensen's list (proteins marked with an asterisk [*] in Supporting Table 1 and Tables 1 and 2).6 Thus, the altered proteins revealed by the present proteomic experiments are very likely to be HSC activation–related.

Bioinformatics Analysis of the Altered Proteins During HSC Activation.

To obtain a comprehensive view of the biological significance of the differentially expressed proteins, these proteins were categorized according to their main biological function by IPA based on the underlying biological evidence from the curated Ingenuity Pathways Knowledge Base. The enriched molecular and cellular functions of up-regulated proteins were mainly related to cellular movement (81 proteins), cell morphology (65 proteins), cellular growth and proliferation (117 proteins), and cellular assembly and organization (93 proteins) (Fig. 2A), whereas the down-regulated proteins were predominantly involved in immune response (56 proteins), cell death (61 proteins), and lipid metabolism (43 proteins) (Fig. 2B). For more details, refer to Supporting Tables 2 and 3.

Figure 2.

Biological function analyses of differentially expressed proteins during HSCs activation. The top biological functions of the up-regulated (A) and down-regulated (B) proteins, as determined by IPA, are shown. The x axis shows the negative log of P value.

IPA was also adopted for grouping proteins into functional networks and/or canonical pathways to determine the altered cellular activities during HSC activation. The top network associated with up-regulated proteins related to skeletal and muscular system development and function, indicative of the myofibroblastic transdifferentiation of activated HSCs. This network was linked by the Rho-ROCK-JNK pathway and calmodulin as predicted by IPA (Fig. 3A). The top network of down-regulated proteins related to immunological disease and/or inflammatory response, which was linked by a nuclear factor of activated T cells (NFAT) complex and extracellular regulated kinase 1/2 (ERK1/2) (Fig. 3B). The top canonical pathway of down-regulated proteins was an antigen presentation pathway, consisting of a panel of molecules involved in antigen processing and presentation, such as major histocompatibility complex 1 (MHC-I), MHC-II, and CD1d1 (Fig. 3C). Generally, except for myofibroblastic transdifferentiation, suppressed immune response emerged as another outstanding characteristic of activated HSCs.

Figure 3.

Top networks and pathway assigned by Ingenuity Pathway Analysis of differentially expressed proteins during HSC activation. (A) The top network of up-regulated proteins is skeletal and muscular system development and function, and this network is linked by the Rho-ROCK-JNK pathway and calmodulin. (B) The top network of down-regulated proteins is related to immunological disease and/or inflammatory response, and this network could be linked by NFAT complex and ERK1/2. (C) Decreased expression of molecules involved in canonical antigen presentation pathway. Proteins shaded in red indicate a 3.0-fold or greater increase, while proteins shaded in green indicate a 3.0-fold or greater decrease in abundance in activated HSCs compared with quiescent HSCs, and the color intensity corresponds to the degree of abundance. Proteins in white are those identified through the Ingenuity Pathways Knowledge Base. The shapes denote the molecular class of the protein. Solid lines indicate direct molecular interactions, and dashed lines indicate indirect molecular interactions.

Validation of the Proteomic Results by Western Blotting.

Differential expression of 13 selected proteins was further evaluated by western blotting, focusing on those involved in the suppressed immune response of activated HSCs. Compared with quiescent HSCs, the expression of eight proteins involved in cellular movement and assembly (biglycan, calponin-1, and TGF-β3), cell proliferation (galectin-1), retinoid metabolism (CRBP1), immune response (B7H3), and cochaperone activity (BAG2 and BAG3) displayed remarkable up-regulation; whereas the five proteins involved in immune response and antigen presentation (B2M, RT1-A, RT1-Ba, CD1d1, and LTA4H) showed a significant down-regulation in activated HSCs (Fig. 4). Among them, RT1-A belongs to the MHC-I family, and RT1-Ba belongs to the MHC-II family. The constitutive protein β-actin exhibited no obvious expression change. The western blotting results confirmed the expression pattern observed in the quantitative proteomics analysis.

Figure 4.

Validation of comparative proteomic results by western blotting. Differential expression of eight up-regulated proteins (BAG2, BAG3, CRBP1, biglycan, galectin-1, calponin-1, TGF-β3, and B7H3 [CD276]) and five down-regulated proteins (B2M, RT1-A [RT1-EC2 A2q], RT1-Ba, CD1d1, LTA4H) was validated by western blotting. β-Actin was served as loading control. Each blot is a representative of three independent experiments. The relative ratios of proteins by isobaric tag for relative and absolute quantitation (iTRAQ) analysis are shown at right. N/A, not available. D2 and D10 represent day 2 quiescent and day 10 activated HSCs.

The Biological Significance of BAG2 and BAG3 in HSC Activation.

In the present study, we report the expression of four of the six members of the BAG family in HSCs for the first time: BAG 1, 2, 3, and 5. The members of BAG family are cochaperones of heat shock protein 70 (Hsp70).10 Until now, however, there were very few reports of BAG family members in HSCs. Given the robust up-regulation of BAG2 and BAG3 (in the top 10 and 20 up-regulated proteins in activated HSCs, respectively) (Table 1) and the critical roles of Hsp70 on the biogenesis and maintenance of proteins, we preliminarily evaluated the biological significance of these two proteins in activated HSCs in siRNA transfection experiments (Fig. 5A). Our data show that knockdown of either Bag2 or Bag3 significantly inhibits the adhesion and migration of activated HSCs, and the inhibitory effects of Bag3 knockdown are more potent than those of Bag2 (Fig. 5B-D), while only the Bag3 knockdown suppresses the proliferation of activated HSCs (Fig. 5E,F).

Figure 5.

Involvement of BAG2 and BAG3 in HSC proliferation, adhesion, and migration. (A) Knockdown efficiency of siRNAs. Passage 2 HSCs were transfected with Bag2- and Bag3-specific siRNA or with NTC siRNA;, after 48 hours, their mRNA and protein levels were determined by quantitative polymerase chain reaction and western blot (inset), respectively. β-Actin was used as a loading control. (B) Knockdown of Bag2 and Bag3 suppresses the adhesion of HSCs. Data represent the percentage change in the absorbance (OD 570 nm) of adherent cells relative to NTC transfected cells at 30 minutes after plating. (C, D) BAG2 and BAG3 are required for HSC migration. (C) HSCs transfected with siRNAs that migrated to the underside of Transwell membranes. HSCs were plated on 8-μm pore size Transwell inserts for 16 hours. The number of migrated cells was counted manually (original magnification ×200). (D) The statistical results of three independent experiments. (E, F) Knockdown of Bag3 suppresses HSCs proliferation. (E) EdU cell proliferation assay. EdU was detected by Alexa Fluor 594 azide (red) and nuclei were counterstained with Hoechst 33258 (blue) (original magnification ×200). (F) Statistical results of three independent experiments. The results are expressed as the labeling index according to the following formula: number of EdU-positive nuclei × 100 / number of total nuclei. N, untransfected normal cells; NTC, nontargeting control siRNA–transfected cells; Bag2-siRNA and Bag3-siRNA, cells transfected with Bag2-siRNA and Bag3-siRNA, respectively. Each image is a representative of three independent experiments. All values are presented as the mean ± SD of three independent experiments. *P < 0.01, #P < 0.05 compared with NTC.

Overexpression of B7H3 in HSCs of Cirrhotic Liver.

Based on the aforementioned in vitro data, the suppressed immune response of activated HSCs was further validated in an in vivo liver fibrosis model. B7H3 was found to be up-regulated in activated HSCs (Table 1 and Fig. 4). On the basis of its inhibitory action on T cell responses,11 B7H3 was chosen for further validation. The chronic CCl4-induced liver fibrosis model was established as described,9 and liver fibrosis was verified by sirius red staining in formalin/paraffin sections (Supporting Fig. 3). Activated HSCs were identified by their location, morphological features, and immunostaining of cytoplasmic α-SMA (Fig. 6A and D). In cirrhotic rat liver, α-SMA–positive cells were mainly found in fibrotic septa (Fig. 6D), while in normal rat liver, α-SMA was only observed in cells in portal vessel walls (Fig. 6A). B7H3 was also visualized by immunostaining (Fig. 6B,E). B7H3-positive cells were rare in the normal liver (Fig. 6C); however, in cirrhotic liver, B7H3 was obviously counterstained with α-SMA in activated HSCs within fibrotic septa (Fig. 6F).

Figure 6.

Double immunostaining of α-SMA and B7-H3 in normal (A-C) and cirrhotic (D-F) rat liver. (A, D) Immunofluorescent staining of α-SMA (fluorescein isothiocyanate, green) (original magnification ×200). (B,E) Immunofluorescent staining of B7H3 (Texas red) (original magnification ×200). (C, F) Merged images of (A), (B) and (D), (E). Nuclei were counterstained with Hoechst 33258 (blue). Each image is a representative of six animals in each group.

Discussion

A Comprehensive View of the Activation of HSCs.

During liver injury, activated HSCs lose their cytoplasmic retinoid lipid droplets, and transdifferentiate into proliferative and fibrogenic myofibroblasts,1 which are the major producers of ECM in the injured liver. The present proteomic study not only exemplified these well-known features of activated HSCs but also identified a number of novel proteins associated with HSC activation and extended our understanding of this process. To our knowledge, this is the first and most comprehensive large-scale proteomic profiling of HSCs, and the data will facilitate future studies of HSCs.

As highlighted by bioinformatics analysis, the most enriched biological function categories of up-regulated proteins in activated HSCs were cellular movement, cell morphology, cellular assembly, and organization (Fig. 2A, Supporting Table 2). The top network of up-regulated proteins was related to skeletal and muscular system development and function (Fig. 3A), which corresponded to increased ECM synthesis, contractility, and migration, exhibiting the myofibroblast transdifferentiation of activated HSCs. In addition to increased expression of fibrillar collagens (COL1A1, COL1A2, COL3A1, and COL51A1), several proteins that are required for proper collagen biosynthesis were simultaneously up-regulated, including prolyl 3-hydroxylase 1 and prolyl 4-hydroxylase.12 Moreover, except for a group of intensively studied ECM components, including fibronectin, laminin, dystroglycan, and SPARC, a number of novel ECM components were found to be up-regulated in activated HSCs, such as biglycan, glypican-1, and glypican-4. Some of these novel ECM components were further validated by western blotting (Fig. 4). These newly identified ECM proteins and proteins involved in ECM synthesis may be useful as potential markers for liver cirrhosis.

Another interesting finding was the involvement of BAG2 and BAG3, two cochaperones of Hsp70, in the motility of activated HSCs (Fig. 5). It has been demonstrated that, by regulating actin folding, stabilizing myofibril structure, and inhibiting myofibrillar degeneration, BAG3 is required for proper cytoskeleton dynamics and cell motility,10 and it has also been reported to be induced during cardiomyoblast differentiation.13 In addition, BAG2 can act as an inhibitor of chaperone-mediated degradation and promote protein maturation.14 In this regard, overexpression of BAG2 and BAG3 could be associated with the robust protein synthesis of activated HSCs, especially of the ECM and cytoskeleton, and cytoskeletal remodeling during the myogenic differentiation of HSCs. This finding could provide new insight into the modulation of HSC activation.

Our observation also certified the critical role of HSCs in liver cirrhosis. Most of the cell structure–associated proteins listed in a recent study searching for novel biomarkers of liver cirrhosis,15 including α-SMA, tropomyosin alpha-4, transgelin, calponin-1, and tropomyosin beta, were found to be markedly up-regulated in activated HSCs (Table 1 and Supporting Table 1). Among them, calponin-1, a specific marker for smooth muscle cell differentiation,16 could serve as a possible marker for HSC activation.

Transdifferentiation of HSCs is primarily regulated by paracrine and autocrine loops of growth factors.1 In the present study, TGF-β3, connective tissue growth factor, and beta-type platelet-derived growth factor receptor were identified as up-regulated proteins during HSC activation (Fig. 4 and Table 1). It should be noted that TGF-β3 is the predominant isoform of TGF-β produced by activated HSCs in liver and has been shown to play an essential role in liver fibrosis by potently stimulating collagen synthesis and promoting myofibroblast differentiation.17, 18 Connective tissue growth factor is an important downstream effector of TGF-β-induced fibrosis.18

Another prominent characteristic of activated HSCs, enhanced proliferation leads to increased numbers of activated HSCs and potently promotes the fibrogenic response of these cells.1 One of the enriched categories of up-regulated proteins was directly associated with cell growth and proliferation (Fig. 2A and Supporting Table 2). During HSC activation, the expression of proteins promoting cell proliferation was up-regulated (CD81, FSTL1, galectin-1, S100-A6/calcyclin, S100-A11/calgizzarin, SPARC, etc.),6 as well as the antiapoptotic proteins (alpha-crystallin B chain, astrocytic phosphoprotein, four and a half LIM domains protein 2, heat shock protein beta-1, etc.).19 In contrast, the abundance of proteins promoting apoptosis, such as caspase-7, was concurrently down-regulated (Supporting Table 1). These findings indicate that the induction of HSC apoptosis represents a promising target to achieve antifibrotic effects.

The loss of cytoplasmic retinoid lipid droplets is also a distinguishing phenomenon during HSC activation,5 but the relevant mechanisms are still poorly understood. Under normal conditions, more than 98% of the retinoid compounds in HSCs are stored as retinyl esters (REs). When dietary retinoid intake is insufficient, these REs undergo hydrolysis, releasing retinol into the blood. Thus, the formation and hydrolysis of REs are key processes in the metabolism of retinoid. In the present study, both CRBP1, a specific carrier for retinol that plays a crucial role in retinol transportation and esterification,20 and carboxylesterase 3 (Ces3 or ES-10), which possesses RE hydrolase activity,21 were identified in HSCs. These findings verified that HSCs have great capacity for the storage and metabolism of retinoid. However, the expression pattern of these proteins is puzzling. In accordance with the reduction of substrate (REs), expression of ES-10 is reduced in activated HSCs. However, as RE reserves are depleted, expression of CRBP1 increases abruptly (14.6577-fold). The induction of CRBP1 during HSC activation might reflect a compensatory mechanism by which the cultured cells try to restore storage of retinoid compounds.22

On the other hand, lipid droplets are a specified organelle for retinoid storage. Retinol esterification and storage, however, depends on the solubilization of newly formed esters in lipid droplets. Our data reveal the abundance of a group of proteins that promote adipocyte differentiation and lipid metabolism is coordinately decreased in activated HSCs, including adipose differentiation–related protein, insulin-like growth factor 2, acyl-COA synthetase, acetyl-CoA acyltransferase, and others (Table 2 and Supporting Table 3).Therefore, in activated HSCs, impaired lipid metabolism would inevitably abate retinol esterification and storage. As Hellemans et al.22 suggested, we also propose that retinoid depletion might result from an inability of the activated HSCs to provide a favored microenvironment for retinol esterification and storage rather than the aberrant retinol-esterification pathway.

Suppressed Immune Response of Activated HSCs.

In normal liver, the majority of pathogens targeting hepatocytes have to cross the perisinusoidal space of Disse. Therefore, HSCs adjacent to hepatocytes are located at a privileged site to sense pathogens attacking liver parenchyma. However, the exact role of HSCs in intrahepatic immune regulation remains uncertain.2, 23

In the present study, we demonstrated that HSCs express a panel of molecules involved in antigen processing and presentation, including those that participate in the MHC-I, MHC-II, and CD1d1 presentation pathways (Figs. 3B,C and 4 and Supporting Table 3). Among them, MHC-II proteins are specifically expressed on professional antigen-presenting cells. Thus, HSCs represent powerful antigen-presenting cells for inducing specific T cell responses in normal liver. More interestingly, almost all of the proteins involved in antigen processing and presentation were down-regulated in activated HSCs, along with proteins promoting immune responses, such as interferon-induced proteins (interferon-γ–inducible protein 1 and interferon-induced protein with tetratricopeptide repeats 2 and 3) and enzymes involved in inflammatory mediator biosynthesis (LTA4H, hematopoietic prostaglandin D synthase, etc.) (Fig. 4 and Supporting Tables 1 and 3). On the other hand, B7H3, a negative regulator for T cell activation and function,11 was up-regulated in activated HSCs (Fig. 4). B7H3 is a member of the coinhibitory molecules of the B7 family that are expressed by professional antigen-presenting cells. Recently, B7H1 and B7H4, another two members of the B7 family, were identified in mouse HSCs and demonstrated to mediate inhibition of intrahepatic T cell responses.2, 24 Therefore, we presumed that the immune response of HSCs was impaired upon activation.

Bioinformatics analysis also highlighted this presumption: the most enriched biological function of down-regulated proteins was involved in the immune response (Fig. 2B), the top canonical pathway of down-regulated proteins was an antigen presentation pathway (Fig. 3C), and the top network of down-regulated proteins was also engaged in the immune response, which could be linked by NFAT and ERK1/2 (Fig. 3B). The NFAT complex can activate the transcription of a large number of genes during an effective immune response,25 and ERK1/2 can effect proinflammatory cytokine production.26 We also noticed that three of the top 10 down-regulated proteins were related to immune response (dipeptidyl peptidase 4, granzyme A, and S100-A9).27, 28 Taken together, these data indicate that the suppressed immune response is another prominent characteristic of activated HSCs. This feature was further validated by confirming the overexpression of B7H3 in HSCs in the rat liver fibrosis model (Fig. 6).

In fibrotic liver, the up-regulation of B7H3 together with the down-regulation of antigen-presenting molecules in activated HSCs would impair intrahepatic T cell responses and establish an immunosuppressive microenvironment, whereby tumor cells could evade antitumor T cell responses.29 Together with ECM remolding30 and loss of retinoid lipid droplets,31 suppressed immune response of activated HSCs may contribute to the establishment of a favorable microenvironment for tumorigenesis. For these reasons, the biological significance of HSC activation in HCC development and prognostic judgment deserves intensive study.

The major limitation of a general proteomic analysis was met in the present study as well: larger and more abundant proteins are more easily detected, whereas low abundance proteins are much less likely to be detected. Those low abundance proteins, especially components of signaling pathways and transcription factors, have not yet been fully presented. Thus, in future proteomic analyses, specific enrichment of low-abundance proteins could be adopted to expand the present proteomic database for HSC activation.

In conclusion, the present quantitative proteomic study provided the most comprehensive proteome profiles of rat HSCs and a list of differentially expressed proteins between quiescent and culture-activated HSCs. Bioinformatics and biological analyses of these altered proteins expanded our understanding of the major characteristics of activated HSCs, such as accumulated ECM components, migration, proliferation, and loss of retinoid lipid droplets. More importantly, the present study cast new light on the suppressed immune response of activated HSCs and provided novel insight into the role of HSCs in intrahepatic immunity during liver injury. The data provided here will promote our understanding of the effects of HSC activation on liver fibrosis and HCC tumorigenesis, but will also benefit the development of more efficient diagnostic and treatment strategies for liver fibrosis, HCC, and other liver diseases.

Acknowledgements

We thank Dongmei Qiu, a graduate student in the Department of Pathology, Medical School of Nantong University, for technical assistance in immunohistochemistry.

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