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.
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.
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
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.
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.
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.
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.
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.
|Accession NO.||Gene Symbol and Name||Accession NO.||Gene Symbol and Name||Accession NO.||Gene Symbol and Name|
|IPI00200134.1||Asam Adipocyte adhesion molecule||IPI00851116.1||Nt5e 5′ nucleotidase, ecto*||IPI003641892||Eif3j Eukaryotic translation initiation factor 3 subunit J|
|IPI00391995.2||Tpm1 Isoform 2 of Tropomyosin alpha-1 chain*||IPI00204078.1||Nradd P75-like apoptosis-inducing death domain protein long isoform||IPI00209789.1||Fkbp3 FK506 binding protein 3, 25kDa|
|IPI00193981.3||Bag2 Bag2 protein||IPI00210187.1||Pdlim5 PDZ and LIM domain protein 5||IPI00361798.2||Igfbp7 Insulin-like growth factor binding protein 7|
|IPI00208118.1||Caldi Non-muscle caldesmon*||IPI00476899.1||Eef1b2 Eukaryotic translation elongation factor 1 beta 2||IPI00480766.1||Acat2 Acetyl-CoA acetyltransferase, cytosolic|
|IPI00231196.5||Tagln Transgelin||IPI00203250.1||Dpysl3 Isoform 2 of Dihydropyrimidinase-related protein 3||IPI00324585.3||Itga1 Integrin alpha-1|
|IPI00366944.2||Col3a1 Collagen alpha-1(lll) chain*||IPI00952140.1||Nedd4 E3 ubiquitin-protein hgase NEDD4||IPI00370411.2||Fbln1 Putative uncharacterized protein Fbln1|
|IPI00198250.1||Akap12 Isoform 2 of A-kinase anchor protein 12||IPI00201858.4||Pbxipi Pre-B-cell leukemia transcription factor-interacting protein 1||IPI00364502.5||Nfu1 NFU1 iron-sulfur cluster scaffold homolog precursor|
|IPI00208221.1||Fkbp10 65kDa FK506-binding protein, isoform CRA_a||IPI00363849.2||Lamc1 laminin, gamma 1||IPI00208306.1||Tpt1 Translationally-controlled tumor protein*|
|IPI00204375.2||Uchl1 Ubiquitin carboxyl terminal hydrolase isozyme L1||IPI00195673.1||Tubb6 Tubulin, beta 6||IPI00371266.1||Naca Nascent-polypeptide-associated complex alpha polypeptide (Predicted), isoform CRA_b|
|IPI00231784.3||Dbn1 Isoform E1 of Drebrin||IPI00202627 1||Scrn1 Secernin-1||IPI00206818.1||Ifitm3 Interferon-inducible protein variant 10|
|IPI00365582.3||Cad Putative uncharacterized protein Cad||IPI00869592.3||Mylk Myosin light chain kinase||IPI00360771.3||Ccdc6 Putative uncharacterized protein Ccdc6|
|IPI00361824.3||Fam114a1 hypothetical protein||IPI00191681.1||Itgb1 Integrin beta-1||IPI00373505.2||Tmod3 Tropomodulin 3|
|IPI00200757.1||Fn1 Isoform 1 of Fibronectin||IPI00930907.1||Sdf4 Isoform 1 of 45 kDa calcium-binding protein||IPI00882548.1||Hdlbp High density lipoprotein binding protein|
|IPI00187731.4||Tpm2 Isoform 2 of Tropomyosin beta chain*||IPI00765344.2||Sfrp1 Secreted frizzled-related protein 1||IPI00957286.1||Sorbs1 sorbin and SH3 domain containing 1|
|IPI00476991.1||Ncam1 Neural cell adhesion molecule 1*||IPI00393975.2||Map4 Isoform 1 of Microtubule-associated protein 4||IPI003391974.1||Pkm2 Isoform M2 of Pyruvate kinase isozymes M1/M2*|
|IPI00188909.3||Col1a1 Collagen alpha-1(l) chain*||IPI00948019.1||Fst1 Ab2-379||IPI00958724.1||Mcc mutated in colorectal cancers|
|IPI00188590.1||Fkbp7 FK506 binding protein 7||IPI00763873.3||Itga11 Integrin, alpha 11||IPI00400579.1||Gaa Lysosomal alpha-glucosidase|
|IPI00203974.2||Bag3 Bcl-2-interacting death suppressor||IPI00326462.1||Enpp3 Ectonucleotide pyrophosphatase/phosphodiesterase: family member 3||IPI00200070.1||Nucb2 Nucleobindin-2|
|IPI00231275.7||Lgals1 Galectin-1*||IPI00205566.1||Cnn3 Calponin-3||IPI00200353.1||Ril PDZ and LIM domain protein 4|
|IPI00214905.3||Tpm4 Tropomyosin alpha-4 chain*||IPI00204748.1||S100a4 Protein S100-A4*||IPI00207037.3||Eif4ebp1 Eukaryotic translation initiation factor 4E-binding protein 1|
|IPI00362131.3||Cdh2 Cadherin-2||IPI00392930.2||Mprip ENSRNOP00000035264||IPI00471525.2||Eef1d Isoform 2 of Elongation factor 1-delta*|
|IPI00763060.2||REVERSED LOC684327 rCG55860-like||IPI00205631.1||Vcam1 Vascular cell adhesion protein 1||IPI00199325.1||Htrai Insulin-like growth factor binding protein 5 protease|
|IPI00957717.1||Plod2 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 isoform 1||IPI00366079.1||LOC100362805 RCG43947||IPI00372259.4||Tpm3 Isoform 1 of Tropomyosin alpha-3 chain*|
|IPI00554194.1||Calu Calumenin isoform b*||IPI00204099.5||Mcfd2 Multiple coagulation factor deficiency protein 2 homolog||IPI00365542.4||Lamb1 Putative uncharacterized protein Lamb1|
|IPI00951503.1||Hspb7 Heat shock protein beta-7||IPI00957368.1||LOC100360976 Peroxisomal biogenesis factor 19-like, partial||IPI00560977.2||Caprin1 Caprin-1|
|IPI00215465.1||Cryab Alpha-crystallin B chain||IPI00212270.7||Gng12 Guanine nucleotide binding protein (G protein), gamma 12||IPI00817070.1||Myl6b;Myl6 myosin, light chain 6, alkali, smooth muscle and non-muscle*|
|IPI00207050.5||Rcn3 Reticulocalbin 3, EF-hand calcium binding domain||IPI00230921.9||S100a10 Protein S100-A10||IPI00210495.1||Rad23b UV excision repair protein RAD23 homolog B|
|IPI00563816.2||Flnc Putative uncharacterized protein Flnc||IPI00944216.1||Plec Plectin isoform 1hij||IPI00199980.1||Psmb7 Proteasome subunit beta type-7|
|IPI00325151 1||Cnn1 Calponin-1||IPI00947746.1||Pawr Putative uncharacterized protein Pawr||IPI00758461.1||Sra1 Isoform 1 of Steroid receptor RNA activator 1|
|IPI00362160.1||Tubb3 Tubulin beta-3 chain||IPI00231107.5||Ptms Parathymosin||IPI00776551.1||Cytsb Sperm antigen with calponin homology and coiled-coil domains 1|
|IPI00188921.1||Col1a2 Collagen alpha-2(l) chain*||IPI00952078.1||Pls3 Putative uncharacterized protein Pls3||IPI00567393.2||Mtap Putative uncharacterized protein Mtap|
|IPI00231566.1||Tpm1 Isoform 6 of Tropomyosin alpha-1chain||IPI00957937.1||LOC100362061 glutathione peroxidase 8-like||IPI00200145.1||LOC100360522:Rplp1 60S acidic ribosomal protein P1|
|IPI00365982.2||Ckap4 Putative uncharacterized protein Ckap4||IPI00555171.3||Tagln2 Transgelin-2||IPI00207068.1||Glg1 Golgi apparatus protein 1|
|IPI00388257.4||Fbln2 Fibulin 2 isoform 1||IPI00206056.1||Fas Tumor necrosis factor receptor superfamily member 6||IPI00207325.1||Lepre1 Prolyl 3-hydroxylase 1|
|IPI00396852.2||Nexn Isoform 2 of Nexilin||IPI00326759.6||Rcn2 Reticulocalbin-2||IPI00951440.1||Tpd52I2 Tumor protein D52-like 2, isoform CRA_h|
|IPI00569885.2||Hspb1 Heat shock protein beta-1*||IPI00230937.5||Pebp1 Phosphatidylethanolamine-binding protein 1*||IPI00327705.5||Dap Death-associated protein 1|
|IPI00949156.1||Lmo7 Putative uncharacterized protein Lmo7||IPI00230902.5||Gyg1 Glycogenin-1||IPI00734740.2||LOC498555 Putative uncharacterized protein ENSRNOP00000054740|
|IPI00204206.1||Tpm1 Tropomyosin 1 alpha chain isoform d||IPI00896144.1||Cav1 Caveolin-1 alpha isoform||IPI00373045.1||Eif4b Eukaryotic translation initiation factor 4B|
|IPI00195384.2||RGD1559896 Similar to RIKENcDNA 2310022B05||IPI00914765.1||Zyx Zyxin||IPI00951329.1||Col14a1 Putative uncharacterized protein Col14a1|
|IPI00949017.1||Csrp2 Csrp2 protein||IPI00766661.2||LOC679129 Peroxisomal biogenesis factor 19-like||IPI00367437.6||Rbm3 Putative RNA-binding protein 3|
|IPI00950740.1||Krt14 Putative uncharacterized protein Krt14||IPI00204483.1||Serpine1 Plasminogen activator inhibitor 1*||IPI00566460.2||Notch2 Putative uncharacterized protein Notch2|
|IPI00777920.1||Cttn Putative uncharacterized protein Cttn||IPI00858359.2||Myl9 Myosin, light polypeptide 9, regulatory||IPI00949471.1||Pin1 Putative uncharacterized protein Pin1|
|IPI00565165.2||Lrrfip1 Putative uncharacterized protein Lrrfip1||IPI00769110.2||Rrbp1 Ribosome binding protein 1 isoform 3||IPI00782070.2||Serpinb6a Serine (Or cysteine) peptidase inhibitor, clade B, member 6a|
|IPI00189424.2||Sparc Secreted protein acidic and rich in cysteine*||IPI00203972.2||Gys1 Glycogen [starch] synthase, muscle||IPI00959247.1||Sept11 Putative uncharacterized protein Sept11|
|IPI00191090.1||Bgn Biglycan||IPI00480840.2||Cdv3 RCG25673, isoform CRA_d||IPI00325146.6||Anxa2 Isoform Short of Annexin A2|
|IPI00210945.7||Tpm1 Tropomyosin 1 alpha chain isoform c||IPI00201868.1||Nenf Neudesin||IPI00203443.3||Ptgr1 Prostaglandin reductase 1|
|IPI00197129.1||Acta2 Actin, alpha-smooth muscle actin*||IPI00767054.2||Nid1 Nidogen 1||IPI00191454.1||Fhl3 Putative uncharacterized protein Fhl3|
|IPI00211206.7||Pdlim1 PDZ and LIM domain protein 1||IPI00764713.3||Kid Isoform A of Kinesin light chain 1||IPI00211709.1||Gpx8 Glutathione peroxidase|
|IPI00471669.1||Gpc4 Glypican 4||IPI00367746.3||Setd7 RCG49977, isoform CRA_b||IPI00565749.1||Rai14 Isoform 1 of Ankycorbin|
|IPI00231825.5||Rbp1 Retinol-binding protein 1*||IPI00476709.1||Vldlr Putative uncharacterized protein Vldlr||IPI00190848.1||Triap1 RCG21156|
|IPI00187707.1||Ppic Peptidyl-prolyl cis-trans isomerase||IPI00831721.1||Gcsh H protein||IPI00337173.1||Cd276 CD276 antigen (B7H3)|
|IPI00201261.2||Nes Isoform 2 of Nestin||IPI00198667.7||Clu Clusterin||IPI00231434.6||Fkbp1a Peptidyl-prolyl cis-trans isomerase FKBP1A|
|IPI00206193.1||Fhl2 Four and a half LIM domains protein 2||IPI00189471.1||Lpl Lipoprotein lipase||IPI00369539.2||Mfap4 Microfibrillar-associated protein 4|
|IPI00373011.3||Limai LIM domain and actin binding 1||IPI00951791.1||Flna Filamin, alpha (Predicted), isoform CRA_a||IPI00555287.3||Sptbn1 Non-erythroid spectrin beta|
|IPI00555213.1||Pea15a Astrocytic phosphoprotein PEA-15||IPI00214192.1||Sh3gl1 Endophilin-A2||IPI00324451.4||Ddb1 DNA damage-binding protein 1|
|IPI00188956.1||Thy1 Thy-1 membrane glycoprotein||IPI00209358.1||Pdlim7 PDZ and LIM domain protein 7||IPI00199861.1||Dcn Decorin|
|IPI00192504.2||Mrc2 C-type mannose receptor 2||IPI00769176.2||Tgfb1i1 Putative uncharacterized protein Tgfb1i1||IPI00191112.1||Ndufab1 Acyl carrier protein|
|IPI00554148.1||S100a11 Protein S100-A11*||IPI00958283.1||LOC100366237 prefoldin subunit 4-like||IPI00480687.2||Marcks Myristoylated alanine-rich C-kinase substrate|
|IPI00782515.1||Akap2 Putative uncharacterized protein Akap2||IPI00392468.5||Cnpy4 Putative uncharacterized protein RGD1307636||IPI00562248.1||App App protein|
|IPI00194930.5||Gpc1 Glypican-1||IPI00949391.1||Sec24d Sec24d protein||IPI00198887.1||P4hb Protein disulfide-isomerase*|
|IPI00776882.1||Calu Calumenin isoform a*||IPI00951259.1||Flnb Putative uncharacterized protein Flnb||IPI00190287.1||Prelp Prolargin|
|IPI00201608.5||Col5a1 Collagen alpha-1(V) chain||IPI00208280.3||Ptgfrn Prostaglandin F2 receptor negative regulator||IPI00369330.1||Ttc1 Tetratricopeptide repeat domain 1|
|IPI00192912.1||Rcn1 Reticulocalbin 1 (Predicted), isoform CRA_a*||IPI00950587.1||Pdgfrb Beta-type platelet-derived growth factor receptor||IPI00200257.1||Cdh13T-cadherin|
|IPI00201300.2||Ptrf Polymerase I and transcript release factor||IPI00768308.2||LOC687057 calponin 2-like||IPI00200898 3||Slc9a3r1 Na(+)/H(+) exchange regulatory cofactor NHE-RF1|
|IPI00782742.2||Map1a Putative uncharacterized protein Map1a||IPI00372789.3||Tnks1bp1 Putative uncharacterized protein Tnks1bp1||IPI00782227.1||Hook3 Hook homolog 3|
|IPI00210119.1||Map6 Isoform 1 of Microtubule-associated protein 6||IPI00763901.2||Upk3b Uroplakin 3B||IPI00563982.3||Zc3h18 ENSRNOP00000048376|
|IPI00194999.1||Tgfb3 Transforming growth factor beta-3||IPI00957850.1||Itga5 Putative uncharacterized protein Itga5||IPI00370450.3||Plxnb2 Putative uncharacterized protein Plxnb2|
|IPI00372009.3||Map1b Microtubule-associated protein 1B||IPI00564409.3||RGD1309537 Myosin regulatory light chain RLC-A*||IPI00213463.2||Actn4 Alpha-actinin-4|
|IPI00215190.1||Fkbp9 Peptidyl-prolyl cis-trans isomerase FKBP9||IPI00358406.2||Ctnna1 Catenin, alpha 1, isoform CRA_b||IPI00188112.1||Psph Phosphoserine phosphatase|
|IPI00365286.3||Vcl Vinculin*||IPI00369995.2||Lrp1 Low density lipoprotein receptor-related protein 1||IPI00207574.1||Slit3 Slit homolog 3 protein|
|IPI00950560.1||- Putative uncharacterized protein ENSRNOP00000058924||IPI00421723.1||Tbca Tubuhn-specific chaperone A||IPI00200661.1||Fasn Fatty acid synthase|
|IPI00388880.5||Yap1 Putative uncharacterized protein Yap1||IPI00215135.2||II6st Ac1055||IPI00211216.4||Eif5a Eukaryotic translation initiation factor 5A-1*|
|IPI00231651.6||Baspi Brain acid soluble protein 1||IPI00326412.4||Eno2 Gamma-enolase||IPI00193171.1||Npc2 Niemann-Pick disease, type C2|
|IPI00204703.5||Serpinh1 Serpin H1||IPI00210351.2||Ak1 Adenylate kinase isoenzyme 1||IPI00203773.3||Mrpl12 Putative uncharacterized protein Mrpl12|
|IPI00199867.2||Emilin1 Putative uncharacterized protein Emilin1||IPI00230927.1||Cltb Isoform Non-brain of Clathrin light chain B||IPI00208154.1||Cd81 CD81 antigen|
|IPI00764966.2||Ahnak2 similar to KIAA2019 protein||IPI00204818.2||S100a6 Protein S100-A6*||IPI00947644.1||Sqrdl 36 kDa protein|
|IPI00607192.1||P4ha2 Prolyl 4-hydroxylase subunit alpha-2||IPI00231615.5||Anxal Annexin A1||IPI00190240.1||Rps27a;LOC100363345 Ubiquitin-40S ribosomal protein S27a*|
|IPI00968512.1||LOC290704 117 kDa protein||IPI00327185.3||Nap1l1 Nucleosome assembly protein 1-like 1||IPI00947893.1||Ltbp2 192 kDa protein|
|IPI00231194.5||Ddahl N(G),N(G)-dimethylarginine dimethylaminohydrolase 1*||IPI00394021 5||Lvrn Putative uncharacterized protein Lvrn||IPI00325912.1||Ctnnb1 Catenin beta-1|
|IPI00199778.1||RGD1305457 Isoform 1 of Inhibitor of nuclear factor kapp-B kinase-interacting protein||IPI009S0067.1||Cdkn2b Cdkn2b protein||IPI00760137.1||Sord Sorbitol dehydrogenase|
|IPI00763134.1||RGD1564327 RGD1564327 protein||IPI00203158.3||Stub1 STIP1 homology and U-Box containing protein 1||IPI00845873.1||Zranb2 Zinc finger, RAN-binding domain containing 2|
|IPI00195803.1||Ugdh UDP-glucose 6-dehydrogenase||IPI00198796.1||Rwddi RWD domain-containing protein 1||IPI00325189.4||Nme2 Nucleoside diphosphate kinase B|
|IPI00204984.1||Bst1 ADP-ribosyl cyclase 2||IPI00515802.1||Pcolce Pcolce protein||IPI002013251||Txndc17 Thioredoxin-like 5 (Predicted), isoform CRA_b|
|IPI00197074.3||Dag1 Dystroglycan 1||IPI00193547.2||Pdcd5 RCG53732, isoform CRA_a||IPI00957217.1||Bin1 Putative uncharacterized protein Bin1|
|IPI00210111 1||Rgc32 Response gene to complement 32 protein||IPI00213015.1||Dctn2 Dynactin subunit 2||IPI00198567.1||Laspi LIM and SH3 domain protein 1|
|IPI00210360.3||Hspg2 394 kDa protein||IPI00231690.5||Csrp1 Cysteine and glycine-rich protein 1||IPI00366399.3||Eny2 RCG59696, isoform CRA_e|
|IPI00209863.2||P4ha1 Prolyl 4-hydroxylase subunit alpha-1*||IPI00870042.1||Tjp1 Tight junction protein 1||IPI00766955.1||LOC687820 Coiled-coil domain-containing protein 6-like|
|IPI00567305.1||- Putative uncharacterized protein ENSRNOP00000039900||IPI00951644.1||FdpsAc2-125||IPI00779594.2||Aldh1l2 Putative uncharacterized protein Aldh1l2|
|IPI00422076.1||Thbs1 Thrombospondin 1||IPI00190701.5||Apoe Apolipoprotein E||IPI00212651.1||Timm13 Mitochondrial import inner membrane translocase subunit Tim13|
|IPI00194087.3||Actc1 Actin, alpha cardiac muscle 1||IPI00211448.2||Ehd2 EH domain-containing protein 2||IPI00361686.5||C1qbp Complement component 1 Q subcomponent-binding protein, mitochondrial|
|IPI00201548.1||Carhsp1 Calcium-regulated heat stable protein 1||IPI00208184.1||Fam136a Protein FAM136A||IPI00209283.3||Vapb Vesicle-associated membrane protein-associated protein B|
|IPI00207199.3||Ctgf Connective tissue growth factor||IPI00210783.4||Fam25a hypothetical protein LOC684972||IPI00555327.1||Clec10a Macrophage galactose N-acetyl-galactosamine specific lectin 1|
|IPI00327143.1||Alpl Alkaline phosphatase, tissue-nonspecific isozyme||IPI00765234.2||Ktn1 Kinectin 1||IPI00367715.3||Flrt2 RCG20814|
|IPI00949586.1||Myh10 Myosin-10||IPI00197553.1||Npm1 Isoform B23.1 of Nucleophosmin*||IPI00951274.1||Dync1i2 Putative uncharacterized protein Dync1i2|
|IPI00209574.3||Leprel2 Leprel2 protein||IPI00388209.2||Prkcsh Protein kinase C substrate 80K-H (Predicted) isoform, CRA_b||IPI00766527.1||LOC683470 growth arrest specific 1-like|
|IPI00766972.1||Mxra7 Matrix-remodelling associated 7-like||IPI00197216.3||M6prbp1 Mannose-6-phosphate receptor binding protein 1-like||IPI00959549.1||Tp53bp1 212 kDa protein|
|IPI00201034.5||Cdh3 Cadherin 3||IPI00360916.3||Gcc2 Putative uncharacterized protein Gcc2||IPI00952449.1||Arfip1 Putative uncharacterized protein Arfip1|
|IPI00896761.2||LOC683788 Fascin||IPI00197711.1||Ldha L-lactate dehydrogenase A chain||IPI00195719.1||Olr1 Oxidized low-density lipoprotein receptor 1|
|IPI00627074.2||Cd99 CD99 antigen||IPI00949745.1||Ece1 Endothelin converting enzyme 1||IPI00958129.1||Col12a1 Putative uncharacterized protein Col12a1|
|IPI00327697.4||Dpepi Dipeptidase 1||IPI00200920.1||Khsrp Far upstream element-binding protein 2||IPI00360246.2||Reep5 Receptor expression-enhancing protein 5|
|IPI00958032.1||LOC100363622;LOC100363145 rCG42396-like isoform 2||IPI00421366.1||RGD1305481 LRRGT00030||IPI00392935.3||Myl6b RCG42490, isoform CRA_f|
|IPI00555275.1||LOC100365629;LOC100362623 Metallothionein||IPI00363265.3||Hspa9 Stress-70 protein, mitochondrial*||IPI00215294.1||Ddah2 N(G),N(G)-dimethylargimne dimethylaminohydrolase 2|
|IPI00207480.3||Crtap Cartilage-associated protein, isoform CRAb*||IPI00471890.1||F3 Tissue factor||IPI00766695.2||Golga4 golgi autoantigen, golgin subfamily a, 4|
|IPI00952031.1||Mfge8 Putative uncharacterized protein Mfge8||IPI00568756.1||Epb4 113 Putative uncharacterized protein Epb4.1l3||IPI00957678.1||LOC100364427 ribosomal protein S12-like*|
|IPI00959858.1||Vcan Versican||IPI00779937.3||LOC100361890 ubiquitin-conjugating enzyme E2H-like isoform 1||IPI00869493.2||Sdf2 Stromal cell derived factor 2|
|IPI00371230.2||Tmemi 19 Transmembrane protein 119||IPI00231660.5||S100g Protein S100-G||IPI00950239.1||Bsg 30 kDa protein|
|IPI00373753.5||Ptk7 102 kDa protein|
|Accession NO.||Gene Symbol and Name||Accession NO.||Gene Symbol and Name||Accession NO.||Gene Symbol and Name|
|IPI00208422.2||Dpp4 Dipeptidyl peptidase 4||IPI00768591.1||Pstpip2 Proline-serine-threonine phosphatase interacting protein 1-like||IPI00370714.1||Umps Uridine monophosphate synthetase|
|IPI00464895.1||LOC298116 Rat alpha-2u-globulin||IPI00464672.1||Abi3 ABI gene family, member 3, isoform CRA b||IPI00914737.1.||Tmtc3 Transmembrane and tetratricopeptide repeat containing i (Predicted), isoform CRA_a|
|IPI00372776.3||Dnajc 10 DnaJ homolog subfamily C member 10||IPI00327398.1||Enpep Isoform 1 of Glutamyl aminopeptidase||IPI00373076.1||Atp6v1a ATPase, H+ transporting, lysosomal V1 subunit A|
|IPI00212697.1||Napsa napsin A aspartic peptidase||IPI00231200.5||Por NADPH--cytochrome P450 reductase||IPI00421601.3||Asah1 Acid ceramidase|
|IPI00192301.2||Gpx1 Glutathione peroxidase 1||IPI00766273.1||LOC684828 Histone cluster 1, H1d-like||IPI00464668.1||Irgm Immunity-related GTPase family M protein|
|IPI00194804.1||Gzma Granzyme A||IPI00210444.5||Hmgcs2 Hydroxymethylglutaryl-CoA synthase, mitochondrial||IPI00193108.1||Pycard Apoptosis-associated speck-like protein|
|IPI00231262.7||S100a9 Protein S100-A9||IPI00373492.2||Lcp1 Lymphocyte cytosolic protein 1||IPI00366190.4||Lmnb2 Putative uncharacterized protein Lmnb2|
|IPI00210644.1||Cps1 Carbamoyl-phosphate synthase, mitochondrial*||IPI00365967.3||Gzmm Granzyme M||IPI00197770.1||Aldh2 Aldehyde dehydrogenase, mitochondrial*|
|IPI00392753.3||LOC298109 RCG32004||IPI00767601.1||LOC100366216 Nuclear antigen Sp100-like||IPI00367063.1||Cndp1 Beta-Ala-His dipeptidase|
|IPI00785608.2||Mup5 Alpha-2u globulin||IPI00369397.5||H2afx Histone H2A||IPI00326972.6||Ces3 Carboxylesterase 3*|
|IPI00213847.3||Grn Granulins isoform a||IPI00213828.1||LOC619574 Uncharacterized protein C17orf62 homolog||IPI00421874.4||Vdad Voltage-dependent anion-selective channel protein 1|
|IPI00230979.1||Nampt Nicotinamide phosphoribosyltransferase||IPI00829505.1||Lap3 Isoform 2 of Cytosol aminopeptidase||IPI00367815 1||MGC108823 Similar to interferon-inducible GTPase|
|IPI00369234.3||Igtp Ac2-233||IPI00551812.1||Atp5b ATP synthase subunit beta, mitochondrial||IPI00370711.3||Epx Putative uncharacterized protein Epx|
|IPI00207390.9||Anxa3 Annexin A3*||IPI00766882.1||Rbmxrt RNA binding motif protein, X-linked-like||IPI00782366.1||Anxa7 Putative uncharacterized protein Anxa7|
|IPI00230874.10||Blvra Biliverdin reductase A||IPI00948721.1||Acaa2 42 kDa protein||IPI00210920.1||1Got2 Aspartate aminotransferase, mitochondrial|
|IPI00231742.5||Cat Catalase||IPI00204359.1||B2m Beta-2-microglobulin||IPI00210280.1||1Comt Isoform 1 of Catechol O-methyltransferase*|
|IPI00364591.2||Plbdi Putative phospholipase B-like 1||IPI00562255.2||Ctla2a Similar to ctla-2-beta protein (141 AA) (Predicted) isoform CRA_a||IPI00214434.3||Rab11a Ras-related protein Rab-11A|
|IPI00364616.2||RGD1309676 Uncharacterized protein C10orf58 homolog||IPI00190499.5||Tpp1 Tripeptidyl-peptidase 1||IPI00204763.4/||Arhgap25 Similar to Rho-GTPase-activating protein 25|
|IPI00195423.1||Ugt2b37 UDP-glucuronosyltransferase 2B37||IPI00949858.1||Ctsc Putative uncharacterized protein Ctsc||IPI00782093.2||Prexi Putative uncharacterized protein Prexi|
|IPI00203054.2||Acsf2 Acyl-CoA synthetase family member 2, mitochondrial||IPI00959089.1||Fth1 Ferritin (Fragment)||IPI00195123.1||Atp5o ATP synthase subunit O, mitochondrial|
|IPI00767419.2||Siae Putative uncharacterized protein Siae||IPI00382226.1||Fam65bAb2-162||IPI00198327.2||Vdac2 Voltage-dependent anion-selective channel protein 2|
|IPI00567836.2||Ptgs1 Ptgs1 protein||IPI00199695.3||Serpinf2 Serine (Or cysteine) peptidase inhibitor, clade F member 2||IPI00205157.1||Hadh Hydroxyacyl-coenzyme A dehydrogenase, mitochondria|
|IPI00206626.1||Hmox1 Heme oxygenase 1||IPI00325975.3||Vamp8 Vesicle-associated membrane protein 8||IPI00210692.1||1Casp7 Caspase-7|
|IPI00760118.1||Hsd11b1 Isoform 11-HSD1B of Corticosteroid 11-beta-dehydrogenase isozyme 1||IPI00763589.2||LOC684681 histone cluster 1, H1c-like||IPI00361732.4||4Diaph2 Diaphanous homolog 2|
|IPI00360317.1||Cd180 Putative uncharacterized protein Cd180||IPI00950371.1||Stat4 88 kDa protein||IPI00198237.1||1Psmb10 Proteasome subunit beta type-10|
|IPI00554206.3||Ugt2b UDP glycosyltransferase 2 family, polypeptide B||IPI00949517.1||Actr3 Actin-related protein 3||IPI00196648 5||5Stx7 Syntaxin-7|
|IPI00958846.1||LOC683399 Igk protein-like isoform 2||IPI00388462.4||- Putative uncharacterized protein 687510||IPI00209184.3||Cd1d1 Antigen-presenting glycoprotein CD1d|
|IPI00895603.1||Pld4 Phospholipase D4||IPI00948996.1||- Putative uncharacterized protein Lpcat2||IPI00198080.1||Pcyox1 Prenylcysteine oxidase|
|IPI00421898.1||Ifi47 Ifi47 protein||IPI00211970.5||Psme2;LOC100364216 Proteasome activator complex subunit 2*||IPI00948302.1||Atp5c1 ATP synthase gamma chain|
|IPI00207947.6||Lta4h Leukotriene A4 hydrotase||IPI00422051.2||RT1-Ba MHC class II antigen RT1.B alpha chain||IPI00951116.1||Hbb 16 kDa protein|
|IPI00948229.1||Gbp2 Putative uncharacterized protein ENSRNOP00000022899||IPI00569009.1||RT1-Bb RT1 class II histocompatibility antigen, B-1 beta chain precursor||IPI00515816.1||Lgmm Legumain|
|IPI00231191.7||Glrxi Glutaredoxin-1||IPI00200806.3||Ada Adenosine deaminase||IPI00373418.3||Dbt dihydrolipoamide branched chain transacylase E2|
|IPI00882532.1||Afmid Arylformamidase||IPI00944815.1||Psmb9 Proteasome subunit beta type-9||IPI00370158.3||Rac2 Ras-related C3 botulinum toxin substrate 2|
|IPI00209291.1||Esyt1 Extended synaptotagmin-1||IPI00480679.4||Krt18 Keratin, type I cytoskeletal 18||IPI00191737.6||Alb Serum albumin|
|IPI00371870.1||Smpdl3a Acid sphingomyelinase-like phosphodiesterase 3a||IPI00781895.1||Fyb Similar to FYN binding protein (Predicted), isoform CRA_b||IPI00206092.1||Akr1b8 Aldose reductase-like protein|
|IPI00209690.1||Ephxi Epoxide hydrolase 1||IPI00193049.1||Sult1a1 Sulfotransferase 1A1*||IPI00564976 3||Fgd2 Putative uncharacterized protein Fgd2|
|IPI00969321.1||RT1-EC2 A2q||IPI00231418.5||Lmnbi Lamin-B1||IPI00198897 1||Ndufa6 NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 6|
|IPI00911233.2||Tapbp TAP-binding protein||IPI00734729.1||Tcirgi V-H+ATPase subunit a3||IPI00360541 4||tgb2 Integrin beta 2|
|IPI00958096.1||LOC100360950 GF20391-like isoform 1||IPI00359383.3||LOC100359680;Lpcat2 Lysophosphatidylcholine acyltransferase 2||IPI00394488.2||Gbas Glioblastoma amplified sequence|
|IPI00476115.1||RT1-Db1 RT1 class II histocompatibility antigen, D-1 beti chain||IPI00382229.1||Stat1;Stat4Ab2-131||IPI00957241.1||Vdac3 Putative uncharacterized protein Vdac3|
|IPI00958624.1||Stab2 Stabilin 2||IPI009589621||Hist1h4b;Hist1h4m histone cluster 1, H4m||IPI00327781 1||Cyp2c11 Cytochrome P450 2C11|
|IPI00231248.5||Hpgds Hematopoietic prostaglandin D synthase||IPI00207663.2||Ctsz Cathepsin Z||IPI00210228.5||Ctss Cathepsin S|
|IPI00758468.2||Pgcp Plasma glutamate carboxypeptidase||IPI00368211.2||Ifit2 Interferon-induced protein with tetratricopeptide repeats 2||IPI00193485 2||Idh2 Isocitrate dehydrogenase [NADP], mitochondrial|
|IPI00324309.4||Cybb Endothelial type gp91-phox||IPI00471577.1||Uqcrc1 Cytochrome b-c1 complex subunit 1, mitochondrial||IPI00213457 1||Atp6v1c1 V-type proton ATPase subunit C 1|
|IPI00372391.2||F5AC2-120||IPI00202580.3||Cyp2d3 Cytochrome P450 2D3||IPI00562259.1||Slc25a3 Phosphate carrier protein, mitochondrial|
|IPI00555265.1||Nnt Nicotinamide nucleotide transhydrogenase||IPI00559910.1||Rbmxrtl Heterogeneous nuclear ribonucleoprotein G||IPI00361887.1||LOC685909 Histone H2A|
|IPI00208382.1||Ppt1 Palmitoyl-protein thioesterase 1||IPI00948938.1||Mrc1 Putative uncharacterized protein Mrc1||IPI00780674.1||Ugt1a7c UDP-glucuronosyltransferase 1-7|
|IPI00371511.4||Creg1 Cellular repressor of E1A-stimulated genes 1||IPI00214461.1||1ll 1rn lnterleukin-1 receptor antagonist protein||IPI00231370.7||S100a8 Protein S100-A8|
|IPI00212635.2||Igf2 insulin-like growth factor 2 precursor||IPI00212219.1||Ssb Lupus La protein homolog||IPI00362072.2||Actr2 Actin-related protein 2|
|IPI00372986.5||Vsig4 V-set and immunoglobulin domain containing 4||IPI00471794.1||Npl N-acetylneuraminate lyase||IPI00876603 1||Hnrpm Heterogeneous nuclear ribonucleoprotein M isoform a|
|IPI00199448.4||Lgals3bp Galectin-3-bmding protein||IPI00948998.1||Naga Putative uncharacterized protein Naga||IPI00205519.5||Uggti UDP-glucose:glycoprotein glucosyltransferase 1|
|IPI004762957||Ahcy Adenosylhomocysteinase||IPI00777022.2||Fcgr2b Low affinity immunoglobulin gamma Fc region receptor II||IPI00196107.1||Atp5f1 ATP synthase subunit b, mitochondrial|
|IPI00210038.1||Lipa Lysosomal acid lipase/cholesteryl ester hydrolase||IPI00198717.8||Mdh1 Malate dehydrogenase, cytoplasmic*||IPI00231780.5||H1f0 Histone H1.0|
|IPI00464497.1||LOC305806 Similar to glutaredoxin 1||IPI00950779.1||Nckap1l Putative uncharacterized protein Nckap1l||IPI00947965 1||Lrmp 25 kDa protein|
|IPI00392676.4||Blvrb Biliverdin reductase B||IPI00366405.2||Fam49b Similar to 0910001A06Rik protein (Predicted), isoform CRA_a||IPI00470288.4||Ckb Creatine kinase B-type*|
|IPI00201891.1||Hk3 Hexokinase-3||IPI00421899.1||Cndp2 Cytosolic non-specific dipeptidase||IPI00208568 3||Plek Pleckstrin|
|IPI00368206.1||Ifit3 Interferon-induced protein with tetratricopeptide repeats 3||IPI00203406.1||Osbpl1a Oxysterol-binding protein-related protein 1||IPI00200466.3||Slc25a5 ADP/ATP translocase 2|
|IPI00210901.1||Cd38 ADP-ribosyl cyclase 1||IPI00192876.1||Mx1 Interferon-induced GTP-binding protein Mx1||IPI00764346.2||REVERSED - 48 kDa protein|
|IPI00365035.2||Adfp Adipose differentiation related protein||IPI00188924.4||Uqcrc2 Cytochrome b-d complex subunit 2, mitochondrial||IPI00213667.1||LOC286987 Hemiferrin|
|IPI00230788.6||Car3 Carbonic anhydrase 3||IPI00954695.1||RGD1309586 RCG20177||IPI00358463.1||Arhgdib Rho, GDP dissociation inhibitor (GDI) beta*|
|IPI00959683.1||Hist2h3c2;Hist1h3f;Hist2h3c;LOC100364555;LOC684762 Histone H3||IPI00231949.5||Cd14 Monocyte differentiation antigen CD14||IPI00565330 1||Acaa1 Putative uncharacterized protein Acaa1|
|IPI00369645.2||Cd68 Cd68 molecule||IPI00949639.1||Pik3r1 Protein||IPI00327330.2||Cd2ap CD2-associated protein|
|IPI00190377.2||Taldo1 Transaldolase||IPI00760125.1||Ndel1 Isoform 2 of Nuclear distribution protein nudE-like 1||IPI00194341.5||Lgals3 Galectin-3|
|IPI00903439.1||RT1-EC2A2b||IPI00951515.1||Paox RCG47968, isoform CRA_a||IPI00189981.1||F2 Prothrombin (Fragment)|
|IPI00951700.1||Cd163 Putative uncharacterized protein Cd163||IPI00327644.5||Lyn Isoform LYN A of Tyrosine-protein kinase Lyn||IPI00365613.2||Snx6 Sorting nexin 6|
|IPI003914422||Stom Putative uncharacterized protein Stom||IPI00371634.1||Bcap31 B-cell receptor-associated protein 31||IPI00194222.1||Cox4i1 Cytochrome c oxidase subunit 4 isoform 1, mitochondrial|
|IPI00776581.2||Gusb Beta-alucuronidase||IPI00896162.1||Fermt3 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.
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.
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.
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).
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).
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.
We thank Dongmei Qiu, a graduate student in the Department of Pathology, Medical School of Nantong University, for technical assistance in immunohistochemistry.