Activation of Hepatic Stellate Cells Requires Dissociation of E-Cadherin–Containing Adherens Junctions with Hepatocytes
2020; Elsevier BV; Volume: 191; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2020.12.007
ISSN1525-2191
AutoresHayato Urushima, Hideto Yuasa, Tsutomu Matsubara, Noriyuki Kuroda, Yaiko Hara, Kouji Inoue, Kenjiro Wake, Tetsuji Sato, Scott L. Friedman, Kazuo Ikeda,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoHepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. In vivo, following carbon tetrachloride–induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. In vitro, reconstitution of E-cadherin–containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-β, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes. Hepatic stellate cells (HSCs) are resident mesenchymal cells in the space of Disse interposed between liver sinusoidal endothelial cells and hepatocytes. Thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. Although HSC activation is initiated largely from stimulation by adjacent cells, isolated HSCs also activate spontaneously in primary culture on plastic. Therefore, other unknown HSC-initiating factors apart from paracrine stimuli may promote activation. The dissociation of adherens junctions between HSCs and hepatocytes as an activating signal for HSCs was explored, establishing epithelial cadherin (E-cadherin) as an adhesion molecule linking hepatocytes and HSCs. In vivo, following carbon tetrachloride–induced liver injury, HSCs lost their spines and dissociated from adherens junctions in the early stages of injury, and were subsequently activated along with an increase in YAP/TAZ expression. After abrogation of liver injury, HSCs reconstructed their spines and adherens junctions. In vitro, reconstitution of E-cadherin–containing adherens junctions by forced E-cadherin expression quiesced HSCs and suppressed TAZ expression. Additionally, increase of TAZ expression leading to the activation of HSCs by autocrine stimulation of transforming growth factor-β, was revealed as a mechanism of spontaneous activation. Thus, we have uncovered a critical event required for HSC activation through enhanced TAZ-mediated mechanotransduction after the loss of adherens junctions between HSCs and hepatocytes. Hepatic stellate cells (HSCs) are resident mesenchymal cells localized within the space of Disse, interposed between liver sinusoidal endothelial cells (LSECs) and hepatocytes. Quiescent HSCs store vitamin A lipid droplets in their cytoplasm and encircle the endothelial cells with their long, branching cellular process cells to form hepatic sinusoids. Characteristically, many thorn-like microprojections, or spines, project out from the cell surface of HSCs, crossing the space of Disse, to establish adherens junctions with neighboring hepatocytes. In response to hepatic injury of any etiology, quiescent HSCs transdifferentiate into activated myofibroblasts to produce extracellular matrix (ECM), including collagen and fibronectin, and proliferate, migrate toward regions of injury, and lose vitamin A lipid droplets. After cessation of injury, activated HSCs either undergo apoptosis or revert to an inactivated phenotype,1Troeger J.S. Mederacke I. Gwak G.Y. Dapito D.H. Mu X. Hsu C.C. Pradere J.P. Friedman R.A. Schwabe R.F. Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.Gastroenterology. 2012; 143: 1073-1083.e22Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar,2Iredale J.P. Benyon R.C. Pickering J. McCullen M. Northrop M. Pawley S. Hovell C. Arthur M.J. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (919) Google Scholar which reduces production of ECM. However, persistent activation of HSCs during chronic liver injury due to viral infection, alcohol, or in nonalcoholic steatohepatitis provokes accumulation of collagen in the space of Disse, leading to capillarization of sinusoids, followed by panlobular fibrosis. Furthermore, cirrhosis, the most advanced form of fibrosis, is a critical risk factor for hepatic carcinogenesis.3Fattovich G. Stroffolini T. Zagni I. Donato F. Hepatocellular carcinoma in cirrhosis: incidence and risk factors.Gastroenterology. 2004; 127 Suppl 1: S35-S50Abstract Full Text Full Text PDF Scopus (1808) Google Scholar Therefore, clarifying the extracellular signals that suppress activated HSCs is an important step toward identifying novel therapeutic targets for cirrhosis and hepatocellular carcinoma, although none has yet been identified. Canonically, HSC activation has been conceived as a two-phase event: initiation followed by perpetuation. Initiation refers to early changes in gene expression and phenotype that render cells responsive to stimulants for perpetuation.4Friedman S.L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.Physiol Rev. 2008; 88: 125-172Crossref PubMed Scopus (1902) Google Scholar The initiation of HSC activation results primarily from paracrine stimulation produced by neighboring cells, including hepatocytes, macrophages, LSECs, inflammatory cells, platelets, and HSCs themselves.5Tsuchida T. Friedman S.L. Mechanisms of hepatic stellate cell activation.Nat Rev Gastroenterol Hepatol. 2017; 14: 397-411Crossref PubMed Scopus (883) Google Scholar After initiation, several features of the perpetuation phase, including proliferation, ECM production, chemotaxis, and increased contractility may occur, depending on the type of stimulus. For instance, platelet-derived growth factor is the most potent HSC mitogen identified,6Borkham-Kamphorst E. van Roeyen C.R.C. Ostendorf T. Floege J. Gressner A.M. Weiskirchen R. Pro-fibrogenic potential of PDGF-D in liver fibrosis.J Hepatol. 2007; 46: 1064-1074Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar,7Pinzani M. PDGF and signal transduction in hepatic stellate cells.Front Biosci. 2002; 7: d1720-d1726Crossref PubMed Google Scholar and transforming growth factor beta (TGF-β) induces the production of collagen and other ECM molecules and down-regulates the degradation of ECM by matrix metalloproteinases in HSCs.8Breitkopf K. Godoy P. Ciuclan L. Singer M.V. Dooley S. TGF-beta/Smad signaling in the injured liver.Z Gastroenterol. 2006; 44: 57-66Crossref PubMed Scopus (143) Google Scholar, 9Gressner A.M. Weiskirchen R. Breitkopf K. Dooley S. Roles of TGF-beta in hepatic fibrosis.Front Biosci. 2002; 7: d793-d807Crossref PubMed Google Scholar, 10Iredale J.P. Murphy G. Hembry R.M. Friedman S.L. Arthur M.J. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver.J Clin Invest. 1992; 90: 282-287Crossref PubMed Scopus (182) Google Scholar However, isolated HSCs activate spontaneously in the absence of these exogenous initiation factors, and culture on uncoated plastic or other stiff matrices.11Friedman S.L. Roll F.J. Boyles J. Arenson D.M. Bissell D.M. Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix.J Biol Chem. 1989; 264: 10756-10762Abstract Full Text PDF PubMed Google Scholar,12Lim Y.S. Lee H.C. Lee H.S. Switch of cadherin expression from E- to N-type during the activation of rat hepatic stellate cells.Histochem Cell Biol. 2007; 127: 149-160Crossref PubMed Scopus (37) Google Scholar This raises the prospect that initiating factors other than paracrine signals may also contribute to cellular activation. Although HSCs surround LSECs in normal liver, their contact with LSECs is smooth and does not constitute direct cell adhesion.13Wake K. Cell-cell organization and functions of 'sinusoids' in liver microcirculation system.J Electron Microsc (Tokyo). 1999; 48: 89-98Crossref PubMed Scopus (24) Google Scholar Conversely, HSCs extend protrusions called spines, that establish adherens junction with hepatocytes.14Wake K. Hepatic stellate cells: three-dimensional structure, localization, heterogeneity and development.Proc Jpn Acad Ser B Phys Biol Sci. 2006; 82: 155-164Crossref PubMed Scopus (31) Google Scholar The current study focused on the biological significance of contact between HSCs and hepatocytes through these adherens junctions. It was found that early activation of HSCs requires dissociation of these adherens junctions after injury to hepatocytes, which constitutes a critical event required for HSC activation through enhanced mechanotransduction. All experiments were conducted in compliance with the guidelines for the care and use of laboratory animals and approved by Osaka City University. Liver injury was induced by intraperitoneal injections of carbon tetrachloride (CCl4) to 6- to 7-week–old Wistar rats or 8- to 10-week–old C57BL/6j mice (0.5 μL/g body weight, dissolved in corn oil at a ratio of 1:3). The tissue blocks prepared with perfusion fixation method were postfixed with phosphate-buffered 2% osmium tetroxide, dehydrated, and embedded in Poly/Bed 812. Thin sections were examined in a transmission electron microscope (model 100CX, JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 80 kV. HSCs were isolated as previously described15Kristensen D.B. Kawada N. Imamura K. Miyamoto Y. Tateno C. Seki S. Kuroki T. Yoshizato K. Proteome analysis of rat hepatic stellate cells.Hepatology. 2000; 32: 268-277Crossref PubMed Scopus (200) Google Scholar from C57BL/6j mice by using pronase/collagenase perfusion followed by Nycodenz gradient centrifugation. Isolated primary HSCs were cultured in Dulbecco's modified Eagle medium (Wako, Osaka, Japan) containing 10% fetal bovine serum, 100 mg/mL penicillin, and 100 U/mL streptomycin. The human HSC line HHSteCs (lot number 10326) was purchased from ScienCell Research Laboratories (Carlsbad, CA). These cells were maintained by using the Stellate Cell Medium set (catalog number 5301) at 37°C in a humidified 5% carbon dioxide atmosphere. To generate an epithelial cadherin (E-cadherin)–coated dish, recombinant E-cadherins (1.5 μg/mL; R&D Systems, Minneapolis, MN) were coated in a 24-well plate with phosphate-buffered saline for 2 hours followed by blocking with 1% bovine serum albumin containing phosphate-buffered saline for 3 hours. Mouse primary HSCs or HHSteCs were then cultured in the plate. HHSteCs or mouse primary HSCs were seeded 5.0 × 103 per well in a 96-well, low attachment spheroid culture plate (Sumitomo Bakelite, Tokyo, Japan). For analysis, at least six spheroids were pooled. For neutralization of E-cadherin, E-cadherin monoclonal antibody (SHE78-7, Thermo Fisher Scientific, Waltham, MA) was added into the culture medium at the concentration of 10 μg/mL. For the analysis of TGF-β signaling in the presence or absence of E-cadherin–mediated adherens junctions, empty vector-transfected (control), or E-cadherin overexpressed HHSteC cells were cultured as spheroid and stimulated by 10 ng/mL of TGF-β. Proteins were then isolated at 0, 5, 10, 30, and 60 minutes after TGF-β stimulation. To generate transient overexpression of E-cadherin or TAZ, HHSteCs were transfected with an empty plasmid, hE-cadherin-pcDNA3 (gift from Barry Gumbiner; plasmid number 45769, Addgene, Cambridge, MA) or HA-TAZ (gift from Kunliang Guan; plasmid number 32839, Addgene) using lipofectamine 3000 (Thermo Fisher Scientific) following the manufacturer's protocol. RNA was extracted from cells by using TRIzol reagent (Thermo Fisher Scientific) and Direct-zol RNA MiniPrep (Zymo Research, Irvine, CA). Real-time quantitative PCR (qPCR) was performed by using cDNA generated from RNA and the SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific). The qPCR reaction was conducted by using the SYBR green PCR master mix (Thermo Fisher Scientific) in the Thermal Cycler Dice Real Time System 2 (Takara Bio, Shiga, Japan). The values were quantified by using the comparative CT method and were normalized to 18S ribosomal RNA. Data are expressed as the ratio to the average of the control group. The primers used in this study are listed in Table 1.Table 1Real-Time Quantitative PCR Primer SequencesGene namePrimer sequencesTgfb1Forward5′-TCGAGGGCGAGAGAAGTTTA-3′Reverse5′-AAAAGAATGTCCCGGCTCTC-3′PdgfrbForward5′-CGCCTGCAAGTGTGAGACAAT-3′Reverse5′-CGAATGGTCACCCGAGCTT-3′TnfaForward5′-TCCCAGGTTCTCTTCAAGGGA-3′Reverse5′-GGTGAGGAGCACGTAGTCGG-3′Il1bForward5′-TTGACGGACCCCAAAAGATG-3′Reverse5′-TGGACAGCCCAGGTCAAAG-3′Il6Forward5′-TGATGCACTTGCAGAAAACA-3′Reverse5′-ACCAGAGGAAATTTTCAATAGGC-3′CtgfForward5′-CCGCCAACCGCAAGATC-3′Reverse5′-ACCGACCCACCGAAGACA-3′Acta2Forward5′-CGAAACCACCTATAACAGCATCA-3′Reverse5′-GCGTTCTGGAGGGGCAAT-3′Col1a1Forward5′-CCAAGGGTAACAGCGGTGAA-3′Reverse5′-CCTCGTTTTCCTTCTTCTCCG-3′Col1a2Forward5′-TGTTGGCCCATCTGGTAAAGA-3′Reverse5′-CAGGGAATCCGATGTTGCC-3′Cdh1Forward5′-GAGGTCTACACCTTCCCGGT-3′Reverse5′-CCACTTTGAATCGGGAGTCT-3′Cdh2Forward5′-CAGGGTGGACGTCATTGTAG-3′Reverse5′-AGGGTCTCCACCACTGATTC-3′Human TGFB1Forward5′-CCCTGGACACCAACTATTGC-3′Reverse5′-GCAGAAGTTGGCATGGTAGC-3′ PDGFRBForward5′-GTGCTCACCATCATCTCCCT-3′Reverse5′-ACTCAATCACCTTCCATCGG-3′ CTGFForward5′-CAGGCTAGAGAAGCAGAGCC-3′Reverse5′-TGGAGATTTTGGGAGTACGG-3′ ACTA2Forward5′-GACCGAATGCAGAAGGAGAT-3′Reverse5′-CACCGATCCAGACAGAGTATTT-3′ COL1A1Forward5′-AAGAGGAAGGCCAAGTCGAG-3′Reverse5′-CACACGTCTCGGTCATGGTA-3′ COL1A2Forward5′-GAAAAGGAGTTGGACTTGGC-3′Reverse5′-AGCAGGTCCTTGGAAACCTT-3′ CDH1Forward5′-GGGTGACTACAAAATCAATC-3′Reverse5′-GGGGGCAGTAAGGGCTCTTT-3′ CDH2Forward5′-CACTGCTCAGGACCCAGAT-3′Reverse5′-TAAGCCGAGTGATGGTCC-3′ SERPINH1Forward5′-AGAGTAGAATCGTGTCGCGG-3′Reverse5′-CTGAGAAGCAGGAGGGAGC-3′ SERPINE1Forward5′-AGAAACCCAGCAGCAGATTC-3′Reverse5′-TGGTGCTGATCTCATCCTTG-3′ SNAI1Forward5′-ACCCCAATCGGAAGCCTAACT-3′Reverse5′-GGTCGTAGGGCTGCTGGAA-3′ MMP2Forward5′-TGATGTCCAGCGAGTGGAT-3′Reverse5′-GGAAAGCCAGGATCCATTTT-3′ TGFBR2Forward5′-AGCATCACGGCCATCTGTG-3′Reverse5′-TGGCAAACCGTCTCCAGAGT-3′ Open table in a new tab Cells were homogenized with radioimmunoprecipitation assay buffer (50 mmol/L Tris-HCl at pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1% SDS) containing the protease inhibitor cocktail cOmplete Mini (Roche, Basel, Switzerland) and phosphatase inhibitors (1 mmol/L sodium fluoride, 1 mmol/L β-glycerol phosphate, and 1 mmol/L sodium vanadate). Protein samples were subjected to 8% to 15% SDS-PAGE and were transferred to polyvinylidene difluoride membranes using standard Western blot techniques. After blocking with 5% skim milk, the membranes were probed with primary antibodies diluted at 1:1000 and horseradish peroxidase–conjugated secondary antibodies diluted at 1:5000. Immunoreactive bands were visualized by using the ImmunoStar Zeta or ImmunoStar LD system and were detected with an LAS3000 or LAS4000 device (GE Healthcare, Milwaukee, WI). WB Stripping Solution (Nacalai Tesque, Kyoto, Japan) was used to remove the antibodies from the Western blot membrane. The quantification of Western blot bands was performed by using ImageJ software version 1.52n (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Data are expressed as means ± SEM of at least three different experiments. Isolated HSCs were stained with Alexa Fluor 647 anti-human/mouse CD324 (DECMA-1; BioLegend, San Diego, CA) or isotype control (RTK2071; BioLegend). HSCs emit autofluorescence under UV excitation due to their vitamin A content.16Mabuchi A. Mullaney I. Sheard P.W. Hessian P.A. Mallard B.L. Tawadrous M.N. Zimmermann A. Senoo H. Wheatley A.M. Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat.J Hepatol. 2004; 40: 910-916Abstract Full Text Full Text PDF PubMed Google Scholar Therefore, the expression of E-cadherin in UV autofluorescence-positive cells was characterized by using FACS LSR II (BD Biosciences, San Diego, CA). For histological evaluation, mouse liver were fixed with 4% paraformaldehyde and embedded into paraffin. Then, mouse liver section were sliced (4 µm) and stained with hematoxylin and eosin. For immunohistochemical analysis, they were deparaffinized, hydrated, heated to 110°C in citrate buffer for 20 minutes, depleted of endogenous peroxidase activity, and then blocked with Blocking One (Nacalai Tesque) for 1 hour. Next, the slides were treated with primary antibodies overnight at 4°C. The slides were incubated with secondary antibody (Bioss, Inc., Woburn, MA) for 1 hour at room temperature, and the reaction was visualized by diaminobenzene substrate (Vector Laboratories, Burlingame, CA). All specimens were counterstained with hematoxylin. For immunofluorescence analysis, after hybridization with primary antibodies, the slides were incubated with Alexa Fluor 488 or 594 conjugated donkey anti-mouse or rabbit IgG (Invitrogen, Carlsbad, CA). DAPI (Dojindo, Kumamoto, Japan) was used as a nuclear marker. For quantitative analysis, three images were taken at every indicated time point, and cells positive for Ly6G per field of each view were counted. For immunoelectron microscopy, 4% paraformaldehyde fixative-fixed, frozen mouse liver slices were cut into 4 μm with a cryostat (HM 560; Microm, Walldorf, Germany). The immunostaining procedure was similar to that described in the previous paragraph. After visualizing by diaminobenzene, they were postfixed with 2% osmium tetroxide (Wako) for 1 hour on ice and dehydrated in an ethanol series before embedding in a Quetol 812 mixture. Ultrathin sections were stained with lead citrate solution and observed in Talos F200C G2 (Thermo Fisher Scientific) with an accelerating voltage of 200 kV. The following antibodies were used for Western blot analysis and immunofluorescence: anti–E-cadherin (24E10), anti–phospho-Smad2 (Ser465/467), anti-Smad2 (D43B4), anti-YAP (D8H1X), anti-TAZ (V386), and anti–platelet-derived growth factor-receptor β (28E1), purchased from Cell Signaling Technology (Danvers, MA). Anti–neural cadherin (H-2), anti–placental cadherin (D-6), anti–vascular endothelial cadherin (F-8), and anti–heat shock protein 47 (G-12) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–α-smooth muscle actin (1A4; DAKO, Glostrup, Denmark), anti-Ly6G (1A8; R&D systems), anti–glyceraldehyde-3-phosphate dehydrogenase (Millipore, Billerica, MA), and anti-cytoglobin (kindly provided by Prof. Norifumi Kawada, Osaka City University) were also used for Western blot and immunohistochemical analyses. E-cadherin or TAZ was overexpressed in HHSteC cells and cultured as spheroids. Forty-eight hours later, the levels of TGF-β in the supernatant of spheroid culture were determined by using a human TGF-β ELISA kit (BioLegend). All data are expressed as means ± SD. All data were analyzed by using analysis of variance with a post hoc Dunnett's test or unpaired t-test. The morphologic changes of adherens junctions between hepatocytes and HSCs in rat liver during CCl4-induced acute liver injury were investigated first. In normal liver, several spine-mediated adherens junctions with hepatocytes projected out both from the surface of the dendritic processes as well as from the cell bodies (Figure 1A). A high magnification of an adherens junction between the spine of an HSC and hepatocyte is shown in Figure 1B. The adhesive power of the junction is illustrated by the subjunctional cytoplasm of the hepatocyte being pulled up to form a spine. Actin-like microfibrils in both cells aligned together within each subjunctional region. At 12 hours, individual HSCs in the injured areas were surrounded by hepatocytes with ballooning degeneration (upper left image of Figure 1C). Some microvilli of the degenerating hepatocytes remained in close contact with HSCs. Importantly, however, neither spines nor adherens junctions were observed (Figure 1C), whereas mitotic figures (Figure 1D) were observed in HSCs. Characteristically, Golgi vacuoles located close to the cell membrane of HSCs were filled with collagen fibrils, some of which appeared to be secreted into the extracellular space (Figure 1E). Based on the findings from the rat experiments, morphologic studies were validated in a mouse CCl4 model. Similar to findings in rat liver, spine-mediated adherens junctions with hepatocytes were also identified between hepatocytes and HSCs in normal mouse liver (Figure 1F), which disappeared within 6 hours after CCl4 injection (Figure 1G). Next examined was the relationship between HSCs and hepatocytes in rat liver 7 days after a single CCl4 injection, at the stage of subsiding injury. Damaged hepatocytes had recovered, and inflammatory cell infiltration had subsided. HSCs reconstituted their adherens junctions with hepatocytes with increased lipid droplets, a feature of HSC quiescence4Friedman S.L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.Physiol Rev. 2008; 88: 125-172Crossref PubMed Scopus (1902) Google Scholar (Figure 2A). Interestingly, the HSCs which had not yet restored their adhesion to hepatocytes, extended regenerating spines and established an adherens junction de novo with a regenerating, mitotic hepatocyte (Figure 2B). In the same region, fibrous structures linking hepatocytes and HSCs were observed at the tip of the spine (Figure 2C). Cadherins constitute a large superfamily of cell–cell adhesion molecules.17Yagi T. Takeichi M. Cadherin superfamily genes: functions, genomic organization, and neurologic diversity.Genes Dev. 2000; 14: 1169-1180PubMed Google Scholar,18Nollet F. Kools P. van Roy F. Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members.J Mol Biol. 2000; 299: 551-572Crossref PubMed Scopus (572) Google Scholar The classic cadherins consist of E-cadherin, neural cadherin, placental cadherin, and vascular endothelial cadherin.19Takeichi M. Morphogenetic roles of classic cadherins.Curr Opin Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1239) Google Scholar Thus, we explored the cadherin(s) responsible for hepatocyte–HSC adherens junctions. Almost all of the isolated mouse HSCs contained lipid droplets (left upper panel of Figure 3A) and expressed cell-specific markers such as cytoglobin20Kawada N. Cytoglobin as a marker of hepatic stellate cell-derived myofibroblasts.Front Physiol. 2015; 6: 329Crossref PubMed Scopus (13) Google Scholar and heat shock protein 47,21Kawada N. Kuroki T. Kobayashi K. Inoue M. Nakatani K. Kaneda K. Nagata K. Expression of heat-shock protein 47 in mouse liver.Cell Tissue Res. 1996; 284: 341-346Crossref PubMed Scopus (51) Google Scholar whereas none of the HSCs expressed of albumin and CK18, the specific markers of hepatocytes (right upper panel of Figure 3A). These data confirmed the high purity of isolated mouse HSCs. In addition, murine HSCs expressed E-cadherin but not other cadherins (bottom panel, Figure 3A). The expression of E-cadherin in HSCs was examined by flow cytometry. Because they contain vitamin A, quiescent HSCs have autofluorescence when excited by UV.22Han Y.P. Zhou L. Wang J. Xiong S. Garner W.L. French S.W. Tsukamoto H. Essential role of matrix metalloproteinases in interleukin-1-induced myofibroblastic activation of hepatic stellate cell in collagen.J Biol Chem. 2004; 279: 4820-4828Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar As shown in the upper panel of Figure 3B, 99.1% of isolated cells were UV-positive, which expressed E-cadherin (middle and bottom panel, Figure 3B). Similar to a previous report of rat HSCs,12Lim Y.S. Lee H.C. Lee H.S. Switch of cadherin expression from E- to N-type during the activation of rat hepatic stellate cells.Histochem Cell Biol. 2007; 127: 149-160Crossref PubMed Scopus (37) Google Scholar this study also confirmed that the expression of cadherin in mouse HSCs switched from E-cadherin to neural cadherin along with morphologic changes and spontaneous activation (Figure 3C). Moreover, desmin23Yokoi Y. Namihisa T. Kuroda H. Komatsu I. Miyazaki A. Watanabe S. Usui K. Immunocytochemical detection of desmin in fat-storing cells (Ito cells).Hepatology. 1984; 4: 709-714Crossref PubMed Scopus (342) Google Scholar or cytoglobin-positive HSCs expressed E-cadherin by immunofluorescence (Figure 3D). Generally, E-cadherin is expressed primarily in epithelial cells, including hepatocytes. Although HSCs are a type of mesenchymal cells, previous studies have shown that E-cadherin co-localizes with glial fibrillary acidic protein, a classic marker of quiescent HSCs in rat normal liver.12Lim Y.S. Lee H.C. Lee H.S. Switch of cadherin expression from E- to N-type during the activation of rat hepatic stellate cells.Histochem Cell Biol. 2007; 127: 149-160Crossref PubMed Scopus (37) Google Scholar,24Cho I.J. Kim Y.W. Han C.Y. Kim E.H. Anderson R.A. Lee Y.S. Lee C.H. Hwang S.J. Kim S.G. E-cadherin antagonizes transforming growth factor [beta]1 gene induction in hepatic stellate cells by inhibiting RhoA-dependent Smad3 phosphorylation.Hepatology. 2010; 52: 2053-2064Crossref PubMed Scopus (58) Google Scholar Therefore, we assessed immunohistochemical staining for E-cadherin in normal mouse liver, and detected it at the surrounding cytoglobin-positive cells as well as between hepatocytes (Figure 3E), indicating that hepatocytes express E-cadherin even on the sinusoidal surface. Considering that cadherins are homophilic cell-adhesion molecules, these findings indicate that E-cadherin might contribute to the adhesion between hepatocytes and HSCs. For a more detailed analysis, the localization of E-cadherin was visualized by using immunoelectron microscopy. As expected, the immunostain for E-cadherin was confirmed at the adherens junctions between hepatocytes and HSCs (Figure 3F) as well as hepatocyte–hepatocyte adherens junctions (Figure 3G). Taken together, our findings support the conclusion that E-cadherin is expressed in HSCs and mediates adhesion with hepatocytes. We next analyzed the temporal relationship between the disappearance of the adherens junctions and the enhancement of exogenous factors for HSC initiation using a mouse CCl4 model. Hematoxylin and eosin staining and immunohistochemical staining for a neutrophil marker, Ly6G, revealed that the infiltration of neutrophils, which secrete exogenous activation factors of HSCs such as reactive oxygen species,25Koyama Y. Brenner D.A. Liver inflammation and fibrosis.J Clin Invest. 2017; 127: 55-64Crossref PubMed Scopus (433) Google Scholar was present at 12 and 24 hours but not at 6 hours after CCl4 when E-cadherin–mediated adherens junctions disappeared (Figure 4, A and B ). As indicated by qPCR, mRNA expression levels in the liver for exogenous stimuli that encode important drivers of HSC activation, including Tgfb1, Pdgf, Tnfa,26Knittel T. Müller L. Saile B. Ramadori G. Effect of tumour necrosis factor-alpha on proliferation, activation and protein synthesis of rat hepatic stellate cells.J Hepatol. 1997; 27: 1067-1080Abstract Full Text PDF PubMed Scopus (70) Google Scholar Il1b,27Reiter F.P. Wimmer R. Wottke L. Artmann R. Nagel J.M. Carranza M.O. Mayr D. Rust C. Fickert P. Trauner M. Gerbes A.L. Hohenester S. Denk G.U. Role of interleukin-1 and its antagonism of hepatic stellate cell proliferation and liver fibrosis in the Abcb4(–/–) mouse model.World J Hepatol. 2016; 8: 401-410Crossref PubMed Scopus (20) Google Scholar,28Yaping Z. Ying W. Luqin D. Ning T. Xuemei A. Xixian Y. Mechanism of interleukin-1[beta]-induced proliferation in rat hepatic stellate cells from different levels of signal transduction.APMIS. 2014; 122: 392-398Crossref PubMed Scopus (21) Google Scholar and Il6,29Xiang D.M. Sun W. Ning B.F. Zhou T.F. Li X.F. Zhong W. Cheng Z. Xia M.Y. Wang X. Deng X. Wang W. Li H.Y. Cui X.L. Li S.C. Wu B. Xie W.F. Wang H.Y. Ding J. The HLF/IL-6/STAT3 feedforward circuit drives hepatic stellate cell activation to promote liver fibrosis.Gut. 2018; 67: 1704-1715Crossref PubMed Scopus (73) Google Scholar were still not changed within 6 hours after CCl4 injection (Figure 4C). In addition, the expression of Acta2, which encodes α-smooth muscle actin, a specific marker of HSC activation, tended to increase 3 hours after CCl4 injection. The rodent experiments revealed that the HSCs had already lost adherens junctions with hepatocytes in the early stage of injury, when few, if any, exogenous initiating factors are produced. However, TGF-β signaling, which is a typical driver of perpetuation of the HSC-activated phenotype, is not always regulated by its level of expression but rather through its activation from its latent form. We therefore analyzed the effect of E-cadherin–mediated adherens junctions on the response to TGF-β signaling. E-cadherin–overexpressed HHSteCs were cultured as spheroids that reconstruct E-cadherin–mediated adherens junctions and were stimulated by TGF-β. Phase-contrast microscopy showed a
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