Altered Notch Ligand Expression in Human Liver Disease
2002; Elsevier BV; Volume: 160; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)61116-9
ISSN1525-2191
AutoresSarbjit Nijjar, Lorraine Wallace, Heather A. Crosby, Stefan G. Hübscher, Alastair J. Strain,
Tópico(s)Developmental Biology and Gene Regulation
ResumoThe Jagged and Delta family of transmembrane proteins are ligands for Notch receptors, which control the proliferation and/or differentiation of many cell lineages. Expression and localization of these ligands in the adult human liver has not been fully elucidated, nor whether dysregulation of these proteins contributes to liver disease processes. We have examined expression of the five known Notch ligands in human liver. Expression of Jagged-1 and Delta-4 mRNA was seen in normal and diseased liver tissue, whereas Jagged-2, Delta-1, and Delta-3 mRNA was undetectable. In primary liver cell isolates, Jagged-1 expression was found in all cell types, whereas Delta-4 was present in biliary epithelial and liver endothelial cells, but absent in hepatocytes. Interestingly, Jagged-1 mRNA expression was significantly up-regulated in diseased liver tissue. By immunohistochemistry, Jagged-1 expression was present on most structures in normal tissue. However in disease, strikingly strong Jagged-1 immunoreactivity was observed on many small neovessels and bile ductules. The expression of downstream modulators and effectors of Notch signaling was also detectable in purified cell isolates. This, together with aberrant Jagged-1 expression suggests that the Notch signaling pathway may play a role in the neovascularization and biliary defects observed in the liver during the development of cirrhosis. The Jagged and Delta family of transmembrane proteins are ligands for Notch receptors, which control the proliferation and/or differentiation of many cell lineages. Expression and localization of these ligands in the adult human liver has not been fully elucidated, nor whether dysregulation of these proteins contributes to liver disease processes. We have examined expression of the five known Notch ligands in human liver. Expression of Jagged-1 and Delta-4 mRNA was seen in normal and diseased liver tissue, whereas Jagged-2, Delta-1, and Delta-3 mRNA was undetectable. In primary liver cell isolates, Jagged-1 expression was found in all cell types, whereas Delta-4 was present in biliary epithelial and liver endothelial cells, but absent in hepatocytes. Interestingly, Jagged-1 mRNA expression was significantly up-regulated in diseased liver tissue. By immunohistochemistry, Jagged-1 expression was present on most structures in normal tissue. However in disease, strikingly strong Jagged-1 immunoreactivity was observed on many small neovessels and bile ductules. The expression of downstream modulators and effectors of Notch signaling was also detectable in purified cell isolates. This, together with aberrant Jagged-1 expression suggests that the Notch signaling pathway may play a role in the neovascularization and biliary defects observed in the liver during the development of cirrhosis. The Notch signaling pathway plays an essential role in cellular specification, proliferation, and differentiation in a wide variety of organisms ranging from worms to man.1Artavanis-Tsakonas S Rand MD Lake RJ Notch signaling: cell fate control and signal integration in development.Science. 1999; 284: 770-776Crossref PubMed Scopus (4820) Google Scholar The Notch gene family encodes large type 1 transmembrane proteins that function as receptors in a highly conserved signal transduction pathway.2Mumm JS Kopan R Notch signaling: from the outside in.Dev Biol. 2000; 228: 151-165Crossref PubMed Scopus (831) Google Scholar In man, four Notch genes have been identified so far (Notch-1, -2, 3, and -4) and they are expressed in numerous tissue types.3Ellisen LW Bird J West DC Soreng AL Reynolds TC Smith SD Sklar J TAN-1, the human homologue of the Drosophila gene, is broken by chromosomal translocations in T lymphoblastic neoplasms.Cell. 1991; 66: 649-661Abstract Full Text PDF PubMed Scopus (1433) Google Scholar, 4Stifani S Blaumueller CM Redhead NJ Hill RE Artavanis-Tsakonas S Human homologs of a Drosophila enhancer of split gene product define a novel family of nuclear proteins.Nat Genet. 1992; 2: 119-127Crossref PubMed Scopus (261) Google Scholar, 5Joutel A Corpechot C Ducros A Vahedi K Chabriat H Mouton P Alamowitch S Domenga V Cecillion M Marechal E Maciazek J Vayssiere C Cruaud C Cabanis E-A Ruchoux MM Weissenbach J Bach JF Bousser MG Tournier-Lasserve E Notch3 mutations in CADASIL, an hereditary adult onset condition causing stroke and dementia.Nature. 1996; 383: 707-710Crossref PubMed Scopus (1644) Google Scholar, 6Li L Huang GM Banta AB Deng Y Smith T Dong P Friedman C Chen L Trask BJ Spies T Rowen L Hood L Cloning, characterization, and the complete 56.8-kilobase DNA sequence of the human Notch4 gene.Genomics. 1998; 51: 51:45-58Crossref Scopus (38) Google Scholar Signaling is activated after receptor binding to type 1 transmembrane ligands expressed on adjacent cells. The ligands are characterized by two conserved motifs in the extracellular region, the DSL (Delta, Serrate, Lag-2) domain, important for receptor binding, and a series of epidermal growth factor-like repeats that may function to stabilize or modify ligand receptor interactions.7Weinmaster G The ins and outs of Notch signaling.Mol Cell Neurosci. 1997; 9: 91-102Crossref PubMed Scopus (338) Google Scholar Two Notch ligand families have been identified in man based on the presence of a cysteine-rich region in the extracellular domain. Those with the cysteine-rich domain belong to the Serrate/Jagged family and include Jagged-1 and -2.8Oda T Elkahloun AG Pike BL Okajima K Krantz ID Genin A Piccoli DA Meltzer PS Spinner NB Collins FS Chandrasekharappa SC Mutations in the human Jagged1 gene are responsible for Alagille syndrome.Nat Genet. 1997; 16: 235-242Crossref PubMed Scopus (904) Google Scholar, 9Luo B Aster JC Hasserjian RP Kuo F Sklar J Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor.Mol Cell Biol. 1997; 17: 6057-6067Crossref PubMed Scopus (172) Google Scholar The Delta family, which lack the cysteine-rich motif, include Delta-like-1, -3, and -4.10Gray GE Mann RS Mitsiadis E Henrique D Carcangiu ML Banks A Leiman J Ward D Ish-Horowitz D Artavanis-Tsakonas S Human ligands of the Notch receptor.Am J Pathol. 1999; 154: 785-794Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 11Bulman MP Kusumi K Frayling TM McKeown C Garrett C Lander ES Krumlauf R Hattersley AT Ellard S Turnpenny PD Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.Nat Genet. 2000; 24: 438-441Crossref PubMed Scopus (309) Google Scholar, 12Shutter JR Scully S Fan W Richards WG Kitajewski J Deblandre GA Kintner CR Stark KL Dll4, a novel Notch ligand expressed in arterial endothelium.Genes Dev. 2000; 14: 1313-1318PubMed Google Scholar The ligands also possess a poorly conserved cytoplasmic domain, the function of which is unknown. However, deletion studies have revealed that this domain is essential for normal wild-type ligand function.13Sun X Artavanis-Tsakonas S The intracellular deletions of Delta and Serrate define dominant negative forms of the Drosophila Notch ligands.Development. 1996; 122: 2465-2474PubMed Google Scholar The ligands all display distinctive expression patterns in human organs and tissues. During early human development the Jagged-1 gene is expressed in various tissues and cell types.14Crosnier C Attie-Bitachi T Encha-Razavi F Audollent S Soudy F Hadchouel M Meunier-Rotival M Vekemans M JAGGED1 gene expression during human embryogenesis elucidates the wide phenotypic spectrum of Alagille syndrome.Hepatology. 2000; 32: 574-581Crossref PubMed Scopus (128) Google Scholar In adult tissues strong Jagged-1 mRNA expression is detectable in heart, placenta, and kidney with lower levels in lung muscle and pancreas.10Gray GE Mann RS Mitsiadis E Henrique D Carcangiu ML Banks A Leiman J Ward D Ish-Horowitz D Artavanis-Tsakonas S Human ligands of the Notch receptor.Am J Pathol. 1999; 154: 785-794Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar Jagged-2 transcripts are also detectable in heart and placenta, together with skeletal muscle.10Gray GE Mann RS Mitsiadis E Henrique D Carcangiu ML Banks A Leiman J Ward D Ish-Horowitz D Artavanis-Tsakonas S Human ligands of the Notch receptor.Am J Pathol. 1999; 154: 785-794Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar The Delta-1 gene is highly expressed in heart and pancreas, with weak expression also detectable in brain and muscle tissue.10Gray GE Mann RS Mitsiadis E Henrique D Carcangiu ML Banks A Leiman J Ward D Ish-Horowitz D Artavanis-Tsakonas S Human ligands of the Notch receptor.Am J Pathol. 1999; 154: 785-794Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar Little is known about the tissue distribution of Delta-3 in adult organs, however during mouse embryogenesis, expression implicates Delta-3 with a role in somitogenesis and neurogenesis.15Dunwoodie SL Henrique D Harrison SM Beddington RS Mouse Dll3: a novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo.Development. 1997; 124: 3065-3076PubMed Google Scholar In contrast, Delta-4 gene expression is seen in most adult and fetal tissues.12Shutter JR Scully S Fan W Richards WG Kitajewski J Deblandre GA Kintner CR Stark KL Dll4, a novel Notch ligand expressed in arterial endothelium.Genes Dev. 2000; 14: 1313-1318PubMed Google Scholar Mutations in two of the five known human Notch ligands have been associated with developmental disorders in man. Lesions in the human Delta-like-3 gene give rise to spondylocostal dysostosis, a group of vertebral malsegmentation syndromes resulting from axial skeletal defects. Spondylocostal dysostosis is a disorder with autosomal-dominant and autosomal-recessive modes of inheritance, which is characterized by multiple hemivertebrae, rib fusions, and deletions with a nonprogressive lateral curving of the spine.11Bulman MP Kusumi K Frayling TM McKeown C Garrett C Lander ES Krumlauf R Hattersley AT Ellard S Turnpenny PD Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.Nat Genet. 2000; 24: 438-441Crossref PubMed Scopus (309) Google Scholar In patients with spondylocostal dysostosis, two mutations in the human Delta-3 gene were identified within the conserved extracellular domain and are predicted to give rise to truncated proteins. A third missense mutation present in a highly conserved glycine residue of the fifth epidermal growth factor-like repeat highlights the functional importance of this domain.11Bulman MP Kusumi K Frayling TM McKeown C Garrett C Lander ES Krumlauf R Hattersley AT Ellard S Turnpenny PD Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.Nat Genet. 2000; 24: 438-441Crossref PubMed Scopus (309) Google Scholar Mutations in the Jagged-1 gene give rise to Alagille syndrome an autosomal-dominant developmental disorder that affects structures in the liver, skeleton, eye, heart, kidney, and other organs.8Oda T Elkahloun AG Pike BL Okajima K Krantz ID Genin A Piccoli DA Meltzer PS Spinner NB Collins FS Chandrasekharappa SC Mutations in the human Jagged1 gene are responsible for Alagille syndrome.Nat Genet. 1997; 16: 235-242Crossref PubMed Scopus (904) Google Scholar, 16Li L Krantz ID Deng Y Genin A Banta AB Collins CC Qi M Trask BJ Lin Kuo W Cochran J Costa T Pierpont MEM Rand EB Picoli DA Hood L Spinner NB Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.Nat Genet. 1997; 16: 243-251Crossref PubMed Scopus (983) Google Scholar, 17Louis AA Van Eyken P Haber BA Hicks C Weinmaster G Taub R Rand EB Hepatic jagged-1 expression studies.Hepatology. 1999; 30: 1269-1275Crossref PubMed Scopus (73) Google Scholar In the liver it is characterized by cholestatic liver disease related to paucity of intrahepatic bile ducts. The disease can vary in its degree of severity from an apparently normal phenotype to chronic cases in which cirrhosis and liver failure result in the requirement for liver transplantation in early childhood. Analysis of DNA samples from Alagille syndrome patients has revealed three frame-shift mutations, two splice donor mutations, and one mutation that abolishes RNA expression from the altered allele. Thus the phenotype observed for Alagille syndrome and spondylocostal dysostosis may be the result of haplo-insufficiency or dominant-negative effects of the Jagged-1 and Delta-like-3 gene products.8Oda T Elkahloun AG Pike BL Okajima K Krantz ID Genin A Piccoli DA Meltzer PS Spinner NB Collins FS Chandrasekharappa SC Mutations in the human Jagged1 gene are responsible for Alagille syndrome.Nat Genet. 1997; 16: 235-242Crossref PubMed Scopus (904) Google Scholar, 11Bulman MP Kusumi K Frayling TM McKeown C Garrett C Lander ES Krumlauf R Hattersley AT Ellard S Turnpenny PD Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.Nat Genet. 2000; 24: 438-441Crossref PubMed Scopus (309) Google Scholar, 16Li L Krantz ID Deng Y Genin A Banta AB Collins CC Qi M Trask BJ Lin Kuo W Cochran J Costa T Pierpont MEM Rand EB Picoli DA Hood L Spinner NB Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.Nat Genet. 1997; 16: 243-251Crossref PubMed Scopus (983) Google Scholar We have previously shown that all four of the human Notch genes are expressed in adult human liver and the expression, together with the distribution of these receptors, is altered in liver disease.18Nijjar S Crosby HA Wallace L Hubscher SG Strain AJ Notch receptor expression in adult human liver: a possible role in bile duct formation and hepatic neovascularisation.Hepatology. 2001; 34: 1184-1192Crossref PubMed Scopus (88) Google Scholar To determine whether these perturbations in Notch receptor expression are the result of altered ligand expression, we have analyzed the expression profile of all of the Notch ligands together with downstream targets and modulators of the Notch pathway in normal and diseased liver tissue. Taken together with our previous data on the localization of Notch receptor gene expression, these results provide valuable insight into possible ligand/receptor interactions occurring in liver cell types in the normal liver as well as during disease processes.18Nijjar S Crosby HA Wallace L Hubscher SG Strain AJ Notch receptor expression in adult human liver: a possible role in bile duct formation and hepatic neovascularisation.Hepatology. 2001; 34: 1184-1192Crossref PubMed Scopus (88) Google Scholar Diseased liver tissue was obtained from the adult liver transplant program at University Hospital Birmingham, National Health Science Trust. Hepatectomy specimens were obtained from patients undergoing transplantation for primary biliary cirrhosis (PBC) (n = 5; age range, 19 to 45 years), primary sclerosing cholangitis (PSC) (n = 5; age range, 27 to 46 years), and alcoholic liver disease (n = 5; age range, 18 to 46 years). Donor tissue excess to surgical requirements, served as normal controls (n = 5; age range, 20 to 43 years). For immunohistochemistry and tissue reverse transcriptase-polymerase chain reaction (RT-PCR), tissue was snap-frozen and stored at −70°C. For cell isolations, tissue was stored in Dulbecco's modified Eagles medium (Gibco, Paisley, UK) at 4°C and used within 48 hours after hepatectomy. Informed consent and local regional ethical committee approval was obtained before tissue collection. Hepatocytes were isolated from human hepatectomy specimens and donor livers by collagenase perfusion.19Strain AJ Ismail T Tsubouchi H Arakaki N Hishida T Kitamura N Daikuhara Y McMaster P Native and recombinant human hepatocyte growth factors are highly potent promoters of DNA synthesis in both human and rat hepatocytes.J Clin Invest. 1991; 87: 1853-1857Crossref PubMed Scopus (188) Google Scholar After isolation, cells were resuspended in William's E medium containing insulin, glutamine, and hydrocortisone and plated as previously described.19Strain AJ Ismail T Tsubouchi H Arakaki N Hishida T Kitamura N Daikuhara Y McMaster P Native and recombinant human hepatocyte growth factors are highly potent promoters of DNA synthesis in both human and rat hepatocytes.J Clin Invest. 1991; 87: 1853-1857Crossref PubMed Scopus (188) Google Scholar Isolation of biliary epithelial cells was performed as previously described.20Joplin R Strain AJ Neuberger JM Immuno-isolation and culture of biliary epithelial cells from normal human liver.In Vitro Cell Dev Biol. 1989; 25: 1189-1192Crossref PubMed Scopus (82) Google Scholar, 21Joplin R Hishida T Tsubouchi H Daikuhara Y Ayres R Neuberger JM Strain AJ Human intrahepatic biliary epithelial cells proliferate in vitro in response to human hepatocyte growth factor.J Clin Invest. 1992; 90: 1284-1289Crossref PubMed Scopus (129) Google Scholar Endothelial cells were selected from the same Percoll gradient fraction as the biliary epithelial cells (BECs), but were further purified by immunomagnetic separation using the monoclonal antibody CD31 (endothelial cell marker). Cells were maintained in culture for a week before harvesting and RNA extraction. Total RNA was isolated from whole tissue or cultured cells using RNazol B (Biogenesis Ltd., Poole, UK) essentially as described by the manufacturer. Briefly, liver tissue (1 g) was homogenized in 6 ml of RNazol B solution. The homogenate was extracted by the addition of 600 μl of chloroform and centrifuged at 12,000 rpm for 15 minutes. The RNA-containing supernatant was then extracted again by the addition of an equal volume of phenol:chloroform:isoamyl alcohol (5:3:1) (Sigma, Poole, UK). Total RNA was then recovered by isopropanol precipitation and centrifugation at 12,000 rpm for 30 minutes. The pellet was washed once with 75% ethanol and dissolved in distilled water treated with diethyl pyrocarbonate. RNA was then incubated with RNase-free DNase I at 37°C for 20 minutes to remove traces of genomic DNA. After heat denaturation to inactivate DNase I activity, total RNA was recovered by isopropanol precipitation, subjected to a 75% ethanol wash, and resuspended in diethyl pyrocarbonate-treated water. Total RNA was extracted from cultured cells by using a scaled-down version of the procedure used for extracting total RNA from tissue. Briefly 1 ml of RNazol B was added directly to culture flasks. Cell lysates were extracted by the addition of 0.1 ml of chloroform and centrifuged at 15,000 rpm for 15 minutes. After DNase I treatment, RNA was recovered by isopropanol precipitation, overnight at −20°C, and centrifugation at 15,000 rpm for 30 minutes. Pellets were then dissolved in diethyl pyrocarbonate-treated water and stored at −70°C until required. Semiquantitative RT-PCR was performed as previously described.22Hudson C Clements D Friday RV Stott D Woodland HR Xsox17alpha and beta mediate endoderm formation in Xenopus.Cell. 1997; 91: 397-405Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, 23Brennan HC Nijjar S Jones EA The specification and growth factor inducibility of the pronephric glomus in Xenopus laevis.Development. 1999; 126: 5847-5856PubMed Google Scholar Briefly, first-strand cDNA was prepared from 1 μg of DNase I-treated total RNA using random hexamers and Superscript II reverse transcriptase (Gibco). Control reactions were also performed in the presence and absence of reverse transcriptase or RNA. The cDNA was then normalized for equivalent template amounts (up to a volume of 1.6 μl) by amplification with primers specific for β-actin in the presence of α-32P dGTP (Amersham-Pharmacia Biotech, Little Chalfont, UK). The cycling parameters used for β-actin amplification were as follows: an initial cycle of 94°C for 3 minutes (denaturation step), 58°C for 1 minute (annealing step), 72°C for 1 minute (extension step). The required number of cycles as specified in Table 1 were performed as for the initial cycle, but with 30 seconds for the 94°C denaturation step. A final extension step of 72°C for 5 minutes was then performed. With the exception of the annealing temperature and the cycle number, which are given in Table 1 for specific primers, these cycling parameters were used for all of the primers sets used. PCR products were then resolved on 6% nondenaturing polyacrylamide gels, dried under vacuum, and subjected to autoradiography. After autoradiography the intensities of amplified bands was quantified using the Gel Doc 2000 densitometry software (BioRad, Hemel Hempstead, UK). In control cDNA synthesis reactions RNA and reverse transcriptase were excluded and the products of these reactions were then used for PCR. In control PCR reactions cDNA was omitted and water added to the subsequent PCR reaction. 0.2 μl, 0.4 μl, 0.8 μl, and 1.6 μl are controls with the implied proportion of cDNA added to four PCR reactions to ensure that amplification was within the linear range.Table 1Primer Sequences for RT-PCR AnalysisGenePrimer sequence (5′–3′)Starting position in cDNAAnnealing temperature (°C)Product amplified (bp)Jagged-1AFw. 5′-AGTCACTGGCACGGTTGTAG-3′178356227(28 cycles)Rv. 5′-TCGCTGTATCTGTCCACCTG-3′2009Jagged-2BFw. 5′-GATTGGCGGCTATTACTGTG-3′135556600(35 cycles)Rv. 5′-AGGCAGTCGTCAATGTTCTC-3′1954Delta-1CFw. 5′-AGACGGAGACCATGAACAAC-3′207955382(35 cycles)Rv. 5′-TCCTCGGATATGACGTACAC-3′2460Delta-3DFw. 5′-GTGAATGCCGATGCCTAGAG-3′71157256(35 cycles)Rv. 5′-GGTCCATCTGCACATGTCAC-3′966Delta-4EFw. 5′-TGACCACTTCGGCCACTATG-3′57655620(27 cycles)Rv. 5′-AGTTGGAGCCGGTGAAGTTG-3′1195Hes-1FFw. 5′-TGGATGCGGAGTCTACGATG-3′21757468(24 cycles)Rv. 5′-TAAGGCCACTTGCCACCTTC-3′684LnFgGFw. 5′-TGGAGTATGACCGCTTCATC-3′23857460(24 cycles)Rv. 5′-ATACCGTAGCTCAGCGTCAC-3′697DeltexHFw. 5′-TGATGCCTGTGAATGGTCTG-3′52957450(23 cycles)Rv. 5′-TCTGCGACATGCTGTTGAAG-3′978β-actinIFw. 5′-CATCACCATTGGCAATGAGC-3′78258284(16 cycles)Rv. 5′-CGATCCACACGGAGTACTTG-3′1065Fw., forward; Rv., reverse; bp, base pairs.Genbank accession numbers: A, U61276; B, AF003521; C, AF003522; D, XM009230; E, AF253468; F, Y07572; G, U94354; H, AF053700; I, X00351. Open table in a new tab Fw., forward; Rv., reverse; bp, base pairs. Genbank accession numbers: A, U61276; B, AF003521; C, AF003522; D, XM009230; E, AF253468; F, Y07572; G, U94354; H, AF053700; I, X00351. Horseradish peroxidase staining was used to visualize antigens on acetone-fixed 5-μm cryostat tissue sections. Briefly, sections were incubated with 20% normal swine serum (DAKO, High Wycombe, UK) for 20 minutes. Sections were then incubated with a Jagged-1 primary goat polyclonal antibody (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA), (diluted 1:100 in 10% normal swine serum) for 1 hour. Sections were washed twice in Tris- buffered saline and then incubated with a rabbit anti-goat peroxidase conjugate (DAKO) (diluted 1:100 in 10% normal swine serum) for 45 minutes. Signals were enhanced by the addition of a goat anti-rabbit peroxidase conjugate (DAKO) (diluted 1:100 in 10% normal swine serum) for a further 45 minutes. Slides were then washed twice in Tris-buffered saline and staining was visualized using the diaminobenzidine color substrate (Sigma, Poole, UK). Before mounting in DEPEX mountant, slides were counterstained with Mayer's hematoxylin (BDH, Lutterworth, UK). In control sections the primary antibody was omitted. Double immunostaining for studies on antigen co-localization was performed with selected antibodies using the fluorescent conjugates alexaFluor 594 (red fluorescence, rabbit anti-mouse 1:200; Cambridge Bioscience) and alexaFluor 488 (green fluorescence, donkey anti-goat, 1:50; Cambridge Bioscience). The primary antibody combinations consisted of either cytokeratin 19 (monoclonal antibody recognizing BECs, 1:100; DAKO) or CD31 (monoclonal antibody recognizing endothelial cells, 1:100; DAKO), coupled with Jagged-1 (goat polyclonal, 1:50; Santa Cruz Biotechnologies, Inc.). Primary antibodies were incubated overnight at 4°C on 5-μm cryostat sections, and visualized using the alexaFluor conjugates on an Axiovert fluorescence microscope (CarlZeiss, UK). Images were captured using a digital camera and Axiovision software (Carl Zeiss, UK). Staining of sections was assessed by two independent observers using a validated semiquantitative scale, where − denotes absence of staining; ±, occasional weak staining on some structures; +, weak staining; ++, moderate staining, and +++, strong staining.24Adams DH Hubscher SG Shaw J Johnson GD Bubbs C Rothlein R Neuberger JM Increased expression of ICAM-1 on bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis.Hepatology. 1991; 14: 426-432Crossref PubMed Scopus (140) Google Scholar To identify which Notch ligand genes were expressed in the adult liver, semiquantitative RT-PCR was performed initially on total RNA extracted from normal and diseased (PBC and PSC) adult liver tissue. The expression of Jagged-1 and Delta-4 mRNA was seen in normal and diseased liver tissue (Figure 1A). However, the expression of Jagged-2, Delta-1, and Delta-3 was not detectable (data not shown). Interestingly, the expression of Jagged-1 seemed to be elevated in liver tissue with PBC and PSC, when compared to normal liver (Figure 1B) and was statistically significant (PBC, P < 0.05; PSC, P < 0.01; paired Student's t-test). The up-regulation in Jagged-1 mRNA expression was confirmed by performing RT-PCR on RNA extracted from a second sample of tissue from the same donor/patient groups (data not shown). We then investigated Jagged-1 and Delta-4 mRNA expression in cell isolates derived from normal and diseased liver tissue. Semiquantitative RT-PCR was performed on total RNA extracted from BECs, liver endothelial cells (LECs), and hepatocytes (Figure 2). The Jagged-1 gene was detectable in all BECs, LECs, and hepatocyte cell isolates. Delta-4 mRNA however displayed a more interesting expression pattern. Although Delta-4 mRNA was undetectable in hepatocytes, it was strongly expressed in LECs, with the exception of one normal liver cell preparation. Delta-4 expression was also seen in BEC isolates although this expression was somewhat variable (Figure 2).Figure 2RT-PCR analysis of Jagged-1 and Delta-4 mRNA expression in cell isolates. RT-PCR analysis was performed on RNA extracted from primary cell cultures. NL, normal liver; AIH, autoimmune hepatitis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To localize the expression of Jagged-1 within the adult liver immunohistochemistry was performed on normal and diseased liver tissue sections. Results are summarized in Table 2. In normal liver most portal tract structures were positive for Jagged-1 immunoreactivity (Figure 3B, Table 2). Weak staining of hepatocyte membranes and the endothelium of hepatic veins was also seen in the parenchyma of normal liver tissue (Figure 3C, Table 2). In diseased liver, the immunolocalization of Jagged-1 in PBC, PSC, and alcoholic liver disease was similar. There was weak to moderate staining of bile ducts and portal vessels, as seen in normal liver. However, in portal and septal regions strong staining of bile ductules (Figure 3, D and F; Table 2) and other structures that appeared to resemble small vessels was evident (Figure 3, E and F; Table 2). In the parenchyma, Jagged-1 immunoreactivity was localized to hepatocyte cell membranes in all three diseases although this appeared to be slightly higher in PBC tissue (Table 2).Table 2Summary of Immunohistochemical Analysis for Jagged-1 Expression in Normal and Diseased Adult Liver TissuePortal tractParenchymaVesselsSinusoidsBile ductsB.D.P.V.H.A.S.N.V.I.C.HepatocytesK cellsSECsHepatic veinsNORMAL+NA++++NA−+−−+PBC+++++++++++++++−+PSC+++++++++++−+−−+ALD+++++++++−+−−−B.D., bile ductules; H.A., hepatic artery; P.V., portal vein; I.C., inflammatory cells; S.N.V., small neovessels; K cells; Kupffer cells; SECS., sinusoidal endothelial cells; NA., not applicable.−, Negative; +/−, occasional weak staining on some structures; +, weak staining; ++, moderate staining; +++, strong staining. Open table in a new tab B.D., bile ductules; H.A., hepatic artery; P.V., portal vein; I.C., inflammatory cells; S.N.V., small neovessels; K cells; Kupffer cells; SECS., sinusoidal endothelial cells; NA., not applicable. −, Negative; +/−, occasional weak staining on some structures; +, weak staining; ++, moderate staining; +++, strong staining. To confirm that Jagged-1 expression was indeed localized to bile ductules in diseased tissue, dual-immunofluorescence staining using cytokeratin 19 (a biliary cell marker) and Jagged-1 was used. In normal tissue, both CK19 and Jagged-1 were found to stain the epithelial cells of bile ducts, but also co-localized to these cells (Figure 4A). In PBC tissue, both markers were co-expressed by bile ductules (Figure 4C). Jagged-1 was also seen to stain numerous structures in portal regions of diseased tissue that resembled small blood vessels (Figure 3E, Table 2). Immunofluorescence staining using CD31 (an endothelial cell marker) and Jagged-1 was performed to confirm that this was the case. In normal tissue, both CD31 and Jagged-1 co-localized to the vascular endothelium in portal regions (Figure 4B). In PBC tissue, CD31 and Jagged-1 were co-expressed on numerous neovessel-like structures (Figure 4D), confirming that they were indeed blood vessels. To gain insights into whether Notch signaling can occur between similar cell types in liver tissue, we have analyzed the expression of two downstream targets of Notch activation (Hes-1 and Deltex) in addition to an e
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