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- W1753444805 abstract "The expression of the β1C integrin, an alternatively spliced variant of the β1 subunit, was investigated in human adult and fetal tissues. In the adult, β1C immunoreactivity was found in nonproliferative, differentiated simple, and/or pseudostratified epithelia in prostate glands and liver bile ducts. In contrast, β1C was undetectable in stratified squamous epithelium of the epidermis and/or in hepatocytes. Luminal prostate epithelial cells expressed β1C in vivo and in vitro, but no β1Cwas seen in basal cells, which are proliferating cells. Fetal prostate expressed β1C in differentiated glands that had a defined lumen, but not in budding glands, indicating that β1C is a marker of prostate epithelium differentiation. The β1C and the common β1Avariants are differentially distributed: β1A was found in luminal and basal epithelial as well as in stromal cells in the prostate. In the liver, β1C and β1Awere coexpressed in biliary epithelium, whereas vascular cells expressed only β1A. Because we found β1C in nonproliferative and differentiated epithelium, we investigated whether β1C could have a causal role in inhibiting epithelial cell proliferation. The results showed that exogenous expression of a β1C, but not of a β1A, cytoplasmic domain chimeric construct, completely inhibited thymidine incorporation in response to serum by prostate cancer epithelial cells. Consistent with these in vitro results, β1C appeared to be downregulated in prostate glands that exhibit regenerative features in benign hyperplastic epithelium. These data show that the presence of β1C integrins in epithelial cells correlates with a nonproliferative, differentiated phenotype and is growth inhibitory to prostate epithelial cells in vitro. These findings indicate a novel pathophysiological role for this integrin variant in epithelial cell proliferation. The expression of the β1C integrin, an alternatively spliced variant of the β1 subunit, was investigated in human adult and fetal tissues. In the adult, β1C immunoreactivity was found in nonproliferative, differentiated simple, and/or pseudostratified epithelia in prostate glands and liver bile ducts. In contrast, β1C was undetectable in stratified squamous epithelium of the epidermis and/or in hepatocytes. Luminal prostate epithelial cells expressed β1C in vivo and in vitro, but no β1Cwas seen in basal cells, which are proliferating cells. Fetal prostate expressed β1C in differentiated glands that had a defined lumen, but not in budding glands, indicating that β1C is a marker of prostate epithelium differentiation. The β1C and the common β1Avariants are differentially distributed: β1A was found in luminal and basal epithelial as well as in stromal cells in the prostate. In the liver, β1C and β1Awere coexpressed in biliary epithelium, whereas vascular cells expressed only β1A. Because we found β1C in nonproliferative and differentiated epithelium, we investigated whether β1C could have a causal role in inhibiting epithelial cell proliferation. The results showed that exogenous expression of a β1C, but not of a β1A, cytoplasmic domain chimeric construct, completely inhibited thymidine incorporation in response to serum by prostate cancer epithelial cells. Consistent with these in vitro results, β1C appeared to be downregulated in prostate glands that exhibit regenerative features in benign hyperplastic epithelium. These data show that the presence of β1C integrins in epithelial cells correlates with a nonproliferative, differentiated phenotype and is growth inhibitory to prostate epithelial cells in vitro. These findings indicate a novel pathophysiological role for this integrin variant in epithelial cell proliferation. Cell interactions with extracellular matrix proteins control proliferation, differentiation, and survival,1Brakebusch C Hirsch E Potocnik A Fassler R Genetic analysis of β1 integrin function: confirmed, new and revised role for a crucial family of cell adhesion molecules.J Cell Sci. 1997; 110: 2895-2904PubMed Google Scholar, 2Damsky CH Werb Z Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information.Curr Opin Cell Biol. 1992; 4: 772-781Crossref PubMed Scopus (482) Google Scholar, 3Ruoslahti E Reed JC Anchorage dependence, integrins and apoptosis.Cell. 1994; 77: 477-478Abstract Full Text PDF PubMed Scopus (964) Google Scholar as well as tumor growth, angiogenesis, and metastasis.4Varner JA Cheresh DA Integrins and cancer.Curr Opin Cell Biol. 1996; 8: 724-730Crossref PubMed Scopus (465) Google Scholar These interactions are predominantly mediated by integrins, cell adhesion receptors composed of an α and a β subunit.5Haas TA Plow EF Integrin-ligand interactions: a year in review.Curr Opin Cell Biol. 1994; 6: 656-662Crossref PubMed Scopus (265) Google Scholar, 6Hynes RO Integrins: versatility, modulation and signaling in cell adhesion.Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8941) Google Scholar, 7Schwartz MA Schaller MD Ginsberg MH Integrins: emerging paradigms of signal transduction.Annu Rev Cell Dev Biol. 1995; 11: 549-599Crossref PubMed Scopus (1456) Google Scholar In addition to mediating physical interactions, integrins signal intracellularly through their cytoplasmic domains.8Fornaro M Languino LR Alternatively spliced variants: a new view of the integrin cytoplasmic domain.Matrix Biol. 1997; 16: 185-193Crossref PubMed Scopus (59) Google Scholar The cytoplasmic domains also control integrin affinity for ligands and subcellular localization.8Fornaro M Languino LR Alternatively spliced variants: a new view of the integrin cytoplasmic domain.Matrix Biol. 1997; 16: 185-193Crossref PubMed Scopus (59) Google Scholar, 9Williams MJ Hughes PE O'Toole TE Ginsberg MH The inner world of cell adhesion: integrin cytoplasmic domains.Trends Cell Biol. 1994; 4: 109-112Abstract Full Text PDF PubMed Scopus (164) Google Scholar, 10Hemler ME Weitzman JB Pasqualini R Kawaguchi S Kassner PD Berdichevsky FB Structure, biochemical properties, and biological functions of integrin cytoplasmic domains.in: Takada Y Integrins: The Biological Problems. CRC Press, Ann Arbor1995: 1-35Google Scholar The cytoplasmic domain of the β1 subunit, in its canonical form (β1A), is highly conserved from fungi and invertebrates to vertebrates.11Marcantonio EE Hynes RO Antibodies to the conserved cytoplasmic domain of the integrin β1 subunit react with proteins in vertebrates, invertebrates and fungi.J Cell Biol. 1988; 106: 1765-1772Crossref PubMed Scopus (216) Google Scholar Experimental modifications of the β1 cytoplasmic domain have been shown to affect cell proliferation,12Fornaro M Zheng DQ Languino LR The novel structural motif Gln795-Gln802 in the integrin β1C cytoplasmic domain regulates cell proliferation.J Biol Chem. 1995; 270: 24666-24669Crossref PubMed Scopus (54) Google Scholar, 13Meredith Jr, J Takada Y Fornaro M Languino LR Schwartz MA Inhibition of cell cycle progression by the alternatively spliced integrin β1C.Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar development,14Baudoin C Goumans M-J Mummery C Sonnenberg A Knockout and knockin of the β1 exon D define distinct roles for integrin splice variants in heart function and embryonic development.Genes Dev. 1998; 12: 1202-1216Crossref PubMed Scopus (76) Google Scholar migration,15Balzac F Retta SF Albini A Melchiorri A Koteliansky VE Geuna M Silengo L Tarone G Expression of β1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.J Cell Biol. 1994; 127: 557-565Crossref PubMed Scopus (66) Google Scholar, 16LaFlamme SE Thomas LA Yamada SS Yamada KM Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly.J Cell Biol. 1994; 126: 1287-1298Crossref PubMed Scopus (205) Google Scholar integrin localization,17Marcantonio EE Guan J-L Trevithick JE Hynes RO Mapping of the functional determinants of the integrin β1 cytoplasmic domain by site-directed mutagenesis.Cell Regul. 1990; 1: 597-604Crossref PubMed Scopus (116) Google Scholar, 18Reszka AA Hayashi Y Horwitz AF Identification of amino acid sequences in the integrin β1 cytoplasmic domain implicated in cytoskeleton association.J Cell Biol. 1992; 117: 1321-1330Crossref PubMed Scopus (240) Google Scholar and mitogen-activated protein kinase activation19Belkin AM Zhidkova NI Balzac F Altruda F Tomatis D Maier A Tarone G Koteliansky VE Burridge K β1D integrin displaces β1A isoform in striated muscles: localization at junctional structures and signaling potential in non-muscle cells.J Cell Biol. 1996; 132: 211-226Crossref PubMed Scopus (188) Google Scholar, 20Wei J Shaw LM Mercurio AM Regulation of mitogen-activated protein kinase activation by the cytoplasmic domain of the α6 integrin subunit.J Biol Chem. 1998; 273: 5903-5907Crossref PubMed Scopus (67) Google Scholar and phosphorylation of focal adhesion kinase or paxillin.15Balzac F Retta SF Albini A Melchiorri A Koteliansky VE Geuna M Silengo L Tarone G Expression of β1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.J Cell Biol. 1994; 127: 557-565Crossref PubMed Scopus (66) Google Scholar, 21Akiyama SK Yamada SS Yamada KM LaFlamme SE Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras.J Biol Chem. 1994; 269: 15961-15964Abstract Full Text PDF PubMed Google Scholar, 22Lukashev ME Sheppard D Pytela R Disruption of integrin function, and induction of tyrosine phosphorylation, by the autonomously expressed β1 integrin cytoplasmic domain.J Biol Chem. 1994; 269: 18311-18314Abstract Full Text PDF PubMed Google Scholar, 23Schaller MD Parsons JT Focal adhesion kinase and associated proteins.Curr Opin Cell Biol. 1994; 6: 705-710Crossref PubMed Scopus (493) Google Scholar Alternative splicing events, by creating variant cytodomains, generate functionally distinct integrin complexes.8Fornaro M Languino LR Alternatively spliced variants: a new view of the integrin cytoplasmic domain.Matrix Biol. 1997; 16: 185-193Crossref PubMed Scopus (59) Google Scholar Alternatively spliced forms of the β (β1, β3, β4, and β5) and α (α3, α6, and α7) integrin cytoplasmic domains have been identified,8Fornaro M Languino LR Alternatively spliced variants: a new view of the integrin cytoplasmic domain.Matrix Biol. 1997; 16: 185-193Crossref PubMed Scopus (59) Google Scholar, 24Zhang H Tan SM Lu J cDNA cloning reveals two mouse β5 integrin transcripts distinct in cytoplasmic domains as a result of alternative splicing.Biochem J. 1998; 331: 631-637PubMed Google Scholar thus adding further complexity to the regulatory pathways mediated by integrins. The integrin β1 variants are known as: the canonical β1A, β1B, β1C, β1C-2, and β1D; they differ in the COOH-terminal sequences.8Fornaro M Languino LR Alternatively spliced variants: a new view of the integrin cytoplasmic domain.Matrix Biol. 1997; 16: 185-193Crossref PubMed Scopus (59) Google Scholar The β1C integrin (formerly β1S)25Languino LR Ruoslahti E An alternative form of the integrin β1 subunit with a variant cytoplasmic domain.J Biol Chem. 1992; 267: 7116-7120Abstract Full Text PDF PubMed Google Scholar is generated by the presence of an unspliced intervening 116-bp sequence (exon C), which causes a frame shift in the 3′ end of the β1 subunit and codes for a unique 48-amino acid COOH-terminal sequence. Unlike β1A, β1C inhibits cell proliferation and is downregulated in cancer cells.12Fornaro M Zheng DQ Languino LR The novel structural motif Gln795-Gln802 in the integrin β1C cytoplasmic domain regulates cell proliferation.J Biol Chem. 1995; 270: 24666-24669Crossref PubMed Scopus (54) Google Scholar, 13Meredith Jr, J Takada Y Fornaro M Languino LR Schwartz MA Inhibition of cell cycle progression by the alternatively spliced integrin β1C.Science. 1995; 269: 1570-1572Crossref PubMed Scopus (117) Google Scholar, 26Fornaro M Tallini G Bofetiado CJM Bosari S Languino LR Down-regulation of β1C integrin, an inhibitor of cell proliferation, in prostate carcinoma.Am J Pathol. 1996; 149: 765-773PubMed Google Scholar We show here that β1C is expressed in a subset of epithelial cells exhibiting a nonproliferating and differentiated phenotype, and that it is lost in regenerative areas of prostate glands. We also provide evidence suggestive of a causal role for the β1C cytodomain in suppressing epithelial cell proliferation. The PC3 human prostatic carcinoma cell line was obtained from American Type Culture Collection (Manassas, VA). PC3 cells were maintained in RPMI (Life Technologies Inc., Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gemini Bioproducts Inc., Calabasas, CA), 2 mmol/L glutamine (Gemini Bioproducts Inc.), 100 μg/ml streptomycin-100 U/ml penicillin (Gemini Bioproducts Inc.), 0.1 mmol/L nonessential amino acids (Life Technologies), and 1 mmol/L sodium pyruvate (Life Technologies). Human prostate luminal epithelial cells, BPH-1,27Hayward SW Cunha GR Bartek J Deshpande N Narayan P Establishment and characterization of an immortalized but not transformed human prostate epithelial cell line: BPH-1.In Vitro Cell Dev Biol Anim. 1995; 31: 14-24Crossref PubMed Scopus (290) Google Scholar were maintained in RPMI supplemented with 2.5% FCS, 2 mmol/L glutamine, and 100 μg/ml streptomycin-100 U/ml penicillin. Rabbit antibodies specific for the β1C or the β1A subunit cytoplasmic domains were generated and affinity purified as previously described.26Fornaro M Tallini G Bofetiado CJM Bosari S Languino LR Down-regulation of β1C integrin, an inhibitor of cell proliferation, in prostate carcinoma.Am J Pathol. 1996; 149: 765-773PubMed Google Scholar The following antibodies were used: fluorescein isothiocyanate-conjugated rat monoclonal antibody to mouse CD4, clone YTS 191.1 (Caltag Laboratories, San Francisco, CA); mouse monoclonal antibody to the human β1 integrin extracellular domain, mAb 13 (Beckton Dickinson, San Jose, CA) and kindly provided by Dr. K. M. Yamada (National Institutes of Health, Bethesda, MD); mouse monoclonal antibodies to basal cytokeratins 1, 5, 10, and 14, clone 34βE12 (Enzo Diagnostic, Farmingdale, NY) or to type I and type II cytokeratins, clone AE1/AE3 (Boehringer Mannheim, Indianapolis, IN); and mouse monoclonal antibody to cytokeratins 8 and 18, clone CAM 5.2 (Becton Dickinson). Fluorescein isothiocyanate-rat immunoglobulin G (IgG) were purchased from Caltag. Samples (autopsy or surgical specimens) were obtained from the files of the Department of Pathology at Yale New Haven Hospital. The following adult tissues were included in the study: benign prostate (62 samples, including 25 benign prostatic hyperplastic samples), normal liver (12 samples), normal gallbladder (4 samples), normal kidney (5 samples), and normal lung (1 sample). Developing tissues from spontaneous or therapeutic abortions corresponding to different gestational ages were also included: prostate (4 samples, 18 to 22 weeks; 9 samples, 23 weeks or later) and liver (3 samples, 16 to 19 weeks). Hematoxylin and eosin sections were analyzed from all samples to assess the integrity of the tissue selected for the evaluation of β1C, β1A, or cytokeratin immunoreactivity. All tissues were obtained under review board-approved protocols. Immunostaining of β1C, β1A, and cytokeratins was performed essentially as described,26Fornaro M Tallini G Bofetiado CJM Bosari S Languino LR Down-regulation of β1C integrin, an inhibitor of cell proliferation, in prostate carcinoma.Am J Pathol. 1996; 149: 765-773PubMed Google Scholar with minor modifications. For immunostaining of liver specimens, nonspecific binding of biotin/avidin was prevented using the Avidin/Biotin blocking kit from Vector Laboratories (Burlingame, CA), according to the manufacturer's instructions. For immunostaining of prostate specimens, endogenous peroxidase was quenched with 3% H2O2 for 5 minutes at room temperature, microwave treatment was avoided, blocking was achieved with either 50% goat or horse serum in Tris-buffered saline containing 0.2% bovine serum albumin for 20 minutes at room temperature; each incubation step was followed by three washes with Tris-buffered saline. The specificity of the affinity-purified antibody to the β1C 785 to 808 peptide12Fornaro M Zheng DQ Languino LR The novel structural motif Gln795-Gln802 in the integrin β1C cytoplasmic domain regulates cell proliferation.J Biol Chem. 1995; 270: 24666-24669Crossref PubMed Scopus (54) Google Scholar in immunohistochemical staining was confirmed as follows: before the immunostaining procedure, the affinity-purified antibody to β1C was preincubated for 30 minutes at 4°C with 10 to 30 μg/ml of either β1C 785 to 808 peptide or a control fibrinogen-derived peptide.28Languino LR Duperray A Joganic KJ Fornaro M Thornton GB Altieri DC Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by ICAM1-fibrinogen recognition.Proc Natl Acad Sci USA. 1995; 92: 1505-1509Crossref PubMed Scopus (171) Google Scholar Frozen prostate tissue, obtained from autopsy specimens, was homogenized using lysis buffer containing 100 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO), 5% sodium dodecyl sulfate (SDS; American Bioanalytical, Natick, MA), 1 mmol/L phenylmethylsulfonyl fluoride (Life Technologies), 10 μg/ml leupeptin (Calbiochem, San Diego, CA), 1 mmol/L benzamidine (Sigma), 1 μmol/L d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (Boehringer Mannheim), 10 μg/ml soybean trypsin inhibitor (Life Technologies), using an OMNI 2000 homogenizer (OMNI International Inc., Gainesville, VA). Insoluble material was removed by centrifugation at 14,000 × g for 30 minutes at 4°C. The protein content in each lysate was quantitated using the BCA protein assay reagent (Pierce, Rockford, IL) according to the manufacturer's instructions. To detect β1C integrin, 200 μg of detergent tissue extract was electrophoresed on a 7.5% SDS-polyacrylamide gel under reducing conditions, transferred to nitrocellulose (Schleicher & Schuell, Keene, NH), and immunostained as described,26Fornaro M Tallini G Bofetiado CJM Bosari S Languino LR Down-regulation of β1C integrin, an inhibitor of cell proliferation, in prostate carcinoma.Am J Pathol. 1996; 149: 765-773PubMed Google Scholar using either 5 μg/ml affinity-purified antibody to β1C; 5 μg/ml nonimmune rabbit IgG; or 2.5 μg/ml mAb 13, monoclonal antibody to β1 integrin. BPH-1 cells were surface iodinated and proteins immunoprecipitated using rabbit antisera to β1C or to β1A or using normal rabbit serum, as previously described.12Fornaro M Zheng DQ Languino LR The novel structural motif Gln795-Gln802 in the integrin β1C cytoplasmic domain regulates cell proliferation.J Biol Chem. 1995; 270: 24666-24669Crossref PubMed Scopus (54) Google Scholar The immunocomplexes were separated on a 7.5% SDS-polyacrylamide gel under reducing conditions and visualized by autoradiography. HindIII DNA fragment, encoding the cytoplasmic domain of β1C (nucleotides 2357 to 2613),25Languino LR Ruoslahti E An alternative form of the integrin β1 subunit with a variant cytoplasmic domain.J Biol Chem. 1992; 267: 7116-7120Abstract Full Text PDF PubMed Google Scholar was isolated from the pBJ1-β1C plasmid. The fragment was inserted into the HindIII site of the Ch2 chimera described by Lukashev et al22Lukashev ME Sheppard D Pytela R Disruption of integrin function, and induction of tyrosine phosphorylation, by the autonomously expressed β1 integrin cytoplasmic domain.J Biol Chem. 1994; 269: 18311-18314Abstract Full Text PDF PubMed Google Scholar which consists of the extracellular domain of murine CD4 joined to the transmembrane domain of the β1 integrin. The resulting construct was designated Chβ1C. Correct assembly of the construct was verified by nucleotide sequencing. The Ch1 chimeric construct, designated here Ch1β1A, containing the extracellular domain of murine CD4 and the transmembrane and cytoplasmic domains of the β1Aintegrin, has been described by Lukashev et al.22Lukashev ME Sheppard D Pytela R Disruption of integrin function, and induction of tyrosine phosphorylation, by the autonomously expressed β1 integrin cytoplasmic domain.J Biol Chem. 1994; 269: 18311-18314Abstract Full Text PDF PubMed Google Scholar Each chimeric construct is expressed under the control of the mouse metallothionein I promoter, which can be induced by addition of ZnSO4 to the growth medium. PC3 cells were electroporated using a Genepulser apparatus set at 250 V and 900 μF using either 10 or 30 μg of the Ch1β1A or the Chβ1Cchimera, respectively. Hygromycin-resistant cells were selected using growth medium containing 0.2 mg/ml hygromycin B (Boehringer Mannheim). Hygromycin-resistant colonies were pooled, and the resultant population was analyzed for cell surface expression of each chimeric construct by fluorescence-activated cell sorting using rat monoclonal antibody to mouse CD4. As negative control antibody, isotype-matched rat IgG was used. Stable transfectants were maintained in growth medium containing 0.2 mg/ml hygromycin B. PC3 stable transfectants were starved for 24 hours in serum-free medium. Surface expression of chimeric constructs was induced by treating the cells with 75 μmol/L ZnSO4 (Sigma) in growth medium for 6 hours at 37°C. Cells were then detached with 0.05% trypsin/0.53 mmol/L ethylenediaminetetraacetic acid (Life Technologies), washed three times in serum-free medium, and analyzed by fluorescence-activated cell sorting using rat monoclonal antibody to mouse CD4 or isotype-matched rat IgG. Positive cells expressing comparable levels of the chimeric constructs were sorted using FACStar (Becton Dickinson). After sorting, the cell transfectants (15 × 103) were resuspended in serum-free medium and added to 96-well microtiter plates (ICN Biomedicals, Costa Mesa, CA) coated with 3 μg/ml fibronectin to ensure comparable attachment. After 1 hour and 30 minutes at 37°C, cells were washed twice, incubated either in the absence or in the presence of 10% FCS for 18 hours at 37°C, and pulsed with 1 μCi[3H]thymidine/well (5.0 Ci/mmol; Amersham Life Sciences, Arlington Heights, IL) during the last 3 hours of the 18-hour culture. Thymidine incorporation was evaluated as described.12Fornaro M Zheng DQ Languino LR The novel structural motif Gln795-Gln802 in the integrin β1C cytoplasmic domain regulates cell proliferation.J Biol Chem. 1995; 270: 24666-24669Crossref PubMed Scopus (54) Google Scholar In each experiment, duplicate or triplicate observations were performed, and the values are reported as mean ± standard error (SE). In parallel, in each experiment, the number of attached cells was evaluated after fixing and staining the cells with 0.5% crystal violet (Sigma), as described.29Languino LR Gehlsen KR Wayner E Carter WG Engvall E Ruoslahti E Endothelial cells use α2β1 integrin as a laminin receptor.J Cell Biol. 1989; 109: 2455-2462Crossref PubMed Scopus (258) Google Scholar The results were evaluated using a 630-nm wavelength filter in a Titertek Multiskan Bichromatic enzyme-linked immunosorbent assay reader (ICN). Group differences were compared using one-way analysis of variance followed by Bonferroni post hoc contrast. Immunohistochemical analysis of adult and fetal human tissue specimens was performed using an affinity-purified antibody to β1C; β1C was detected in a subset of epithelial cells. Specifically, simple or pseudostratified epithelia in prostate, liver, gallbladder, lung, and kidney were positive for β1C (see Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 8) in all the analyzed cases, whereas stratified squamous epithelium of the epidermis was negative (Figure 1E). In the prostate, β1C was preferentially expressed in the luminal glandular epithelium (Figure 1A). In contrast, the basal epithelium, composed of proliferating cells30Mao P Angrist A The fine structure of the basal cells of human prostate.Lab Invest. 1966; 15: 1768-1782PubMed Google Scholar identified by an antibody to basal cell-specific cytokeratins 1, 5, 10, and 14 (Figure 1D), did not reveal detectable levels of β1C(Figure 1A). Similarly, β1C immunoreactivity was undetectable in stromal and endothelial cells in all the analyzed tissues (see Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6). The specificity of the immunoreactivity of the antibody to β1C (Figure 2A) was confirmed by 1) inhibition by the β1C peptide, 785 to 808, used as the antigen (Figure 2B), 2) immunoblotting of detergent lysate of prostate tissue (Figure 2E), and 3) immunoprecipitation of surface-iodinated BPH-1 luminal epithelial cells (Figure 2F). The immunoblotting and immunoprecipitation both revealed bands with an electrophoretic mobility similar to that of β1A (Figure 2, E and F).12Fornaro M Zheng DQ Languino LR The novel structural motif Gln795-Gln802 in the integrin β1C cytoplasmic domain regulates cell proliferation.J Biol Chem. 1995; 270: 24666-24669Crossref PubMed Scopus (54) Google ScholarFigure 2In vivo and in vitro expression of β1C in prostate cells. A through D, a representative case of adult normal prostate tissue is shown. β1C immunostaining (A) was specifically inhibited by antibody absorption on the β1C 785 to 808 peptide (10 μg/ml) used as antigen (B) but not on an irrelevant peptide (C). The affinity-purified antibody to β1C (A throughC) or nonimmune rabbit IgG (D) were used at a final concentration of 5 μg/ml. E: newborn (1-month-old) prostate tissue detergent extract was electrophoresed on 7.5% SDS-polyacrylamide gel under reducing conditions, transferred to nitrocellulose membranes, and immunostained using 5 μg/ml affinity-purified antibody to β1C(a-β1C,lane 1); 5 μg/ml nonimmune rabbit IgG (nonimmune IgG,lane 2); or 2.5 μg/ml mAb 13, monoclonal antibody to β1(a-β1 mAb,lane 3). Proteins were visualized by enhanced chemiluminescence. Prestained marker proteins in kd are shown. The bracket (E) indicates the expected electrophoretic mobility of β1C and β1A. F: BPH-1 cells were surface iodinated and proteins immunoprecipitated using rabbit antisera either to β1C (a-β1C,lane 1) or β1A(a-β1A,lane 3), or normal rabbit serum (non immune,lane 2). The immunocomplexes were separated on a 7.5% SDS-polyacrylamide gel under reducing conditions and visualized by autoradiography. Arrowheads indicate β1C and β1A. Magnification, ×200 (A through D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Expression of β1C in luminal epithelium of fetal prostate. The immunohistochemical analysis was performed on a 20-week-old fetus using 1.85 μg/ml affinity-purified antibody to β1C (A andC); 1.85 μg/ml nonimmune rabbit IgG (B); or 0.2 μg/ml 34βE12, monoclonal antibody to cytokeratins 1, 5, 10, and 14 (D); or 5 μg/ml AE1/AE3, monoclonal antibody to type I and type II cytokeratins (E). The β1C immunostaining was specifically inhibited by antibody absorption on the β1C 785 to 808 peptide (30 μg/ml) used as antigen (C). Magnification, ×100. Note that β1Cexpression is undetectable in the basal layer of the fetal prostate gland (A).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Expression of β1C in prostate budding glands. The immunohistochemical analysis was performed on a 20-week-old fetus using 1.85 μg/ml affinity-purified antibody to β1C(A); 1.85 μg/ml of nonimmune rabbit IgG (B); or 1.25 μg/ml CAM 5.2, monoclonal antibody to cytokeratins 8 and 18 (C). Magnification, ×200. The expression of β1Ccoincides with a differentiated phenotype of the prostate gland: note that β1C is detectable only when a lumen is well defined in the gland.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5The β1C integrin is expressed in adult and fetal liver bile ducts but not in hepatocytes. Immunoperoxidase staining of β1C was performed on adult (A throughF) and 19-week-old fetal (Gand H) liver using 5 μg/ml affinity-purified antibody to β1C (A,C through E, G, andH) or 5 μg/ml nonimmune rabbit IgG (B). The β1C immunostaining (D) was specifically inhibited by antibody absorption on the β1C785 to 808 peptide (10 μg/ml) used as antigen (E), not on an irrelevant peptide (F). Magnification, ×100 (A andB); ×200 (C throughF), and ×400 (G andH). Note strong β1Cimmunoreactivity in the bile ducts (A, D,F, G, and H) and not in hepatocytes (C and G).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Differential expression of β1C and β1A in adult liver. Immunoperoxidase staining of β1C and β1A in liver with mild parenchyma alterations (steatohepatitis) was performed using 5 μg/ml affinity-purified antibodies to β1C (A and B) or to β1A (C andD). Nonimmune rabbit IgG (5 μg/ml) was used as a negative control (E). Magnification, ×200. Note, β1C is selectively expressed in biliary epithelium (B), whereas β1A is found in bile ducts (D), as well as in sinusoids (C). Arrows(A and C) point to sinusoids.View Large Image Figure ViewerDownload" @default.
- W1753444805 created "2016-06-24" @default.
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- W1753444805 date "1998-10-01" @default.
- W1753444805 modified "2023-10-10" @default.
- W1753444805 title "β1C Integrin in Epithelial Cells Correlates with a Nonproliferative Phenotype" @default.
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