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• W2078571502 abstract "Background & Aims: Muc3 intestinal mucin contains an extracellular cysteine-rich domain with 2 epidermal growth factor (EGF)-like motifs. The aim of this study was to determine the functional properties of Muc3 proteins. Methods: Glutathione S-transferase-fusion proteins containing both Muc3 EGF-like domains (m3EGF1,2) or truncated versions (m3EGF1 and m3EGF2) were purified from Escherichia coli. Mouse colon (young adult mouse colon) and human A431 and LoVo cells were examined for migration and tyrosine phosphorylation in response to recombinant proteins. LoVo cells were transfected with a human MUC3A transmembrane-EGF1,2 construct and a stable clone was isolated (LhM3c14). Endogenous MUC3A in LoVo was inhibited by specific small interfering RNA transfection. Apoptosis was quantitated by nuclear morphology or terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick-end labeling assay. Colitis was induced in mice by oral 5% dextran sodium sulfate or rectal 5% acetic acid, followed by enema treatments. Results: m3EGF1,2 stimulated cell migration in all cell lines, but did not induce proliferation. Migration was inhibited by a tyrosine phosphorylation inhibitor, genistein, but not by the EGF receptor inhibitor, tyrphostin (AG1478). Inhibition of endogenous MUC3A in LoVo reduced baseline migration. Tyrosine phosphorylation of ErbB receptors was not observed after treatment of cells with m3EGF1,2. LoVo cells pretreated with m3EGF1,2 and transfected LhM3c14 cells showed reduced apoptosis in response to tumor necrosis factor α or Fas-receptor stimulation. Administration of m3EGF1,2 per rectum significantly reduced mucosal ulceration and apoptosis in experimental acute colitis. Truncated proteins m3EGF1 and m3EGF2 had no effect. Conclusions: The Muc3 mucin cysteine-rich domain plays an active role in epithelial restitution, and represents a potential novel therapeutic agent for intestinal wound healing. Background & Aims: Muc3 intestinal mucin contains an extracellular cysteine-rich domain with 2 epidermal growth factor (EGF)-like motifs. The aim of this study was to determine the functional properties of Muc3 proteins. Methods: Glutathione S-transferase-fusion proteins containing both Muc3 EGF-like domains (m3EGF1,2) or truncated versions (m3EGF1 and m3EGF2) were purified from Escherichia coli. Mouse colon (young adult mouse colon) and human A431 and LoVo cells were examined for migration and tyrosine phosphorylation in response to recombinant proteins. LoVo cells were transfected with a human MUC3A transmembrane-EGF1,2 construct and a stable clone was isolated (LhM3c14). Endogenous MUC3A in LoVo was inhibited by specific small interfering RNA transfection. Apoptosis was quantitated by nuclear morphology or terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate biotin nick-end labeling assay. Colitis was induced in mice by oral 5% dextran sodium sulfate or rectal 5% acetic acid, followed by enema treatments. Results: m3EGF1,2 stimulated cell migration in all cell lines, but did not induce proliferation. Migration was inhibited by a tyrosine phosphorylation inhibitor, genistein, but not by the EGF receptor inhibitor, tyrphostin (AG1478). Inhibition of endogenous MUC3A in LoVo reduced baseline migration. Tyrosine phosphorylation of ErbB receptors was not observed after treatment of cells with m3EGF1,2. LoVo cells pretreated with m3EGF1,2 and transfected LhM3c14 cells showed reduced apoptosis in response to tumor necrosis factor α or Fas-receptor stimulation. Administration of m3EGF1,2 per rectum significantly reduced mucosal ulceration and apoptosis in experimental acute colitis. Truncated proteins m3EGF1 and m3EGF2 had no effect. Conclusions: The Muc3 mucin cysteine-rich domain plays an active role in epithelial restitution, and represents a potential novel therapeutic agent for intestinal wound healing. Mucins are a family of large secreted and membrane-bound glycoproteins expressed by most epithelial tissues, where they are directed to the cell surface and are thought to play a protective role.1Ho S.B. Shekels L.L. Mucin and goblet cell function.in: Koch T.R. Colonic diseases. Humana Press, Totowa, NJ2004: 53-72Google Scholar, 2Hollingsworth M.A. Swanson B.J. Mucins in cancer: protection and control of the cell surface.Nat Rev Cancer. 2004; 4: 45-60Crossref PubMed Scopus (1376) Google Scholar Mucin structure consists of a central domain of tandemly repeated units rich in serine and threonine residues that are O-glycosylated, flanked by unique carboxyl and amino terminal domains. Membrane-bound mucins are characterized by a short amino terminal domain followed by a large glycosylated tandem-repeat domain, an extracellular domain that is characterized in some cases by cysteine-rich areas with similarity to an epidermal growth factor (EGF)-like motif, a transmembrane segment, and a small cytoplasmic domain. The secreted mucins have cysteine-rich amino and carboxyl terminal domains with similarity to von Willebrand’s factor, which allows for polymerization through disulfide bonding and results in highly viscous mucous secretions. Gastrointestinal tract tissues typically express high levels of both secreted and membrane-bound mucins. Several different membrane-bound mucins (MUC1, MUC3A/B, MUC4, MUC12, MUC13, and MUC17) have been described in intestinal and colonic mucosa, localized to the apical membranes of columnar and goblet cells. Membrane-bound mucins may form a static external barrier at the cell surface, or they may be cleaved or shed into the lumen; however, the specific sites and mechanisms of this cleavage have not been well described.2Hollingsworth M.A. Swanson B.J. Mucins in cancer: protection and control of the cell surface.Nat Rev Cancer. 2004; 4: 45-60Crossref PubMed Scopus (1376) Google Scholar Splice variants of transcribed membrane-bound mucin genes have been reported that lack the transmembrane domain, and hence are considered soluble or secreted forms of these mucins.3Williams S.J. Munster D.J. Quin R.J. Gotley D.C. McGuckin M.A. The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.Biochem Biophys Res Commun. 1999; 261: 83-89Crossref PubMed Scopus (74) Google Scholar, 4Crawley S.C. Gum J.R. Hicks J.W. Pratt W.S. Aubert J.P. Swallow D.M. Kim Y.S. Genomic organization and structure of the 3’ region of human MUC3: alternative splicing predicts membrane-bound and soluble forms of the mucin.Biochem Biophys Res Commun. 1999; 263: 728-736Crossref PubMed Scopus (69) Google Scholar Posttranslational processing of membrane-bound mucins also may occur, resulting in cleavage from the membrane-spanning domain, resulting in a secreted form that may be stored in goblet-cell vacuoles.5Moniaux N. Escande F. Batra S.K. Porchet N. Laine A. Aubert J.P. Alternative splicing generates a family of putative secreted and membrane-associated MUC4 mucins.Eur J Biochem. 2000; 267: 4536-4544Crossref PubMed Scopus (88) Google Scholar, 6Komatsu M. Arango M.E. Carraway K.L. Synthesis and secretion of Muc4/sialomucin complex: implication of intracellular proteolysis.Biochem J. 2002; 368: 41-48Crossref PubMed Scopus (35) Google Scholar The mouse Muc3 mucin contains a cysteine-rich extracellular region with 2 EGF-like domains,7Shekels L.L. Hunninghake D.A. Tisdale A.S. Gipson I.K. Kieliszewsji M. Kozak C.A. Ho S.B. Cloning and characterization of mouse intestinal Muc3 mucin: 3’ sequence contains epidermal-growth-factor-like domains.Biochem J. 1998; 330: 1301-1308Crossref PubMed Scopus (48) Google Scholar and is similar in sequence and chromosomal localization to the human MUC3A, MUC3B, and MUC17 genes.3Williams S.J. Munster D.J. Quin R.J. Gotley D.C. McGuckin M.A. The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.Biochem Biophys Res Commun. 1999; 261: 83-89Crossref PubMed Scopus (74) Google Scholar, 4Crawley S.C. Gum J.R. Hicks J.W. Pratt W.S. Aubert J.P. Swallow D.M. Kim Y.S. Genomic organization and structure of the 3’ region of human MUC3: alternative splicing predicts membrane-bound and soluble forms of the mucin.Biochem Biophys Res Commun. 1999; 263: 728-736Crossref PubMed Scopus (69) Google Scholar, 8Pratt W.S. Crawley S. Hicks J. Ho J. Nash M. Kim Y.S. Gum J.R. Swallow D.M. Multiple transcripts of MUC3: evidence for two genes, MUC3A and MUC3B.Biochem Biophys Res Commun. 2000; 275: 916-923Crossref PubMed Scopus (71) Google Scholar Other membrane-bound mucins that contain EGF-like domains include human MUC49Moniaux N. Nollet S. Porchet N. Degand P. Laine A. Aubert J.-P. Complete sequence of the human mucin MUC4: a putative cell membrane-associated mucin.Biochem J. 1999; 338: 325-333Crossref PubMed Scopus (220) Google Scholar; the rodent homologue of MUC4, termed sialomucin complex, or ascites sialoglycoprotein ASGP1,2,10Carraway K. Fregien N. Carraway 3rd, K. Carraway C. Tumor sialomucin complexes as tumor antigens and modulators of cellular interactions and proliferation.J Cell Sci. 1992; 103: 299-307PubMed Google Scholar MUC12,11Williams S.J. McGuckin M.A. Gotley D.C. Eyre H.J. Sutherland G.R. Antalis T.M. Two novel mucin genes down-regulated in colorectal cancer identified by differential display.Cancer Res. 1999; 59: 4083-4089PubMed Google Scholar and MUC13.12Williams S.J. Wreschner D.H. Tran M. Eyre H.J. Sutherland G.R. McGuckin M.A. Muc13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells.J Biol Chem. 2001; 276: 18327-18336Crossref PubMed Scopus (255) Google Scholar The MUC1 membrane-bound mucin is ubiquitous in all epithelial tissues but lacks cysteine-rich extracellular regions.2Hollingsworth M.A. Swanson B.J. Mucins in cancer: protection and control of the cell surface.Nat Rev Cancer. 2004; 4: 45-60Crossref PubMed Scopus (1376) Google Scholar The function of membrane-bound mucins containing cysteine-rich EGF-like domains in the intestine and their possible role in mucosal protection has not been determined. Alterations in mucin proteins have been noted in conditions such as gastritis, peptic ulcer disease, intestinal neoplasia, Crohn’s disease, and ulcerative colitis. The membrane-bound mucin gene cluster (MUC3A/B, MUC12, and MUC17) located on chromosome 7q22 has been implicated in a large population survey as a possible site for a genetic susceptibility to inflammatory bowel disease.13Satsangi J. Parkes M. Louis E. Two-stage genome-wide search in inflammatory bowel disease: evidence for susceptibility loci on chromosomes 3, 7 and 12.Nat Genet. 1996; 14: 199-202Crossref PubMed Scopus (677) Google Scholar, 14Satsangi J. Jewell D.P. Bell J.I. The genetics of inflammatory bowel disease.Gut. 1997; 40: 572-574PubMed Google Scholar Kyo et al15Kyo K. Muto T. Nagawa H. Lathrop G.M. Nakamura Y. Associations of distinct variants of the intestinal mucin gene MUC3A with ulcerative colitis and Crohn’s disease.J Hum Genet. 2001; 46: 5-20Crossref PubMed Scopus (73) Google Scholar reported that rare alleles of the MUC3A mucin gene were more common in patients with ulcerative colitis compared with controls. They also examined single nucleotide polymorphisms over 3400 bp of the MUC3A and MUC3B genes in patients with ulcerative colitis or Crohn’s disease.15Kyo K. Muto T. Nagawa H. Lathrop G.M. Nakamura Y. Associations of distinct variants of the intestinal mucin gene MUC3A with ulcerative colitis and Crohn’s disease.J Hum Genet. 2001; 46: 5-20Crossref PubMed Scopus (73) Google Scholar One polymorphism that replaces a tyrosine of the MUC3A cytoplasmic domain with an asparagine or histidine was shown to occur with a higher frequency in patients with a family history of Crohn’s disease compared with patients without a family history. Muc3 gene expression has been shown to be up-regulated in small intestinal villi in a mouse model of acute enteritis,16Shekels L.L. Anway R.E. Lin J. Lawrence C.E. Garside P. Kennedy M.W. Ho S.B. Coordinated Muc2 and Muc3 mucin gene expression in Trichinella spiralis infection in wild-type and cytokine deficient mice.Dig Dis Sci. 2001; 46: 1757-1764Crossref PubMed Scopus (38) Google Scholar and mouse Muc3 and human MUC3A promoter activity can be regulated by cytokines and growth factors.17Shekels L.L. Ho S.B. Characterization of the mouse Muc3 membrane bound intestinal mucin 5’ coding and promoter regions: regulation by inflammatory cytokines.Biochim Biophys Acta. 2003; 1627: 90-100Crossref PubMed Scopus (25) Google Scholar, 18Gum Jr, J.R. Hicks J.W. Crawley S.C. Dahl C.M. Yang S.C. Roberton A.M. Kim Y.S. Initiation of transcription of the MUC3A human intestinal mucin from a TATA-less promoter and comparison with the MUC3B amino terminus.J Biol Chem. 2003; 278: 49600-49609Crossref PubMed Scopus (21) Google Scholar These data suggest that MUC3A mucins may play an important role in intestinal mucosal defense and susceptibility for chronic inflammation. Cysteine-rich EGF-type domains, also known as G-modules, are common structural features of proteins that participate in protein–protein interactions, including both growth factors and structural proteins.19Baron M. Norman D. Campbell I. Protein modules.Trends Biochem Sci. 1991; 16: 13-17Abstract Full Text PDF PubMed Scopus (180) Google Scholar We hypothesized that the cysteine-rich domains of the Muc3 mucin also may play a role in protein–protein interactions. Therefore, the purpose of this study was to begin to delineate the functional significance of the extracellular cysteine-rich EGF-like domains of the Muc3 intestinal mucin. Synthesis of full-length mucin proteins is not feasible because of their large size. One strategy is to study individual mucin protein domains synthesized as recombinant fusion proteins. This has been used to study the interactions and phosphorylation of the cytoplasmic domain of the MUC1 mucin.20Ren J. Li Y. Kufe D. Protein kinase C delta regulates function of the DF3/MUC1 carcinoma antigen in beta-catenin signaling.J Biol Chem. 2002; 277: 17616-17622Crossref PubMed Scopus (131) Google Scholar Thus, we report here the activity of recombinant Muc3 EGF-like domain peptides in relation to cell migration, proliferation, stimulation of EGF receptors, apoptosis, and intestinal wound healing. The extracellular region of mouse Muc3 including both EGF-like domains (m3EGF1,2) was amplified from mouse intestinal complementary DNA (cDNA). In addition, products corresponding to only the first EGF-like domain (m3EGF1) or only the second EGF-like domain (m3EGF2) also were amplified, as described previously.7Shekels L.L. Hunninghake D.A. Tisdale A.S. Gipson I.K. Kieliszewsji M. Kozak C.A. Ho S.B. Cloning and characterization of mouse intestinal Muc3 mucin: 3’ sequence contains epidermal-growth-factor-like domains.Biochem J. 1998; 330: 1301-1308Crossref PubMed Scopus (48) Google Scholar The resulting fragments were cloned into the pGEX-2TK vector (Amersham, Piscataway, NJ), sequenced, and introduced into Escherichia coli strain BL21 (Invitrogen, Carlsbad, CA). Glutathione S-transferase (GST)-fusion proteins then were expressed in E coli by induction with .5 mmol/L isopropylthio-β-D-galactoside (IPTG; Fisher, Pittsburgh, PA) and purified by affinity chromatography using glutathione agarose (Sigma Chemical Co, St. Louis, MO). Figure 1A shows the spacing of the cysteine residues in mouse Muc3 and related peptides. The specific amino acid sequences of mouse Muc3 and human MUC3A EGF-like domains and linker region are indicated in Figure 1B. The fusion proteins are diagrammed in Figure 1C. Mouse and human cells known to contain EGF-family receptors were used. A431 cells, an immortalized human epidermoid carcinoma cell line, were obtained from American Type Culture Collection (Manassas, VA). A431 cells express high levels of EGF (ErbB1) receptor and migrate in response to EGF.21Gill G.N. Lazar C.S. Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells.Nature. 1981; 293: 305-307Crossref PubMed Scopus (323) Google Scholar, 22Kawahara E. Nakada N. Hikichi T. Kobayashi J. Nakanishi I. EGF and beta1 integrin convergently regulate migration of A431 carcinoma cell through MAP kinase activation.Exp Cell Res. 2002; 272: 84-91Crossref PubMed Scopus (21) Google Scholar A431 cells lack human MUC3A messenger RNA (mRNA) as measured by reverse transcription and polymerase chain reaction (PCR) amplification (data not shown). LoVo cells are a human colon adenocarcinoma cell line described previously and express ErbB1 and low-level ErbB2 receptors.23Magne N. Fischel J.L. Dubreuil A. Formento P. Poupon M.F. Laurent-Puig P. Milano G. Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumour cells on the antiproliferative effect of ZD1839 (“Iressa”).Br J Cancer. 2002; 86: 1518-1523Crossref PubMed Scopus (154) Google Scholar, 24Nyati M.K. Maheshwari D. Hanasoge S. Sreekumar A. Rynkiewicz S.D. Chinnaiyan A.M. Leopold W.R. Ethier S.P. Lawrence T.S. Radiosensitization by pan ErbB inhibitor CI-1033 in vitro and in vivo.Clin Cancer Res. 2004; 10: 691-700Crossref PubMed Scopus (80) Google Scholar LoVo cells previously have been shown to express an additional truncated form of human MUC3A that lacks a portion of the EGF2 domain and the entire transmembrane domain.3Williams S.J. Munster D.J. Quin R.J. Gotley D.C. McGuckin M.A. The MUC3 gene encodes a transmembrane mucin and is alternatively spliced.Biochem Biophys Res Commun. 1999; 261: 83-89Crossref PubMed Scopus (74) Google Scholar The LoVo cells used in this study have this truncated form as the major mRNA species, and also have a lesser amount of the intact MUC3A with both EGF domains, as determined by reverse-transcription PCR using primers targeting the cytoplasmic domain and extracellular EGF domains (data not shown). Cells were grown in 24-well plates for cell migration and proliferation experiments or T-25 flasks for immunoblotting experiments using Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) + 50 U penicillin/mL and .05 μg streptomycin/mL (Invitrogen). Cells were cultured at 37°C, 5% CO2, 10% FCS until the desired confluence was reached. Then the monolayers were washed with phosphate-buffered saline (PBS) 24 hours before experiments and switched to serum-free media for cell migration and immunoblotting experiments or media containing .5% serum for cell proliferation experiments. Young adult mouse colon (YAMC) cells are conditionally immortalized mouse colon cells grown in RPMI 1640 supplemented with 5% FCS + 50 U penicillin/mL and .05 μg streptomycin/mL, as described previously.25Kaiser G.C. Polk D.B. Tumor necrosis factor alpha regulates proliferation in a mouse intestinal cell line.Gastroenterology. 1997; 112: 1231-1240Abstract Full Text PDF PubMed Scopus (114) Google Scholar, 26Frey M.R. Golovin A. Polk D.B. Epidermal growth factor-stimulated intestinal epithelial cell migration requires Src family kinase-dependent p38 MAPK signaling.J Biol Chem. 2004; 279: 44513-44521Crossref PubMed Scopus (111) Google Scholar YAMC cells lack Muc3 mRNA as measured by reverse-transcription PCR (data not shown). A431 or LoVo cells grown to 90%–100% confluency in 24-well plates were cultured overnight in serum-free medium, the medium was replaced with PBS, and the monolayers were wounded mechanically using a single-edged razorblade as previously described.27Burk R.R. A factor from a transformed cell line that affects cell migration.Proc Natl Acad Sci U S A. 1973; 70: 369-372Crossref PubMed Scopus (146) Google Scholar Purified recombinant human EGF was used as a positive control for cell migration (Sigma), which had an effective storage shelf-life of 3 months. During inhibition experiments, cells were pre-incubated with 150 nmol/L tyrphostin AG1478 (Sigma) or 55.5 μmol/L genistein (Sigma) for 30 minutes at 37°C and then washed with PBS before wounding. After wounding, cells were rinsed twice with PBS and further incubated with the peptide of interest in Dulbecco’s modified Eagle medium for 18–24 hours (37°C, 5% CO2, 0% FCS). During inhibition experiments, cells were treated with the inhibitor and the peptide of interest for 18–24 hours. After fixation and staining, those cells that had migrated from the wounded edge were counted at 100×, using an inverted light microscope. Two successive fields were counted and averaged within 1 well, and 3–12 wells were averaged for each condition in each experiment. YAMC cells were grown to confluency and then a rotating disc was used to scrape cells from an area within a 24-well plate. At 24 hours, the time necessary for closure of the wound when treated with the positive control EGF, the area of wound remaining was measured, as described previously.26Frey M.R. Golovin A. Polk D.B. Epidermal growth factor-stimulated intestinal epithelial cell migration requires Src family kinase-dependent p38 MAPK signaling.J Biol Chem. 2004; 279: 44513-44521Crossref PubMed Scopus (111) Google Scholar The absolute amount of cell migration measured varies from experiment to experiment primarily because of differences in the degree of initial confluency of the cells, which affected the baseline levels of migration in serum-free medium. Other variables included the specific time of incubation, and the storage time of the purified EGF positive control. To control for these variations, each experiment was performed with appropriate positive (purified recombinant human EGF) and negative (no recombinant protein) controls. To simplify comparisons between experiments, the data were normalized according to the baseline migration in serum-free medium, with the baseline migration in serum-free medium set at 100% in each experiment. Data are represented as percentage of baseline cell migration. MUC3A expression was knocked down using a small interfering RNA (siRNA) technique.28Hannon G.J. Rossi J.J. Unlocking the potential of the human genome with RNA interference.Nature. 2004; 431: 371-378Crossref PubMed Scopus (961) Google Scholar SMARTpool MUC3A siRNA reagent was obtained from Dharmacon (Chicago, IL). A nonspecific control pool of siRNA also was obtained from Dharmacon and used as a negative control. LoVo cells were transfected with the siRNA SMARTpool reagent and Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. After 48 hours cells were changed to serum-free media for cell migration assays or harvested for RNA isolation using Trizol (Invitrogen). MUC3A and actin mRNA levels were determined by reverse-transcription and PCR amplification using MUC3A and actin-specific primers (MUC3A primers: forward: 5’ tccggatggtggggcgg 3’; reverse: 5’ acactgaggacgaggt 3’; actin primers: forward: 5’ agcaagagaggcatcctcaccctgaagtac 3’; reverse: 5’ gcacagcttctccttaatgtcacgcacgat 3’). RNA levels also were determined by slot-blot analysis. One microgram of total RNA was applied to a BioRad (Hercules, CA) Zeta Probe membrane and the membrane was prehybridized and hybridized in Rapid-hyb buffer (Amersham) as directed by the manufacturer. Radiolabeled MUC3A and actin cDNA probes were prepared by random primer labeling. After hybridization, the membrane was washed once with 2 × standard saline citrate and .1% sodium dodecyl sulfate (SDS) for 30 minutes at room temperature, followed by washing twice with .1 × standard saline citrate and .1% SDS for 30 minutes at 55°. The washed blots were exposed to Kodak (Atlanta, GA) radiograph film at −70°. Hybridization signals were quantitated by densitometry. Cells were cultured in 24-well plates until 60% confluent and then switched to control medium containing .5% serum for 24 hours. After the monolayers were rinsed with PBS, they were incubated with the peptide of interest in Dulbecco’s modified Eagle medium for 24 hours. Cells were quantitated by trypan blue staining.25Kaiser G.C. Polk D.B. Tumor necrosis factor alpha regulates proliferation in a mouse intestinal cell line.Gastroenterology. 1997; 112: 1231-1240Abstract Full Text PDF PubMed Scopus (114) Google Scholar Two counts were averaged from each well; 6 wells were averaged per treatment. Proliferation for each treatment was represented as a percentage relative to the control medium. Cells also were grown in 96-well plates and cell numbers were estimated by a tetrazolium-based colorimetric assay using dimethylthiazole diphenyltetrazolium bromide (Sigma), as described previously.29Shekels L.L. Beste J.E. Ho S.B. Tauroursodeoxycholic acid protects in vitro models of human colon cancer cells from cytotoxic effects of hydrophobic bile acids.J Clin Lab Med. 1995; 127: 57-66Abstract Full Text PDF Scopus (39) Google Scholar Cell monolayers were washed with PBS and then lysed in cell lysis buffer containing 150 mmol/L NaCl, 1% NP40, .5% deoxycholic acid, .1% SDS, 1 mmol/L phenylmethylsulfonyl fluoride, and 50 mmol/L Tris pH 7.5. Cells were scraped from the flask and the lysate was incubated on ice for 10–15 minutes. After vortexing for 20 seconds, the lysate was centrifuged at 14,000 rpm for 10 minutes. Membranes were prepared from cells grown in T-75 flasks by the addition of a membrane lysis buffer containing 20 mmol/L Tris HCl pH 8.0, 2 mmol/L ethylenediaminetetraacetic acid, and 1 mmol/L β-mercaptoethanol. Protease and phosphatase inhibitors were added before use. The monolayers were scraped in lysis buffer, put into ice-cold centrifuge tubes, and sheared using a 28-gauge needle. The lysate was centrifuged at 1000 rpm for 5 minutes and then the supernatant was centrifuged at 15,000 rpm for 30 minutes. The pellet containing the membranes was resuspended in 100 μL of RIPA lysis buffer and sheared using a 28-gauge needle. Reagents were purchased from Sigma. For immunoprecipitation, cell lysates or membrane preparations were incubated with either anti–EGF-receptor antibody, anti-ErbB2 antibody, or anti-ErbB3 antibody (all from Cell Signaling, Beverly, MA), at a 1:100 dilution overnight at 4°C; after which protein A beads (30 μL/300 μL lysate) were added for 2 hours. Immunoprecipitates were recovered by centrifugation and washed 3 times in lysis buffer. Pellets were resuspended in 2 × SDS sample buffer and vortexed for 30 seconds. Immunoprecipitates were denatured for 5 minutes at 100°C and separated by SDS polyacrylamide gel electrophoresis before transfer to nitrocellulose membrane. After blocking for 2 hours with 5% nonfat dried milk in Tris-buffered saline and washing 2 × 5 minutes with .05% Tween in Tris-buffered saline, Western blotting was conducted using an antiphosphotyrosine monoclonal antibody (Cell Signaling) at a 1:2000 dilution overnight at 4°C. To confirm equal loading, samples were immunoprecipitated with receptor-specific antibodies and then blotted with the same receptor-specific antibody. The membranes were washed twice with .05% Tween in Tris-buffered saline and then incubated for 1 hour with the peroxidase-conjugated secondary antibody (Sigma) at a 1:2000 dilution. After washing 4 × 5 minutes, proteins were visualized by chemiluminescence detection using Pierce Supersignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). Immunoblotting was performed in a similar fashion on samples of cell lysates or membrane preparations without prior immunoprecipitation, using antiphosphotyrosine monoclonal antibody (Cell Signaling). Determination of free cysteines in recombinant mucin proteins was performed using a method modified from Singh et al,30Singh R. Blattler W.A. Collinson A.R. Assay for thiols based on reactivation of papain.Methods Enzymol. 1995; 251: 229-237Crossref PubMed Scopus (26) Google Scholar as previously published. The Thiol and Sulfide Quantitation Kit from Molecular Probes (Eugene, OR) was used. Briefly, recombinant mucin protein or control peptide was incubated with inactive papain-SSCH3. Free thiols in the protein reduce the papain-SSCH3 to an active form. The activity of the reduced papain is measured using the chromogenic papain substrate, L-BAPNA (N-benzoyl-L-arginine, p-nitroanilide). By using the same method, a standard curve is prepared using a known concentration of L-cysteine. This standard curve is used to calculate the free thiol in the recombinant protein. A peptide corresponding to a tandem repeat sequence of the mouse Muc5AC was used as a control peptide containing no cysteines (KQTSSPNTGKTSTISTT). EGF also was used as a control peptide that has no free thiols; with 6 cysteines that all are involved in disulfide bonds. A peptide corresponding to a nonrepetitive portion of the mouse Muc5AC was used as a control peptide containing 2 free thiols (CKNELCNWTNWLDGSYPGSGRNSGD). Primers corresponding to the human MUC3A EGF1,2 domain were synthesized and used to amplify human colon cDNA. The 936-bp human MUC3A EGF1,2 PCR product encoded the 2 human MUC3A EGF-like domains, the MUC3A transmembrane region, and 20 amino acids of the MUC3A cytoplasmic domain (Figure 1D). The MUC3A PCR fragment was ligated to pFLAG-CMV-3 (Sigma). This vector encodes for the preprotrypsin leader sequence, allowing for secretion of expressed proteins. The preprotrypsin leader sequence is followed by the FLAG tag at the amino terminus of the expressed protein of interest. The MUC3A transmembrane sequence targets the protein for insertion into the cell membrane. Confirmation of sequence and orientation of the insert was achieved by DNA sequencing. LoVo cells were transfected with the human MUC3A transmembrane-EGF1,2 construct using Lipofectamine 2000 (Invitrogen). Forty-eight hours after the start of transfection, cells were cultured in the presence of 800 μg/mL G418 (InvivoGen, San Diego, CA). G418-resistant clones were isolated using sterile cloning rings. Clone LhM3c14 was used for apoptosis assays. LoVo cells also were transfected with empty vector to generate a stable mock-transfected clone (Lmock). The transfectants were maintained in selective medium containi" @default.
• W2078571502 created "2016-06-24" @default.
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• W2078571502 date "2006-11-01" @default.
• W2078571502 modified "2023-10-16" @default.
• W2078571502 title "Cysteine-Rich Domains of Muc3 Intestinal Mucin Promote Cell Migration, Inhibit Apoptosis, and Accelerate Wound Healing" @default.
• W2078571502 cites W1496993956 @default.
• W2078571502 cites W1529922125 @default.
• W2078571502 cites W1576174931 @default.
• W2078571502 cites W1590994864 @default.
• W2078571502 cites W1598843348 @default.
• W2078571502 cites W1855226200 @default.
• W2078571502 cites W1963629707 @default.
• W2078571502 cites W1965097026 @default.
• W2078571502 cites W1968598371 @default.
• W2078571502 cites W1973024773 @default.
• W2078571502 cites W1975106258 @default.
• W2078571502 cites W1976162686 @default.
• W2078571502 cites W1978596592 @default.
• W2078571502 cites W1981341773 @default.
• W2078571502 cites W1982087729 @default.
• W2078571502 cites W1989897633 @default.
• W2078571502 cites W1994830329 @default.
• W2078571502 cites W2000843748 @default.
• W2078571502 cites W2001805903 @default.
• W2078571502 cites W2008037959 @default.
• W2078571502 cites W2018620221 @default.
• W2078571502 cites W2020475637 @default.
• W2078571502 cites W2022357421 @default.
• W2078571502 cites W2029580509 @default.
• W2078571502 cites W2033757431 @default.
• W2078571502 cites W2033802532 @default.
• W2078571502 cites W2034457272 @default.
• W2078571502 cites W2042838875 @default.
• W2078571502 cites W2043000199 @default.
• W2078571502 cites W2050484748 @default.
• W2078571502 cites W2052312685 @default.
• W2078571502 cites W2052330087 @default.
• W2078571502 cites W2052884329 @default.
• W2078571502 cites W2056189319 @default.
• W2078571502 cites W2063220206 @default.
• W2078571502 cites W2073893705 @default.
• W2078571502 cites W2078244407 @default.
• W2078571502 cites W2089084934 @default.
• W2078571502 cites W2090548042 @default.
• W2078571502 cites W2094381106 @default.
• W2078571502 cites W2099599782 @default.
• W2078571502 cites W2110883638 @default.
• W2078571502 cites W2121618677 @default.
• W2078571502 cites W2131192078 @default.
• W2078571502 cites W2134181529 @default.
• W2078571502 cites W2144233480 @default.
• W2078571502 cites W2149881105 @default.
• W2078571502 cites W2150312232 @default.
• W2078571502 cites W2189565186 @default.
• W2078571502 cites W2196240791 @default.
• W2078571502 cites W2398138655 @default.
• W2078571502 cites W2516194524 @default.
• W2078571502 cites W4236697171 @default.
• W2078571502 cites W4243638039 @default.
• W2078571502 cites W4251858006 @default.
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